• High precision and sensitivity: The use of duplex, hydrolysis probe-based assays allows for highly precise detection of mutations. The presence of both mutant and wild-type probes in the same reaction ensures that researchers can detect and quantify minor genetic variations with great accuracy, crucial for studies in heterogeneous cancer samples where only a few cells may carry the mutation.
• Enhanced specificity: The integration of LNA into the probes increases the binding affinity and specificity towards the target sequences, minimizing the risk of non-specific bindings and improving the overall reliability of the assays.
• Multiplexing capability: Each assay is capable of detecting mutations using two different fluorescent dye combinations, allowing for the simultaneous analysis of mutant and wild-type alleles within the same reaction. This multiplexing ability is particularly useful in applications requiring the analysis of multiple targets, such as assessing co-occurring mutations in cancer.
• Flexibility in sample analysis: By dividing the reaction across multiple wells, even greater sensitivity can be achieved, facilitating the detection of extremely rare mutations. This is especially valuable in cancer research, where detecting low-frequency mutations can inform prognosis and treatment strategies.
• Streamlined workflow: Supplied in a single-tube format with ready-to-use primer pairs and probes, these assays simplify the experimental setup, enabling efficient and straightforward integration into existing research workflows.

EGFR, or epidermal growth factor receptor, is a receptor tyrosine whose mutations are implicated in the uncontrolled cell proliferation characteristic of non-small cell lung cancer (NSCLC). These specific EGFR gene variants play a critical role in the cascade of signals that lead to cellular growth and division, marking them as prime targets for therapies tailored to halt this progression. The identification and targeting of these mutations have been pivotal in improving patient outcomes, as they are central to the development and advancement of the disease and respond well to targeted treatments that specifically inhibit the aberrant EGFR signaling in NSCLC.

• Exon 19 Deletions: Deletions in exon 19 generally result in the loss of four amino acids from the EGFR kinase domain, which alters the configuration of the protein, enhancing tyrosine kinase activity. This increase in enzymatic activity leads to unchecked cell signaling pathways that drive tumorigenesis and tumor growth. Molecular models suggest that this deletion affects the dynamic equilibrium of the kinase domain, shifting it towards an active state.
• L858R Point Mutation in Exon 21: The L858R mutation changes the hydrophobic core of the kinase domain, leading to a conformation that mimics the active state of the EGFR protein. This mutation stabilizes the active conformation even in the absence of ligand binding, promoting constant downstream signaling. Structural analysis reveals that the arginine residue at position 858 forms new stabilizing interactions that are absent in the wild-type kinase, which increases the affinity of the kinase domain for ATP.
• T790M Point Mutation in Exon 20: The T790M mutation introduces a bulkier methionine residue into the ATP-binding pocket, which can affect drug binding. This mutation is associated with acquired resistance to first- and second-generation TKIs because it increases the binding affinity for ATP, making it more competitive against these drugs. The steric hindrance caused by the methionine also interferes with the proper binding of some TKIs.
• Exon 20 Insertions: Insertions in exon 20 lead to the addition of amino acids within or near the C-helix of the kinase domain, disrupting the normal kinase activity regulation and causing constitutive activation. These insertions can lead to unique structural changes that may affect the binding of ATP and TKIs differently compared to other EGFR mutations, explaining the resistance to certain therapies.
• G719X Point Mutation in Exon 18: Mutations at codon 719, typically resulting in substitution of glycine by cysteine (G719C), alanine (G719A), or serine (G719S), alter the ATP-binding pocket of the EGFR kinase domain. These changes alter the charge and steric properties of the pocket, increasing the kinase's intrinsic activity. The G719X mutations are thought to affect the conformational dynamics of the kinase activation loop, enhancing its activity even without ligand stimulation.

   

   

KRAS, or Kirsten rat sarcoma viral oncogene homolog, is a gene encoding a GTPase that functions as a molecular switch within the RAS/MAPK signaling pathway, regulating cell division, differentiation, and apoptosis. In lung cancer, particularly NSCLC, mutations in the KRAS gene represent a common oncogenic driver, resulting in constitutive activation of downstream signaling pathways that promote cellular proliferation and survival independent of external growth signals. These mutations are often associated with resistance to EGFR-targeted therapies and are considered a hallmark of poor prognosis. The identification of KRAS mutations provides critical insight into the pathophysiology of lung cancer and guides therapeutic decision-making, although direct targeting remains challenging.

• G12C Mutation: The G12C mutation results from a single nucleotide change, leading to the substitution of glycine with cysteine at codon 12 in the KRAS protein. This alteration disrupts the GTPase activity, locking KRAS in its active GTP-bound state, and perpetuates the growth signal. This specific variant is noteworthy because it is targetable by covalent inhibitors that bind to the cysteine residue, offering a therapeutic opportunity for patients with KRAS G12C-mutant lung cancer.
• G12D and G12V Mutations: Both the G12D and G12V mutations involve the substitution of glycine at codon 12 with aspartic acid and valine, respectively. These changes further reduce the intrinsic GTPase activity of KRAS, leading to sustained activation of the protein. These mutations are less amenable to targeted therapy compared to G12C, but they remain important biomarkers for prognosis and are the subject of ongoing drug development efforts.
• G13 Mutations: Mutations at codon 13, most commonly resulting in a glycine-to-aspartic acid substitution (G13D), similarly impair the GTPase activity of KRAS, contributing to continuous activation of signaling pathways. The impact of G13 mutations on prognosis and treatment response is an active area of research, with some studies suggesting differential responses to certain chemotherapy agents.

   

   

BRAF, or v-Raf murine sarcoma viral oncogene homolog B, is a proto-oncogene that encodes a protein kinase involved in the MAPK/ERK signaling pathway, which regulates cell growth, differentiation, and survival. In lung cancer, mutations in the BRAF gene can lead to constitutive activation of the MAPK pathway, driving tumorigenesis and cancer progression. These mutations are less common than KRAS or EGFR mutations but are important because they represent another piece of the lung cancer genomic puzzle and are amenable to targeted therapy. The discovery of specific BRAF mutations has expanded the arsenal of molecularly targeted treatments, providing tailored options that can significantly impact clinical outcomes.

• V600E Mutation: The V600E mutation involves the substitution of valine with glutamic acid at position 600, which is located in the activation segment of the BRAF kinase domain. This mutation leads to a high level of kinase activity that stimulates the MAPK/ERK pathway without the need for upstream activation. BRAF V600E is the most common BRAF mutation in lung cancer and is actionable with inhibitors that have demonstrated efficacy in treating melanoma, leading to interest in their use for lung cancer harboring the same mutation.
• Non-V600 Mutations: Non-V600 BRAF mutations, such as G469A and D594G, occur in regions of the kinase domain other than the activation segment and result in intermediate to low kinase activity. While these mutations are less common and less well-understood than the V600E mutation, they can still contribute to oncogenesis through aberrant MAPK pathway signaling. Their therapeutic implications are currently an area of active investigation, with a potential for combination therapies to target the altered signaling cascade effectively.

   

   

ALK, or anaplastic lymphoma kinase, is a gene that can create fusion proteins through chromosomal rearrangements. These ALK fusion proteins are oncogenic drivers in a subset of NSCLC and are effectively targeted by ALK inhibitors.

MET, or MET proto-oncogene, receptor tyrosine kinase, is a gene that encodes the MET protein, which plays a role in cell growth, survival, and metastasis. MET amplifications or mutations can contribute to the progression of both NSCLC and SCLC, making it a potential target for therapy.

PIK3CA, or phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, is a gene that is part of the PI3K/AKT pathway, a critical pathway for cell growth and survival. Mutations in PIK3CA are implicated in NSCLC and can contribute to cancer progression and resistance to therapies.