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Description

Problem Definition

Non small cell lung cancer (NSCLC) is one of the most common types of lung cancer, accounting for 85% of all lung cancer cases. It got its name from its appearance under the microscope, which is different to small cell lung cancer which looks like small, circular or oval structures. Because NSCLC takes up such a large portion of all lung cancer cases, and lung cancer is the leading cause for cancer deaths worldwide, this poses a significant threat. It is also extremely widespread, with approximately 3200 cases per year, this is reflected in the fact that it is one of the top 4 diagnosed cancers in Europe.

Pathophysiology

NSCLC is caused by the amplification of oncogenes and the failing of tumor suppressing genes. Oncogenes are mutated genes that push cells to grow and divide, even when they shouldn’t. They are mutated proto-oncogenes, which are normal genes found in DNA. Tumor suppressing genes are effectively the opposite of proto-oncogenes, as their primary function is to suppress cell growth.

Oncogenes are amplified when the DNA containing them is duplicated through normal cell processes. The combination between rapid, uncontrolled cell growth from failing tumor suppressing genes combined with the mutated oncogenes promotes a rapid and practically irreversible spread of the mutation.

The most common oncogenes to be mutated are in the ras family, which is composed of H-ras, N-ras, and K-ras oncogenes. These oncogenes code for the protein p21, a protein in the inner cell membrane, which has guanosine triphosphatase (GTPase) activity. Mutations in this family often cause blockage of GTPase activity, allowing the cells to proliferate uncontrollably.

Mutation of the K-ras oncogene is the one most associated with exposure to asbestos or tobacco. Additionally, mutations of this oncogene, specifically at codon 12, have been found to cause patients with it to have a relative risk of death of 5.6 compared to patients without it.

NSCLC can be classified into 3 types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. These types share similar treatments yet have different ways of manifesting and each have their own histological and clinical characteristics.

Adenocarcinoma

Adenocarcinoma is the most common type of lung cancer found in non-smokers. It starts in the glands that line the lungs, which secrete mucus. When cancer causes the glands to grow and expand out of control, there is a high risk of tumors.

This type of cancer can spread throughout the body despite it starting in the glands. When it spreads there are 2 types that it can be classified into:

Invasive adenocarcinoma: Cancer cells spread through the tissue to surrounding lymph nodes.

Metastatic adenocarcinoma: Cancer cells break away from the tumor and travel to distant parts of the body via the bloodstream and the lymphatic system.

Squamous Cell Carcinoma

Squamous cell carcinoma accounts for 30% of all lung cancers and begins in the squamous cells—wide, flat cells that line the inside of the lungs and bronchial tubes—that look like fish scales. Squamous cell tumors generally occur in the center of the lung or in the bronchi. This is the type of cancer that is most associated with smoking.

Large Cell Carcinoma

Large cell carcinoma usually takes the form of large peripheral mass on chest radiograph. Unlike adenocarcinoma and SCC, it shows no evidence of keratinization or gland formation. This type of NSCLC is usually grouped with adenocarcinomas for clinical trials.

Symptoms

Symptoms can vary depending on the patient, but they mostly occur in the lungs. However, if the cancer has metastasized, it could cause symptoms in other parts of the body. The main symptoms that can be found in most patients are the following:

  1. Coughing that gets worse or doesn’t go away.
  2. Chest pain.
  3. Shortness of breath.
  4. Wheezing.
  5. Coughing up blood.
  6. Feeling very tired all the time.
  7. Weight loss with no known cause.

EGFR

The epidermal growth factor receptor (EGFR) mutations can be detected in the bloodstream because it is a circulating free tumour derived DNA (ctDNA) and thus can be used in detecting tumour presence in patients with advanced non-small-cell lung cancer (NSCLC).

At the moment research suggest that accurate results can be gathered from using pre-analytical plasma processing, ctDNA extraction and mutation detection methods. The detection of EGFR can be especially helpful for tyrosine kinase inhibitor (TKI) therapy, targeting resistance mutations.

Despite this there is evidence that suggests that ctDNA doesn’t allow for detection of EGFR mutations in all patients with mutation-positive NSCLC. Even though it can be inaccurate and tumour tissue sampling is more accurate, ctDNA testing can be used as a preliminary diagnostic in areas that have less accessibility to biopsy centers.

Additionally, biopsies can pose too great of a health risk to 27–31% of NSCLC patients, and in these cases non-invasive detections are proposed.

The EGFR gene was first discovered in 1995, and in 2006 the clinical walrus of ctDNA was uncovered when the mutations in a serum were associated with a response to gefitinib, a cancer drug, in a study.

It has been hypothesized that ctDNA comes from necrotic neoplastic cells that have been phagocytized by macrophages and as well, apoptotic cells. Additionally, ctDNA can be attributed to the breakdown of circulating tumour cells, but this has been reevaluated because in patients with multiple malignancies (excluding lung) the levels of ctDNA appear to be higher than that of circulating updating tumour cells. Finally, it has been suggested that tumour cells actually secrete DNA fragments. This phenomenon has been observed in lymphocytes for patients with NSCLC through exosomes.

Meta-analyses have shown promising results that EGFR mutations using ctDNA can be good for diagnostic purposes. However, experimental results fluctuate: many studies suggest that it is more difficult to detect EGFR mutations in ctDNA in plasma samples versus tumour tissue. Generally, ctDNA is highly diluted and as a result very sensitive techniques are needed to detect the mutation in the samples.

At the moment, there are multiple methods used for detecting ctDNA at its low concentration of mutant alleles <1% of total DNA. In order to sufficiently detect the ctDNA, sensitive and precise devices need to be used; due to their precision they are prone to false positives. For this reason, it might be wise to use higher specificity with the sensitivity sacrificed for the sake of more targeted results.

Models such as next-generation sequencing (NGS) and enhanced PCR techniques like droplet PCR (ddPCR) and BEAMing are used to detect EGFR in ctDNA plasma. All the methods listed are costly and require a time delay for processing, with ddPCR being the fastest and BEAMing the most cost-effective.

Product Summary

Our project aims to develop a simple, affordable, and user-friendly prelimenary diagnostic kit for the detection of EGFR mutations associated with non-small cell lung cancer (NSCLC) in circulating tumor DNA (ctDNA). The system uses recombinase polymerase amplification (RPA) combined with CRISPR-Cas9 amplicon cleavage and a lateral flow assay (LFA) readout to create a one-pot, isothermal assay that produces a clear, visual result without the need for specialized laboratory equipment.

This design is intended to act as a low-cost screening tool for NSCLC, making early molecular diagnosis more accessible—especially in resource-limited settings.

Importance of Early Diagnosis

Lung cancer is one of the leading causes of cancer-related death worldwide, and NSCLC accounts for approximately 85% of cases. Unfortunately, NSCLC is often diagnosed at an advanced stage, when treatment options are limited and survival rates are poor. Early detection of driver mutations such as EGFR can significantly improve patient outcomes by allowing clinicians to initiate targeted therapies at a stage where they are most effective.

However, conventional diagnostic approaches such as PCR and next-generation sequencing are expensive, require specialized equipment, and are not widely available in low-resource regions. This leads to delayed diagnosis, higher treatment costs, and poorer prognoses. Our project addresses this challenge by offering a low-cost, accessible, and rapid diagnostic solution that could be deployed as a screening tool for early detection, reducing healthcare burdens and improving survival rates.

Importance of Screening and Our Project

In medical diagnostics, screening means checking for disease in people who do not yet show symptoms, so it can be caught and treated earlier.

Our project aims to use a ctDNA-based test to identify people at high risk for NSCLC (those with the EGFR deletion mutation at exon 19 for the moment) before they show symptoms.

Screening is different from diagnosis because it is fast, cheap, large-scale, and sometimes less precise. By contrast, a diagnosis is specific and confirmatory.

For lung cancer, we know that patients who are given treatment at earlier stages have significantly longer lifespans. In NSCLC, therefore, screening is very important to be able to efficiently screen at-risk individuals in impoverished regions so they may be able to receive treatment as soon as possible.

Our test is not intended as a population-wide screening tool, but rather as an early test for high-risk individuals, particularly in regions where CT scans are not accessible.

Screening with ctDNA presents several challenges. Most ctDNA assays in clinical use today are applied for monitoring in known cancer patients, while their use for early detection remains limited and largely experimental. [3] Current approaches typically depend on PCR or sequencing, which require expensive instruments, trained personnel, and centralized laboratories. In addition, ctDNA levels in early-stage patients are extremely low, making sensitivity a major technical hurdle.

To overcome these limitations, we chose to use Recombinase Polymerase Amplification (RPA) instead of PCR. RPA is a rapid, isothermal amplification technique that operates at low temperatures, making it well suited for portable testing. This enables amplification of small amounts of ctDNA from a simple blood sample without a thermocycler, allowing for more accessible and decentralized screening.

Implementing ctDNA screening also raises important ethical and social considerations. In regions with limited follow-up care, a positive result could cause distress if patients cannot access further testing or treatment. False positives may also strain already limited health services. For these reasons, our test is designed not to replace diagnosis, but to help flag high-risk individuals so they can be prioritized for confirmatory testing and earlier treatment when resources permit.

Recombinase Polymerase Amplification (RPA)

Recombinase polymerase amplification is an isothermal nucleic acid amplification method that operates at a constant, low temperature (typically 37–42 °C). Recombinase proteins pair primers with their complementary sequence in the target DNA, allowing primers to invade double-stranded DNA and initiate synthesis by a strand-displacing polymerase. Significant DNA amplification is achieved within 20 minutes under these mild conditions.

Because it does not require thermocyclers or complex instrumentation, RPA is highly suitable for point-of-care diagnostics and can be implemented in low-resource settings. It offers a rapid turnaround time, is robust to inhibitors found in biological samples, and can be easily integrated into portable testing kits.

Cas9 Enzyme

The newest development in our project would be the inclusion of Cas9 endonuclease enzyme to enhance accuracy of the lateral flow assay readout. We found in our experiments that the RPA was consistently amplifying the wildtype, and the mutation. For the readout to be accurate, only the mutation should be amplified. To mitigate this issue, Cas9 enzyme will be introduced to the sample before the RPA; this way it will cut any amplified wildtype DNA and the mutation DNA will be left whole. The Cas9 is specified to the wildtype DNA and will only cut the wildtype and not the mutation DNA so a more accurate dye result will be obtained.

If the individual only has the wildtype sequence, Cas9 will recognize and cut the DNA at a specific site. This cut separates the two tags attached to the DNA, for example, a dye (like FAM) and biotin. In a lateral flow assay, this means the dye-labeled piece will move past the test line and not be captured, while only the biotin-tagged piece is retained, resulting in no visible line or a color change at a different spot.

However, if the mutation is present, Cas9 will not cut the DNA. The intact DNA still has both tags, so when it flows through the lateral flow strip, it gets captured at the test line, and the colored dye stays visible.

Lateral Flow Assay (LFA) Readout

Following amplification and probe processing, the reaction mixture is applied directly to a lateral flow strip. The probes are labeled with two distinct antigens (such as FAM and biotin), and successful amplification and cleavage result in dual-labeled products. As the sample migrates along the strip, these products are captured at a test line coated with anti-FAM antibodies, forming a visible band.

This readout method is:

  • Simple – no need for expensive fluorescence detectors.
  • Fast – results can be read within minutes.
  • Portable – suitable for clinics, screening programs, and field settings.

By combining amplification, sequence verification, and readout into a single one-pot assay, this approach lowers costs, reduces complexity, and enables widespread screening for lung cancer–associated mutations, potentially improving early detection rates and patient outcomes worldwide.

From Last Year to This Year

Last year, our team developed a diagnostic concept based on rolling circle amplification (RCA) followed by a split lettuce aptamer fluorescence readout. While this design demonstrated proof-of-concept for EGFR mutation detection at a low cost, several practical limitations were identified:

Complex workflow – multiple enzymatic steps, including ligation and amplification, required careful timing and temperature control. Specialized equipment requirement – a fluorescence detector was needed to quantify results, limiting accessibility.

Based on these findings, we redesigned our diagnostic system with the goal of simplifying the workflow, reducing equipment requirements, and lowering the overall cost per test.

This year, we have shifted to a method that integrates:

  • Recombinase Polymerase Amplification (RPA) – a rapid, isothermal amplification technique that does not require a thermocycler.
  • CRISPR-Cas9 amplicon cleavage – for enhanced specificity and reduced false positives.
  • Lateral Flow Assay (LFA) readout – producing a clear, visual result that can be interpreted without specialized instruments.

This new design allows all steps to take place in a single reaction tube, reducing contamination risk and operator error. It also provides a low-cost, portable, and rapid diagnostic platform that is better suited for point-of-care screening, especially in low-resource settings.

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