Summary a non-invasive screening method for non-small cell lung cancer (NSCLC)

Abstract

The invention is a non-invasive screening method for non-small cell lung cancer (NSCLC), developed for use in the healthcare and medical device manufacturing sectors. Its features include: preparing serum samples by taking blood from patients for use in spectroscopic analysis; Performing molecular structure analyses using Raman spectroscopy; measuring molecular bond vibrations using FT-IR spectroscopy; processing spectral data, evaluating results, and establishing a decision mechanism for NSCLC detection by performing PCA, LDA, and ROC analyses using data analysis software.

Description

[0001]DESCRIPTION A NON-INVASIVE SCREENING METHOD FOR NON-SMALL CELL LUNG CANCER (NSCLC) Technical Area The invention relates to a non-invasive screening method for non-small cell lung cancer (NSCLC) developed for use in the healthcare and medical device manufacturing sectors. The invention relates in particular to a non-invasive screening method for non-small cell lung cancer (NSCLC), which is particularly suitable for use in large-scale screening programmes due to its potential for early diagnosis and low cost. The Known State of the Art Non-small cell lung cancer (NSCLC) is one of the most common types of cancer that develops in the lungs. Lung cancer has two main types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Non-small cell lung cancer accounts for approximately 85-90% of lung cancers. NSCLC is a type of cancer that develops from large cells or epithelial cells in the lungs and generally grows more slowly and can be less aggressive than small cell lung cancer. However, these types of cancer can also complicate the treatment process when they reach advanced stages. Squamous cell carcinoma, adenocarcinoma, and large cell carcinoma are the main subtypes of NSCLC. Symptoms of NSCLC include cough, chest pain, shortness of breath, bloody sputum, and weight loss. Treatment options include surgery, radiotherapy, chemotherapy, and targeted therapy. The treatment plan is usually determined based on the stage of the cancer, the patient's overall health, and other factors. Non-small cell lung cancer (NSCLC) diagnosis is made using various medical tests and methods. As a first step, the doctor assesses the patient's medical history, symptoms, and lifestyle and performs a physical examination. At this stage, sensitivity or abnormalities in the chest area are sought. The main imaging tests used in diagnosis include chest X-ray, computed tomography (CT) scan, and positron emission tomography (PET) scan. Chest X-ray is used to initially assess abnormalities or tumours in the lungs. A CT scan determines the size, location, and extent of the tumour in more detail. A PET scan assesses whether the cancer has spread throughout the body. A biopsy is usually performed to make a definitive diagnosis. Types of biopsy include bronchoscopy, needle biopsy, and surgical biopsy. Bronchoscopy is used to obtain tissue samples from the airways and lungs using a tube. Needle biopsy involves obtaining tissue samples from the chest wall or other areas using a needle. Surgical biopsy involves surgically obtaining tissue samples from the tumour or suspicious area. The tissue sample is examined under a microscope to determine the presence, type, and grade of cancer cells. In addition, various blood tests may be performed. These tests are used to assess the spread of cancer and monitor the patient's overall health. By combining these methods, the accurate diagnosis of NSCLC and the development of an appropriate treatment plan are ensured. Current non-small cell lung cancer (NSCLC) screening and diagnostic methodologies face various technical and clinical limitations. Invasive procedures, particularly biopsy, carry significant morbidity risks for patients and constitute a psychological stress factor. Advanced imaging techniques, especially positron emission tomography (PET) and computed tomography (CT), are considered suboptimal options for large-scale screening programmes due to their high costs and radiation exposure. The time required to obtain diagnostic results from these methods can delay the clinical decision-making process. Conventional screening modalities show limited sensitivity in detecting early-stage tumours. Inter-observer variability and sampling errors observed in the interpretation of biopsy results can negatively affect diagnostic accuracy. The high cost and invasive nature of current methods limit the feasibility of population-based screening programmes. The limited number of specific and reliable biomarkers for NSCLC complicates the molecular characterisation of the disease and the development of personalised treatment strategies. Current approaches generally adopt the principle that ‘one size fits all,’ which carries the risk of overlooking the individual characteristics of a heterogeneous disease such as NSCLC. Furthermore, longitudinal monitoring of treatment response and disease progression requires repeated applications of existing invasive methods, which can have negative effects on patient compliance and quality of life. These multifaceted limitations highlight an urgent need to develop new methodologies that are less invasive, cost-effective, and highly sensitive for the early diagnosis, characterisation, and monitoring of NSCLC. Today, numerous studies have been conducted and new methods developed for the diagnosis of lung cancer. One such study is the invention covered by patent application US10627413B2. The invention relates to a method for determining cancer biomarkers using xenograft animal models to diagnose a disease, determine its progression, and assess response to treatment. In practice, a tumour taken from a patient is transplanted into an animal model, such as a mouse. The expression levels of disease-related biomarkers in the serum of the animal model are evaluated. A personalised gene expression profile or biomarker gene signature can be obtained from the expression levels of the biomarkers. This profile or signature can be used to determine disease recurrence, progression, and response to treatment. The application describes a method for detecting lung cancer using Raman spectroscopy in respiratory samples. In the method described in the invention, respiratory samples are used instead of serum. Another study concerns the invention covered by patent application US2024248032A1. The invention comprises a method used to detect lung cancer in a human subject at high risk or with a high probability of developing lung cancer. This method involves performing FTIR (Fourier Transform Infrared) spectral analysis on a sputum sample obtained from the subject and, optionally, comparing the spectrum with a control, for example. Furthermore, the invention provides for the use of this method in selecting a treatment process for lung cancer and for use in connection with a method incorporating this treatment method, as well as a set of components for use in this method. Another study concerns the invention covered by patent application GB2494580A. The invention relates to a method for diagnosing lung cancer in a subject or identifying a subject at risk of developing lung cancer. This method involves performing FTIR (Fourier Transform Infrared) spectral analysis on a sputum sample obtained from the subject and comparing the spectrum obtained with the FTIR spectrum of a normal control. In addition, methods for detecting early and late stage cancer, methods for monitoring the progression of lung cancer in a subject, and kits for use in the described methods are provided. Although FTIR spectroscopy is frequently used in current applications, there is no method that combines it with Raman spectroscopy. This situation can reduce diagnostic accuracy and scope due to the lack of information and sensitivity that each method alone cannot provide in the diagnosis of lung cancer. Consequently, the need for a non-invasive screening method for non- small cell lung cancer (NSCLC) that eliminates the disadvantages of the current technique, coupled with the inadequacy of existing solutions, has necessitated development in this technical field. Brief Description of the Invention The present invention relates to a non-invasive screening method for small cell lung cancer (SCLC), developed for use in the healthcare services and medical device manufacturing sectors, which meets the aforementioned requirements, eliminates all disadvantages, and provides additional advantages. Based on the current state of the art, the objective of the invention is to provide a non-invasive, high-sensitivity screening methodology for small cell lung cancer (SCLC) by utilising the synergistic combination of Raman and Fourier Transform Infrared (FTIR) spectroscopy techniques, thereby overcoming the various limitations encountered by existing diagnostic methods. -invasive, high- sensitivity screening methodology for small cell lung cancer (SCLC) and to overcome the various limitations encountered by existing diagnostic methods. The purpose of the invention is to increase patient comfort and safety through a minimally invasive method based on the spectroscopic analysis of serum samples, while eliminating the risks associated with invasive procedures such as biopsies. Another objective of the invention is to ensure speed and cost-effectiveness in the diagnostic process through the integration of spectroscopic analysis and advanced data processing algorithms. Another objective of the invention is that the diagnostic performance of the method, as evidenced by the Area Under the Curve (AUC) value of 0.89 obtained in the Receiver Operating Characteristic (ROC) analysis, demonstrates high sensitivity and specificity, potentially offering a paradigm shift in the early detection of NSCLC. Another objective of the invention is to ensure increased reproducibility and reliability of results through standardised spectroscopic protocols and objective data analysis. Another objective of the invention is to increase the feasibility of large-scale population screening programmes, thanks to its low-cost and rapid results, thereby changing the epidemiological profile of NSCLC and creating a widespread impact on public health. Another objective of the invention is to enable the identification of new molecular biomarkers for NSCLC through characteristic spectral signatures detected by Raman and FT-IR spectroscopy, while also contributing to a deeper understanding of the molecular pathogenesis of the disease and the identification of potential therapeutic targets. Another objective of the invention is to pave the way for personalised medicine applications in the future through the detailed molecular profile obtained by spectral analysis, and to enable the optimisation of screening, diagnosis and treatment strategies based on each patient's unique molecular characteristics. Another objective of the invention is to facilitate the longitudinal follow-up of patients, enable more effective monitoring of treatment response and disease progression, and improve clinical decision- making processes, thanks to the non-invasive and repeatable nature of the method. Another objective of the invention is to enable a more comprehensive understanding of the complex biology of the disease by providing a holistic perspective on NSCLC diagnosis and management through a multidisciplinary approach that integrates the fields of medicine, physics, biochemistry, and data science. Another objective of the invention is to provide a paradigm-shifting potential in the screening and diagnosis of NSCLC. The structural and characteristic features of the invention and all its advantages will be more clearly understood by means of the detailed description provided below, which refers to the accompanying drawings and refers to these drawings. Therefore, the evaluation must be made taking into account these drawings and the detailed description. Brief Explanation of the Figures In order to best understand the structure of the present invention and its advantages together with additional elements, it must be evaluated in conjunction with the figures described below. Figure-1; graph showing the comparative FT-IR spectra of healthy and diseased samples, Figure-2; graph showing the comparative Raman spectra of healthy and diseased samples, Figure-3; 2D PCA graph of FT-IR data, Figure-4; LDA density graph of FT-IR data, Figure 5: Receiver operating characteristic (ROC) curve (FT-IR), Figure 6: Precision-Recall Curve: A graph showing the performance of the FT-IR-based NSCLC screening method, Figure 7: 2D PCA plot of Raman Data graph, Figure-8; LDA density plot (Raman spectroscopy data), Figure-9; Receiver Operating Characteristic (ROC) curve (Raman spectroscopy data), Figure-10; Precision-Recall Curve: Performance graph of Raman-based NSCLC screening method. Detailed Description of the Invention This detailed explanation describes a non-invasive screening method for non-small cell lung cancer (NSCLC), developed for use in the healthcare and medical device manufacturing sectors, solely as an example to aid understanding of the subject and without imposing any restrictive effect. The invention encompasses spectroscopic analysis methods used for the diagnosis and molecular profiling of non-small cell lung cancer (NSCLC). The method described in the invention utilises a combination of Raman spectroscopy and Fourier transform infrared (FT-IR) spectroscopy for the early diagnosis and molecular characterisation of NSCLC. Non-small cell lung cancer (NSCLC) is a type of cancer that is usually diagnosed at a late stage and has a high mortality rate. Traditional diagnostic methods are invasive and are generally effective at late stages. There is a need for non- invasive and sensitive diagnostic methods that can identify changes at the molecular level. Spectroscopic techniques offer a potential solution to meet this need. The subject of the invention is a screening method; the preparation of serum samples by taking blood from patients for spectroscopic analysis; the analysis of molecular structures in the 400-4000cm ¹ range using Raman spectroscopy; measuring molecular bond vibrations in the 400-4000 cm ¹range using FT-IR spectroscopy; processing spectral data, evaluating results, and establishing a decision mechanism for NSCLC detection by performing PCA, LDA, and ROC analyses using data analysis software. The study on the screening method in question was conducted using a prospective case-control design and comprised 37 histopathologically confirmed NSCLC patients and 13 healthy controls. Samples were obtained from thoracic surgery and medical oncology clinics. Serum samples were collected from patients who had not undergone chemotherapy or surgical intervention. Venous blood samples were collected 12 hours after fasting, using standard phlebotomy techniques from the antecubital vein into vacuum tubes without EDTA. The samples were centrifuged at 3000 g for 15 minutes within 30 minutes of collection. The resulting serum was transferred to sterile polypropylene tubes and stored at -80°C until analysis. All samples were analysed within a maximum storage period of 6 months. Spectroscopic analyses were performed using Raman spectroscopy with the Renishaw inVia Reflex Raman Microscope and Spectrometer (Raman spectrometer) and FT-IR spectroscopy with the Shimadzu IRAffinity 1S ATR equipped FT-IR (FT-IR spectrometer). Measurements were takenin the 400-4000 cm ¹ range for Raman spectroscopy and in the same spectral range for FT-IRspectroscopy. Characteristic peaks of carotenoid group substances and phenylalanine were analysed in Raman spectroscopy, while Amide I and Amide II bands belonging to protein groups were analysed in FT-IR spectroscopy. Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) were used for data analysis. According to the PCA results, PCA Component 1 explains 68.49% and Component 2 explains 4.65% of the variance for Raman data, while Component 1 explains 65.54% and Component 2 explains 20.40% of the variance for FT-IR data. In the LDA analysis, histograms and density plots were created using LDA to distinguish between the patient and healthy groups. The ROC analysis results showed an AUC (Area Under Curve) value of 0.85 for Raman spectroscopy and 0.89 for FT-IR spectroscopy.In the Raman Spectrum analysis results; characteristic peaks at 1511, 1154, and 1004 cm ¹ wereobserved in NSCLC patients, along with distinct intensity differences and frequency shifts betweenpatient and healthy samples in the 750–1000 cm ¹ and 1400–1600 cm ¹ ranges. FT-IR spectrumanalysis results showed characteristic peaks at 1641 and 1548 cm-1 belonging to protein groups, and significant intensity differences and frequency shifts between patient and healthy samples in the 1500-1700 cm-1 and 2800-3000 cm-1 ranges. FT-IR infrared spectroscopy measures the bond vibration frequencies of molecules based on the absorption of infrared radiation by the sample. Raman spectroscopy performs molecular structure analyses based on the scattering of laser light sent to the sample. Both spectroscopy methods showed significant spectral differences between NSCLC and healthy controls. PCA and LDA analyses found the distinction between the groups to be statistically significant. ROC analysis confirmed the method's high screening performance. FTIR spectroscopy data showed significant changes in patients with non-small cell lung cancer(NSCLC). The intensity increase observed in the Amide II band (1548 cm ¹) was measured as64%; the intensity of this band was 0.18 ± 0.02 a.u. in NSCLC patients, while it was 0.11 ± 0.01 a.u. in healthy individuals. This increase can be considered a specific biomarker for NSCLC and plays an important role in characterising the tumour microenvironment by reflecting changes in protein secondary structures. A 53% increase in intensity was observed in the Amide I band (1641cm ¹); this band was measured at 0.23 ± 0.03 a.u. in NSCLC patients and 0.15 ± 0.02 a.u. inhealthy individuals. This increase is a characteristic peak for NSCLC, indicating changes in protein conformations and increased protein synthesis in tumour cells. Changes were also observed in thelipid bands: a 25% increase in intensity was detected in the 2925–2931 cm ¹ range, and a 17%increase in the 2854–2878 cm ¹ range. These increases are considered characteristic, reflectingchanges in lipid metabolism in NSCLC. According to Raman spectroscopy data, significant increases were observed in the carotenoidbands. The intensity increase in the 1511–1516 cm ¹ region was measured as 75%, while theincrease in the 1154 cm ¹ region was 67%. The intensities of these bands in NSCLC patients werefound to be 0.35 ± 0.04 a.u. and 0.25 ± 0.03 a.u., respectively, while in healthy individuals they were 0.20 ± 0.03 a.u. and 0.15 ± 0.02 a.u. These increases stand out as biomarkers with high specificity for NSCLC and reflect increased oxidative stress levels, which may play a critical role in the early diagnosis of NSCLC. A 40% increase in intensity was observed at the phenylalanine peak(1004 cm ¹); this peak was measured at 0.14 ± 0.02 a.u. in NSCLC patients and 0.10 ± 0.01 a.u. inhealthy individuals. This peak is characteristic of NSCLC, characterising changes in amino acid metabolism and the metabolic profile of tumour cells. Furthermore, a 38% increase in intensity wasobserved in the 1300-1400 cm ¹ region. Around 1340 cm ¹, the intensity of this region was found tobe 0.11 ± 0.02 a.u. in NSCLC patients and 0.08 ± 0.01 a.u. in healthy individuals. These changes are considered characteristic, reflecting the general metabolic profile of NSCLC. Spectral frequency shifts observed in non-small cell lung cancer (NSCLC) samples are important biophysical indicators reflecting the complex molecular-level changes associated with the disease.In FT-IR spectroscopy, the 4 ± 0.5 cm ¹ bathochromic shift observed in the Amide I band (1654cm ¹ → 1650 cm ¹) indicates a transition in the secondary structure of proteins in NSCLC cellsfrom an α-helix conformation to a β-sheet conformation. This points to specific changes in the protein expression profile and functional protein conformations of tumour cells. Concurrently, the 3± 0.5 cm ¹ hypochromic shift in the Amide II band (1548 cm ¹ → 1545 cm ¹) reflects alterations inthe hydrogen bonding characteristics of peptide bonds, suggesting potential modifications inprotein-protein interactions. The 2.5 ± 0.3 cm ¹ hypsochromic shift observed in the lipid region(2800-3000 cm ¹) reveals changes in the lipid organisation and fluidity of the plasma membrane inNSCLC cells. This change is considered a critical factor that could affect cell signalling and the functions of membrane proteins. In Raman spectroscopy, the 3.5 ± 0.5 cm -1 bathochromic shift(880 cm ¹→ 876.5 cm ¹) observed in the indole ring vibration band belonging to tryptophanresidues indicates the transition of tryptophan residues to a more hydrophobic microenvironment. This points to significant changes in the tertiary structure of proteins in NSCLC cells and potentially to changes in the intracellular redox state. In the nucleic acid and protein backbone vibration region(1400–1600 cm ¹), a 4.5 ± 0.5 cm ¹ hypochromic shift (1485 cm ¹ → 1489. 5 cm ¹) observed in thenucleic acid and protein backbone vibration region (1400-1600 cm ¹) reflects changes in the π-electron system of nucleic acid bases and modifications in DNA / RNA-protein interactions. This finding indicates potential anomalies in gene expression and replication mechanisms in NSCLC cells. When evaluated as a whole, these spectral shifts reveal extensive changes in the intracellular and extracellular microenvironment of NSCLC cells. These alterations in pH, ion concentration, and hydrophobicity / hydrophilicity balance directly affect cellular metabolism and signalling pathways. The observed frequency shifts contribute to a deeper understanding of the molecular pathogenesis of NSCLC and offer potential biomarkers for early diagnosis of the disease. Figure 1 shows the Fourier Transform Infrared (FT-IR) spectra of serum samples from non-small cell lung cancer (NSCLC) patients and healthy controls. The X-axis represents wave numbers inthe 400–4000 cm ¹ range, while the Y-axis shows intensity (a.u.). Figure 2 shows the Ramanspectra of serum samples from non-small cell lung cancer (NSCLC) patients and healthy controls.The X-axis represents the Raman shift in the range of 0–4000 cm ¹, while the Y-axis representsthe intensity (a.u.). Figure 3 shows the 2D PCA graph of FT-IR data, where the X-axis represents PCA Component 1 (65.54% variance) and the Y-axis represents PCA Component 2 (20.40% variance). Light-coloured points indicate NSCLC patient samples, while dark-coloured points indicate healthy controls. The graph shows a clear separation between patient and healthy samples. Patient samples are generally clustered in the upper right part of the graph, while healthy samples are concentrated in the lower left part. This separation indicates that FTIR spectroscopy could potentially be used as a biomarker in NSCLC screening. Ellipses represent confidence intervals for each group and emphasise the separation between groups. Figure 4 shows the LDA density plot of the FT-IR data, where the curve for patient samples is clearly shifted to the left (negative LDA values), while the curve for healthy samples is shifted to the right (positive LDA values). The clear separation of the two groups' curves demonstrates that FTIR spectroscopy is highly effective in distinguishing NSCLC from healthy samples. This distinction highlights the method's screening power and indicates that it could potentially be used as a screening tool for NSCLC. Figure 5 shows the receiver operating characteristic (ROC) curve (FT-IR), where the X- axis represents the false positive rate and the Y-axis represents the true positive rate. The solid line represents the ROC curve, while the dashed line represents the reference line. The area under the curve (AUC) was calculated as 0.89. This high AUC value indicates that the method has excellent discriminatory power. The proximity of the ROC curve to the upper left corner indicates that the test has high sensitivity and specificity. This result proves that the developed FTIR-based screening method is highly reliable and effective in detecting NSCLC. Figure 6 is a graph showing the precision-recall performance of the screening method. The X-axis represents Recall, while the Y-axis represents Precision. The AP (Average Precision) value indicated at the top of the graph is 0.75. This value demonstrates that the method possesses both high precision and high sensitivity. The steep and rightward-sloping curve highlights the method's robust performance. This result indicates that the NSCLC screening method has low false positive and false negative rates, suggesting that the method is promising for clinical applications. Figure 7 shows the Principal Component Analysis (PCA) results of the Raman spectroscopy data. The X-axis represents PCA Component 1 (68.49% variance), and the Y-axis represents PCA Component 2 (4.65% variance). Light-coloured points represent NSCLC patient samples, while dark-coloured points represent healthy controls. A clear separation between patient and healthy samples is observed, indicating that Raman spectroscopy could be effective in NSCLC screening. Figure 8 shows the results of Linear Discriminant Analysis (LDA) of Raman data. The X-axis represents the LDA component, while the Y-axis represents the intensity. The upper curve represents NSCLC patient samples, while the lower curve represents healthy controls. The curve for patient samples is clearly shifted to the left, while the curve for healthy samples is shifted to the right. This clear distinction demonstrates that Raman spectroscopy is a powerful discriminator in NSCLC screening methods. Figure 9 shows the ROC curve evaluating the performance of the Raman spectroscopy-based screening method. The X-axis represents the false positive rate, and the Y-axis represents the true positive rate. The area under the curve (AUC) was calculated as 0.85, indicating the method's high screening accuracy. Figure 10 shows the sensitivity and precision performance of the Raman spectroscopy-based screening method. The X-axis represents sensitivity (recall), and the Y-axis represents precision. The average precision (AP) value was calculated as 0.72, indicating that the method has both high sensitivity and high precision. When these spectral changes are evaluated together, a unique and comprehensive ‘spectral signature’ emerges for NSCLC. In particular, the superiority of FT-IR in detecting changes in protein structures and the ability of Raman to identify specific molecular bonds are complementary. This synergistic approach offers the potential for a paradigm-shifting advancement in the early detection and definitive screening of NSCLC. Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) results demonstrate that this spectral signature can distinguish NSCLC and healthy samples with high accuracy. The high AUC values obtained in the ROC analysis (0.89 for FTIR, 0.85 for Raman) prove the method's screening potential. The subject of the invention, the screening method, provides a powerful screening tool for the non- invasive, rapid, and potentially early diagnosis of NSCLC. In particular, the carotenoid bands inRaman spectroscopy (1511–1516 cm ¹ and 1154 cm ¹) and the Amide II band in FTIRspectroscopy (1548 cm ¹) stand out as biomarkers demonstrating high specificity and sensitivity forNSCLC. The spectroscopic screening method described also provides valuable insights into the molecular pathogenesis of NSCLC. Changes in protein metabolism, lipid structure, oxidative stress, and amino acid metabolism create new opportunities for understanding the biology of NSCLC and identifying potential therapeutic targets. The findings of our study provide strong evidence that the integration of FT-IR and Raman spectroscopy techniques can be used as a highly sensitive, minimally invasive, and economically viable methodology for NSCLC diagnosis. This innovative approach has the capacity to overcome the current limitations of conventional screening methods and holds the potential to optimise the prognosis and quality of life of NSCLC patients. The translation of the screening method in question into clinical practice could represent a significant advancement in the field of oncology and guide the development of future cancer diagnostic strategies.

Claims

CLAIMS 1. Developed for use in the healthcare services and medical device manufacturing sectors, it is a non-invasive screening method for non-small cell lung cancer (NSCLC), characterised by: - Preparation of serum samples by drawing blood from patients for spectroscopic analysis; - Conducting molecular structure analyses using Raman spectroscopy; - Measuring molecular bond vibrations using FT-IR spectroscopy; - Processing spectral data, evaluating results, and establishing a decision mechanism for NSCLC detection by performing PCA, LDA, and ROC analyses using data analysis software.

2. This is a scanning method compliant with Requirement 1, characterised by including the process step of performing molecular structure analysis in the 400–4000 cm ¹ range.

3. A scanning method compliant with Requirement 1, characterised by including the process step of measuring molecular bond vibrations in the 400–4000 cm ¹ range.

4. It involves the combined use of Raman spectroscopy and Fourier transform infrared (FT-IR) spectroscopy for the diagnosis of non-small cell lung cancer (NSCLC).

5. A screening method suitable for Requirement 1, characterised by the collection of venous blood samples from NSCLC patients using vacuum tubes without EDTA after a 12-hour fast.

6. A screening method compliant with Requirement 1, characterised by the process step of centrifuging the collected blood samples at 3000 g for 15 minutes, separating the serum, and storing it at -80°C.

7. A screening method suitable for Request 1, characterised by the process step of performing molecular structure analyses in serum samples in the 400-4000 cm^-1 range using Raman spectroscopy.

8. A screening method suitable for Requirement 1, characterised by: the process step of determining the characteristic peaks of carotenoid group substances and phenylalanine.

9. A screening method suitable for Requirement 1, characterised by: the process step of separating NSCLC patients from healthy samples using Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) methods based on Raman spectroscopy data.

10. A screening method suitable for Requirement 1, characterised by: the process step of measuring molecular bond vibrations in the 400-4000 cm ¹ range in serum samples usingFT-IR spectroscopy.

11. A scanning method compliant with Requirement 1, characterised by: the process step of analysing the Amid I and Amid II bands belonging to protein groups and lipid bands.

12. A scanning method compliant with Requirement 1, characterised by: the process step of separating NSCLC patients from healthy samples using PCA and LDA methods based on FT-IR spectroscopy data.

13. A screening method suitable for Requirement 1, characterised by: the processing of spectroscopic data using PCA (Principal Component Analysis) and LDA (Linear Discriminant Analysis), and the evaluation of the distinction between groups.

14. A screening method suitable for Request 1, characterised by: measuring screening performance using ROC (Receiver Operating Characteristic) analysis and including the process step of calculating AUC (Area Under Curve) values.

15. A screening method suitable for Requirement 1, characterised by: the use of intensity changes in the carotenoid bands (1511-1516 cm ¹ and 1154 cm ¹) and the phenylalaninepeak (1004 cm ¹) in Raman spectroscopy as biomarkers for NSCLC.

16. A screening method suitable for Claim 1, characterised by: the process step of using intensity changes in the Amide I band (1641 cm ¹) and Amide II band (1548 cm ¹) in FT-IRspectroscopy and changes in the lipid bands as biomarkers for NSCLC.

17. A screening method suitable for Claim 1, characterised by: the process step of correlating the bathochromic and hypochromic shifts observed in the Amide I and Amide II bands in FT-IR spectroscopy with protein conformations and lipid organisation in NSCLC cells.

18. A screening method suitable for Claim 1, characterised by: the process step of using frequency shifts in Raman spectroscopy associated with tryptophan residues and nucleic acid bases in relation to molecular changes in NSCLC cells.

19. A screening method suitable for Requirement 1, characterised by the process step of evaluating spectral analyses as a whole.

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