SERUM-BASED MULTIPLE BIOMARKER PANEL FOR GLIOMA DIAGNOSIS
Patent Information
- Authority / Receiving Office
- TR · TR
- Patent Type
- Applications
- Current Assignee / Owner
- GAZI UNIVERISTESI
- Filing Date
- 2026-06-09
- Publication Date
- 2026-06-22
Abstract
Description
SERUM-BASED MULTIPLE BIOMARKER PANEL FOR GLIOMA DIAGNOSIS Technical Area The invention provides for the in vitro diagnostic evaluation of gliomas, which are tumors of the central nervous system. It relates to the field of technologies. More specifically, the invention involves the detection of GAL-8 in a serum sample obtained from an individual. Quantitative determination of ITGβ-1 and HIF-1α biomarkers and their combined effects This assessment determines the presence of glioma, glioma risk, low / high stage differentiation, prognosis, or treatment response. serum-based multibiomarker panels for providing information about, and kits using this panel, It relates to biosensor platforms and computer-aided decision systems. State of the Art Gliomas are primary lesions originating from glial cells that can have an aggressive course within the central nervous system. These are brain tumors. Current clinical evaluation involves imaging methods, histopathological examination, Immunohistochemical analyses and molecular pathology tests play an important role, but these methods are not universal. In this case, an early, non-invasive and easily repeatable liquid biopsy-based evaluation. It does not provide. Known approaches use various serum samples to diagnose glioma, assess its prognosis, or evaluate treatment response. plasma, tissue, genetic variant, mRNA, miRNA, exosome / microvesicle, or protein biomarkers It has been suggested. However, the known technique involves detecting GAL-8, ITGβ-1, and HIF-1α biomarkers together in serum. to measure and this triple combination as a single quantitative panel for glioma assessment Based on its use, it is a non-invasive and versatile clinical decision support tool that can be converted into a kit format. It does not present a multi-parameter solution with sufficient clarity. Therefore, hypoxia, angiogenesis, cell adhesion, migration, and immunity in glioma biology are important. Applicable to serum samples, reflecting modulation processes together, with reference / threshold values. There is a need for a new biomarker panel that can be interpreted and, when necessary, generate a software-assisted risk score. there are. Purpose of the Invention The aim of the invention is to aid in the non-invasive evaluation of gliomas. a study based on the combined measurement of GAL-8, ITGβ-1, and HIF-1α levels in a serum sample obtained from an individual The aim is to provide a method for obtaining in vitro information. Another objective of the invention is to compare the aforementioned three-part biomarker panel with reference or threshold values. A method, kit, or biosensor that produces an output about the presence or probability of glioma by comparison. to provide a platform or computer-aided decision system. Another aim of the invention is to include GAL-1, GAL-3, and MMP-2 panels in addition to the GAL-8, ITGβ-1, and HIF-1α panels. Distinguishing between low-grade and high-grade gliomas using 35 and / or MMP-9 biomarkers. 1. Helps in making distinctions, evaluating prognosis, or monitoring treatment response. The goal is to provide an expanded panel. Brief Description of the Invention The invention is based on measuring the levels of GAL-8, ITGβ-1, and HIF-1α in a serum sample obtained from an individual. quantitative determination and these levels are determined against predetermined reference values, threshold values or multiples The comparison is made using a multivariate classification model. The comparison results show the presence of glioma. An outcome is generated regarding the likelihood of glioma, low / high stage differentiation, prognosis, or treatment response. In one application, the thresholds were 8.28 ng / mL for GAL-8, 436.34 pg / mL for ITGβ-1, and 1411.00 pg / mL for HIF-1α. The measured values are used. By comparing the measured values with these threshold values, the serum sample of the individual is determined. An in vitro data output is provided regarding whether or not it is associated with glioma. In one application, a triple panel combined classification derived from GAL-8, ITGβ-1 and HIF-1α levels. This is converted into a score. A classification score of 0.595 or higher indicates that the serum sample has glioma. This is considered an outcome indicating an increased probability of compatibility. This value is not limiting and may vary for different patients. They can be recalibrated according to cohorts, measurement platforms, or validation studies. The invention also includes antibodies, antibody fragments, aptamers, and ligands suitable for measuring the aforementioned biomarkers. or kits containing other biospecific binding reagents; ELISA, solid-phase immunoassay, immunochromatographic test, lateral-flow test, electrochemical biosensor, and origami-based horizontal flow test platforms that process measurement data and generate risk scores or clinical decision support outputs; It relates to supported systems. Definitions Within the scope of this specification, the term "GAL-8" refers to the protein Galectin-8 or LGALS8 and the serum levels of this protein. It refers to measurable forms. The term “ITGβ-1” refers to Integrin beta-1 or ITGB1 protein, which can be measured in serum. It refers to the forms. The term “HIF-1α” refers to Hypoxia-inducible factor-1 alpha protein and its measurable form in serum. It expresses their forms. The term "serum sample" refers to a blood sample taken from an individual after processes such as clotting and centrifugation. It refers to an extracorporeal sample obtained. The method of the invention is a direct application to the individual's body. It is not a diagnostic or treatment method, but is based on the in vitro analysis of a serum sample obtained from an individual. The terms “reference value” or “threshold value” refer to healthy control groups, glioma patient groups, and low-grade glioma. a group, a high-grade glioma group, or patients from previously validated patient cohorts and a These refer to the values used for interpreting biomarkers. Threshold values, ROC analysis, Youden index, Logistic regression can be determined using machine learning models or clinical validation data. 2. Detailed Description of the Invention Preparation of serum sample In one procedure, blood samples are taken from individuals, placed in serum tubes, and transported to the laboratory under a cold chain. and centrifuged to obtain serum. For example, blood samples can be centrifuged at 4500 rpm for 15 minutes. The collected serum can be aliquoted in suitable tubes and stored at -80 °C until measurement. These parameters are limiting. not any suitable clinical laboratory for preparing serum for biomarker measurement. This procedure can be used. The serum sample may have been obtained from a human individual suspected of having glioma. Measurement of biomarkers Within the scope of this invention, GAL-8, ITGβ-1 and HIF-1α levels are determined quantitatively. The measurement is performed using ELISA. sandwich ELISA, chemiluminescence immunoassay, fluorescence immunoassay, electrochemical immunoassay, surface plasmon resonance, immunochromatographic test, lateral-flow test or any test based on biospecific binding This can be accomplished using a suitable method. Measurements can be performed individually or in multiplex format. In one application, GAL-8, ITGβ-1 and HIF-1α were immobilized separately or on the same solid phase. Antibodies are used. Alternatively, aptamers, peptide binders, and receptor-specific agents are used for these biomarkers. Fragments or other biospecific binding molecules can be used. Measurement results are given in ng / mL, pg / mL. Absorbance can be obtained as a unit of current, potential, fluorescence intensity, or normalized signal, and This can be converted to a concentration value using a calibration curve. Triple biomarker panel and interpretation. In the central application of the invention, serum GAL-8, ITGβ-1 and HIF-1α levels are evaluated together. This Three biomarkers represent related but distinct mechanisms in glioma biology: GAL-8, galectin Tumor microenvironment, immune modulation, cell adhesion and migration via the family; ITGβ-1, integrin mediated cell-matrix interaction and invasion; HIF-1α, on the other hand, is involved in hypoxic response, angiogenesis, and aggressive tumor phenotype. It is related to this. Therefore, the triple panel provides more comprehensive biological information compared to single biomarker measurements. It can provide. The following threshold values are used in an application: Biomarker Preferred threshold value Unit Interpretation example GAL-8 8.28 ng / mL. Values above the threshold indicate a higher probability of glioma. It is used as a panel input to show that it has increased. ITGβ-1 436.34 pg / mL. Values above the threshold indicate a higher probability of glioma. It is used as a panel input to show that it has increased. HIF-1α 1411.00 pg / mL. Values above the threshold indicate a higher probability of glioma. It is used as a panel input to show that it has increased. GAL-1 41.83 ng / mL Optional extended panel input. available. MMP-9 450.08 ng / mL as an optional extended panel input. available. 3. Values above the specified range indicate an increased probability of the presence of glioma. It is evaluated. In one application, GAL-8, ITGβ-1 and HIF-1α values are determined and a composite is formed from these values. The classification score is calculated. The classification score is used in, for example, logistic regression, decision tree, and support vector calculations. The score can be calculated using a machine, artificial neural network, or another statistical / machine learning model. The score is 0.595. A reading of 100% or higher could be used as an indication that the likelihood of compatibility with, for example, glioma is increased. The method is an in vitro method that determines the presence and stage of glioma in a serum sample obtained from an individual. It provides information about the prognosis or treatment response through the following steps. a) Galectin-8 (GAL-8), Integrin β-1 (ITGβ-1) and Hypoxia-Inducible Substances were detected in the serum sample in question. Factor-1α (HIF-1α) levels are measured, b) measured GAL-8, ITGβ-1 and HIF-1α levels compared with one or more reference values and / or threshold values compared, and c) According to the comparison result, the presence of glioma in the individual from whom the sample was taken, low-grade glioma / high-grade glioma Differentiating between stages of glioma provides an outcome regarding prognosis or treatment response. Measurements are performed using binding agents specific to GAL-8, ITGβ-1, and HIF-1α. Binding agents include antibodies, antibody fragments, aptamers, peptide binders, ligands, or combinations thereof. Selectable methods include ELISA, sandwich immunoassay, multiple immunoassay, and chemiluminescence immunoassay. This can be performed using fluorescence immunoassay, electrochemical immunoassay, or biosensor-based analysis. Expanded panel In one application of the invention, the basic triple panel may also include GAL-1, GAL-3, MMP-2 and / or MMP-9. This is expanded with biomarkers that allow for the measurement of low levels of gliomas. Differentiating between low-stage and high-stage subgroups, evaluating tumor aggressiveness, additional information that will help predict the risk of disease progression or monitor the response to treatment This can be achieved using a method with a GAL-1 level threshold of 41.83 ng / mL and / or an MMP-9 level. It is applied by comparison with the threshold value of 450.08 ng / mL. In one application, GAL-8 and / or MMP-9 levels are used to differentiate between low-grade and high-grade gliomas. Useful inputs for and in the form of indicators of tumor progression risk, prognosis, or treatment response. It is used as such. However, the contribution of each biomarker in the expanded panel varies depending on the patient used. The cohort, measurement method, validation model, and clinical question are all considered together. 4. Kit and device applications The invention can be implemented as a kit. The kit is suitable for the detection of GAL-8, ITGβ-1 and HIF-1α in serum samples. biospecific binding reagents, calibrators, positive / negative control materials, signal generators It may include reagents, buffers, washing solutions, solid-phase carriers, and usage instructions. The instructions for use include comparing the measured biomarker levels with reference / threshold values and / or This may include the calculation of a combined panel score. The detection agents in question are antibodies, antibody fragments, aptamers, The peptide binder, ligand, or combination thereof is selected. The kit also includes GAL-1, GAL-3, MMP-2 and / or It may include additional detection agents for measuring MMP-9 levels. In one application, the kit is designed as a microplate-based ELISA kit. In another application, the kit is designed as a lateral- It is organized in flow or immunochromatographic strip format. In another application, the kit is mobile device compatible. It is designed as an origami-based electrochemical horizontal flow test platform. In this application, the serum sample is placed on paper. or advanced along a microfluidic-based carrier, biomarkers are captured by binding reagents and The signal is read electrochemically and transmitted to a mobile device or connected reader. The detection agents in question may be immobilized on a solid support. The solid support plate, membrane, microfluidic channel, electrode surface, paper-based support, horizontal flow test strip, or origami-based It is characterized by its foldable test platform. The test platform is suitable for electrochemical signals, optical signals, and colorimetric signals. The signal is suitable for generating fluorescent signals or chemiluminescent signals. Computer-aided decision system The invention can also be implemented as a computer-aided system for processing measurement data. The system is GAL-8. An input module that receives ITGβ-1 and HIF-1α levels can use these levels as reference / threshold values or for classification. A processor that compares the model with glioma probability, class label, risk group, staging, prognosis or treatment. It may include an output module that provides the response. The system compares data obtained from serial serum samples taken from the same individual over time. It can also provide trend information about treatment response or disease progression. In such an application Increases, decreases, or stable levels of biomarkers, predetermined clinical decision-making rules, or This can be interpreted using machine learning models. In this in vitro method, the biomarker panel includes the biomarkers GAL-8, ITGβ-1, and HIF-1α. The panel provides information about the presence of glioma, glioma stage, prognosis, or treatment response. The panel also includes GAL-1 and GAL-3. It may contain at least one of the MMP-2 and / or MMP-9 biomarkers. As an in vitro decision support system, this system measures: a) GAL-8, ITGβ-1 and HIF-1α values. a) a data input module configured to receive the measurement values, b) the measurement values in question as reference values and / or thresholds a) a processor structured for comparison with values, and c) presence of glioma, glioma stage, prognosis or treatment It includes an output module structured to provide output about the response. 35 Examples of Experimental Applications Example 1 - Study groups and serum measurement In a validation study, 50 patients with low-grade glioma, 50 patients with high-grade glioma, and 50 healthy individuals were included. A cohort of 150 people, including a control group, was used. Patients were newly diagnosed and had been measured prior to the procedure. Adults who had not yet started radiotherapy or chemotherapy were selected. Serum samples were analyzed for GAL- Levels of GAL-1, GAL-3, GAL-8, ITGβ-1, HIF-1α, MMP-2, and MMP-9 were determined using the ELISA method. Example 2 - Threshold values for the presence of glioma In ROC analysis, sample threshold values for the presence of glioma were 8.28 ng / mL for GAL-8 and 436.34 ng / mL for ITGβ-1. It was determined as 1411.00 pg / mL for HIF-1α and 41.83 ng / mL for GAL-1 in the expanded panel. For MMP-9, threshold values of 450.08 ng / mL can be used. These threshold values are based on the measurement platform and the patient. It can be recalibrated according to the population. Example 3 - Combined triple panel performance In the combined panel using GAL-8, ITGβ-1, and HIF-1α biomarkers, the AUC value was 0.959. It was found that when the optimal classification threshold was determined as 0.595, the panel's sensitivity was 85%. The specificity was calculated as 94%. These results are based on a three-panel analysis of glioma patients and healthy controls. This supports the idea that it can demonstrate high diagnostic performance in differentiation. Example 4 - Low / high phase distinction Serum GAL-1, GAL-3, GAL-8, ITGβ-1, HIF-1α, are used in differentiating between low-grade and high-grade gliomas. MMP-2 and MMP-9 levels can be evaluated. In one application, GAL-8 and MMP-9, low-stage and high-stage It showed higher AUC values in terms of differentiating staged gliomas. Therefore, the expanded panel, It can be particularly useful in staging and prognosis assessment. Industrial Applicability of the Invention The invention has applications in clinical biochemistry laboratories, neuro-oncology centers, hospital laboratories, In biotechnology diagnostic kit production, automated analysis devices, lateral-flow or electrochemical biosensors. It can be implemented in platforms and digital health / clinical decision support systems. It is serum-based and in vitro. The fact that it is an analysis ensures that the invention is repeatable, scalable, and adaptable to a commercial kit format. 6
Claims
1. Presence of glioma, glioma stage, prognosis, or treatment response in a serum sample obtained from an individual. It is an in vitro method to provide information about; a) Galectin-8 (GAL-8), Integrin β-1 (ITGβ-1) and Hypoxia were detected in the serum sample in question. Measuring inducible factor-1α (HIF-1α) levels, b) measured GAL-8, ITGβ-1 and HIF-1α levels compared to one or more reference values and / or thresholds comparison with the value, and c) According to the comparison result, the presence of glioma in the individual from whom the sample was taken, and low-grade glioma. Differentiating between glioma and high-grade glioma, providing an outcome regarding prognosis or treatment response. It is characterized by its inclusion.
2. The method is as per Claim 1, and the serum sample in question is taken from a human individual suspected of having glioma. It is characterized by having been obtained.
3. The method is according to claim 1 or 2, and the measurement in question is specific to GAL-8, ITGβ-1 and HIF-1α. It is characterized by being performed using binding agents.
4. The method is according to claim 3, and the binding agents in question are antibodies, antibody fragments, aptamers, It is characterized by the selection of peptide binders, ligands, or combinations thereof.
5. The method is according to any of the previous requirements, and the measurement in question is ELISA, sandwich immunoassay, multiple immunoassay, chemiluminescence immunoassay, fluorescence immunoassay, It is characterized by being performed via electrochemical immunoassay or biosensor-based analysis.
6. The method is based on any of the previous requirements, with a GAL-8 level threshold of 8.28 ng / mL. with, ITGβ-1 level threshold value of 436.34 pg / mL and HIF-1α level threshold value of 1411.00 pg / mL It is characterized by comparison with.
7. The method according to claim 6, where GAL-8 level is above 8.28 ng / mL and ITGβ-1 level is above 8.28 ng / mL. A glioma level above 436.34 pg / mL and / or a HIF-1α level above 1411.00 pg / mL indicates glioma. It is characterized by being considered an indicator of increased probability of its existence.
8. A method according to any of the previous requests, from GAL-8, ITGβ-1 and HIF-1α levels. calculation of a combined biomarker score and the classification of that combined biomarker score It is characterized by comparison with the classification threshold.
9. The method is according to claim 8, and the threshold value of the combined biomarker score in question is 0.
595. It is characterized by comparison.
10. The method is based on any of the previous requests, and the said method is also GAL-1, GAL- 3. It is characterized by involving the measurement of MMP-2 and / or MMP-9 levels. 7 11. The method according to claim 10, with a GAL-1 level threshold of 41.83 ng / mL and / or MMP-9 It is characterized by comparing its level to the threshold value of 450.08 ng / mL.
12. This method is based on any of the previous requests, and the outputs in question are related to the presence of glioma, glioma Absence, likelihood of low-grade glioma, likelihood of high-grade glioma, risk of tumor progression, prognosis It is characterized by being provided as an indicator of treatment response or treatment response.
13. The method is based on any of the previous requests, and the method in question is used at different times. Comparison of GAL-8, ITGβ-1 and HIF-1α levels in serum samples taken at various points It is characterized by providing information about disease progression or treatment response.
14. The method is based on any of the previous requests, and the serum sample in question contains GAL-8, ITGβ- An automated analyzer for measuring HIF-1α and HIF-1α levels, microfluidic platform, multiple analysis platform, portable analysis platform or laboratory type immunoassay system It is characterized by its implementation.
15. This is a method based on any of the previous requirements, involving comparison and output generation. steps of a computer-aided decision support algorithm executed by a processor It is characterized by its implementation.
16. The method is according to claim 15, and the decision support algorithm in question is GAL-8, ITGβ-1 and HIF-1α. It takes measurement values as input, and uses these values against a reference data set and / or threshold values. comparison and classification of glioma presence, stage, prognosis, or treatment response. It is characterized by its ability to produce output.
17. It is a kit for in vitro methods to determine the presence of glioma, glioma stage, prognosis, or treatment response. the chitin in question; a) At least one primary detection agent for measuring GAL-8 levels, b) At least one second detection agent for measuring ITGβ-1 levels, c) At least one third detection agent for measuring HIF-1α levels, and d) the reference values and / or threshold values of the GAL-8, ITGβ-1 and HIF-1α levels in question A kit characterized by containing instructions for comparison.
18. The kit is in accordance with Claim 17 and the detection agents in question are antibodies, antibody fragments, aptamers, peptides. It is characterized by the selection of a binder, ligand, or combination thereof.
19. The kit is in accordance with Claim 17 or 18, and said kit also includes GAL-1, GAL-3, MMP-2 and / or MMP- It is characterized by containing additional detection agents for measuring levels 9. 8 20. The kit is according to any of the claims 17 to 19, and the kit in question is an ELISA kit, multiple immunoassay kit. in the form of a kit, microfluidic analysis kit, biosensor-based analysis kit, or portable analysis kit. It is characterized by its being.
21. The kit is based on any of claims 17 to 20, and requires strict support for the detection agents in question. It is characterized by being immobilized.
22. The kit is in accordance with claim 21 and the said solid support consists of a plate, membrane, microfluidic channel, electrode. surface, paper-based support, horizontal flow test strip, or origami-based foldable test platform It is characterized by its being.
23. The kit is based on claim 22 and the test platform in question measures electrochemical signals, optical signals, Suitable for generating colorimetric signals, fluorescent signals, or chemiluminescent signals. It is characteristic.
24. Used for in vitro methods regarding the presence of glioma, glioma stage, prognosis, or treatment response. It is a biomarker panel, and the said panel consists of GAL-8, ITGβ-1 and HIF-1α biomarkers. A panel of biomarkers characterized by their presence or inclusion.
25. According to claim 24, it is a biomarker panel, and said panel also includes GAL-1, GAL-3, MMP-2 and / or is characterized by containing at least one of the MMP-9 biomarkers.
26. Detection agents specific to GAL-8, ITGβ-1 and HIF-1α, according to any of claims 1 to 16. Its use in preparing an in vitro diagnostic or prognostic kit for use in the method.
27. It is an in vitro decision support system, and the system in question; a) A data input module configured to receive GAL-8, ITGβ-1 and HIF-1α measurement values, b) structured to compare the measured values in question with reference values and / or threshold values. a processor, and c) Structured to provide output regarding the presence of glioma, glioma stage, prognosis, or treatment response. an output module a system characterized by its inclusion. It relates to platforms and computer-aided decision systems. 9