A biomarker combination for predicting immune therapy efficacy in nsclc patients with EGFR gene mutations and its applications

Abstract

The invention belongs to the technical field of biology, and particularly relates to a group of markers for predicting the immunotherapy effect of EGFR gene mutation NSCLC patients and application of the markers. The biomarker disclosed by the invention is simple and convenient to sample and quick in result reporting. The biomarker disclosed by the invention is high in accuracy: the AUC of CCL4 is equal to 0.771, the sensitivity is 0.64, the specificity is 0.67 ([95% CI: 0.62-0.93]); plt; 0.05) of the substrate (1); the AUC of the PD-L1 is equal to 0.720, the sensitivity is 0.60, the specificity is 0.82 ([95% CI: 0.55-0.89]); plt; 0.05) of the method. In a screening queue, a combined diagnosis ROC curve of CCL4 and PD-L1 is as follows: AUC is equal to 0.907, the sensitivity is 0.667, and the specificity is 1.000 ([95% CI: 0.696-1.000]); plt; 0.05) of the substrate (1); in the verification queue, the AUC of the combined diagnosis of CCL4 and PD-L1 is equal to 0.833, the sensitivity is 0.69, the specificity is 0.91, and the AUC is less than [95% CI: 0.70-0.96]; plt; 0.05) of the substrate (1); therefore, the efficiency of combined diagnosis of the CCL4 and the PD-L1 is higher than that of diagnosis by independently using the CCL4 or the PD-L1; the marker is high in diagnosis efficiency, the immunotherapy curative effect of the NSCLC patient with EGFR gene mutation is predicted, and clinical guidance is provided.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511922185.6 (22) Application Date 2025.12.18 (71) Applicant: Beijing Chest Hospital Affiliated to Capital Medical University Address: No. 9, Beiguan Street, Tongzhou District, Beijing 101149 (72) Inventors: Ma Li, Chen Ran, Dong Youhong, Yang Xinyue, Lin Gen, Qian Zhe, Li Jiaxin (74) Patent Agency: Beijing Xianghe Intellectual Property Agency (General Partnership) 11893 Patent Attorney: Feng Mingyan (51) Int.Cl. G01N 33 / 5752 (2026.01) G01N 33 / 575 (2026.01) G01N 33 / 68 (2006.01) (54) Invention Title: A combination of biomarkers for predicting the efficacy of immunotherapy in patients with EGFR gene mutation NSCLC and their application (57) Abstract: This invention belongs to the field of biotechnology, specifically relating to a set of biomarkers for predicting the efficacy of immunotherapy in patients with EGFR gene mutation NSCLC and their application. The biomarkers of this invention are easy to sample and report results quickly. The biomarkers of this invention have high accuracy: AUC=0.771 for CCL4, sensitivity 0.64, specificity 0.67, ([95% CI: 0.62–0.93]; p<0.05); AUC=0.720 for PD-L1, sensitivity 0.60, specificity 0.82, ([95% CI: 0.55–0.89]; p<0.05). In the screening cohort, the combined diagnostic efficacy of CCL4 and PD-L1 was AUC=0.907, sensitivity 0.667, and specificity 1.000 ([95% CI: 0.696–1.000]; p<0.05); in the validation cohort, the combined diagnostic efficacy of CCL4 and PD-L1 was AUC=0.833, sensitivity 0.69, and specificity 0.91 ([95% CI: 0.70–0.96]; p<0.05). This demonstrates that the combined diagnostic efficacy of CCL4 and PD-L1 is higher than that of CCL4 or PD-L1 alone. The high diagnostic efficacy of these biomarkers predicts the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations, providing guidance for clinical practice. Claims (1 page), Description (11 pages), Drawings (5 pages), CN 121856551 A 2026.04.14 CN 1 21 85 65 51 A 1. A combination of biomarkers for predicting the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations, said biomarkers being a combination of CCL4 and PD-L1. 2.1. As described in claim 1, when the biomarker is a combination of CCL4 and PD-L1, and the CCL4 expression level in the patient's biosample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biosample is greater than or equal to 50%, the immunotherapy efficacy in NSCLC patients with EGFR gene mutations is good. 2. As described in claim 2, wherein the patient's biosample is selected from the primary tumor tissue of a patient with EGFR gene mutations in NSCLC. 3. The use of the biomarker as described in claim 1 in the preparation of reagents for predicting the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations; wherein the biomarker is a combination of CCL4 and PD-L1. 4. As described in claim 4, when the biomarker is a combination of CCL4 and PD-L1, and the CCL4 expression level in the patient's biosample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biosample is greater than or equal to 50%, the immunotherapy efficacy in NSCLC patients with EGFR gene mutations is good. 6. The application of claim 5, wherein the patient's biosample is selected from the primary tumor tissue of a patient with EGFR gene mutation-positive NSCLC. 7. A kit for predicting the efficacy of immunotherapy in patients with EGFR gene mutation-positive NSCLC, the kit containing a reagent for detecting the expression level of the biomarker of claim 1; the biomarker is a combination of CCL4 and PD-L1. 8. The kit of claim 7, wherein when the biomarker is a combination of CCL4 and PD-L1, the CCL4 expression level in the patient's biosample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biosample is greater than or equal to 50%, the immunotherapy efficacy in patients with EGFR gene mutation-positive NSCLC is good. 9. The kit of claim 8, wherein the patient's biosample is selected from the primary tumor tissue of a patient with EGFR gene mutation-positive NSCLC. 10. The kit of claim 7, wherein the kit is an ELISA detection kit or a colloidal gold detection kit; the diagnostic methods of the kit include direct methods, indirect methods, double-antibody sandwich methods, and competitive methods. Claims 1 / 1 page 2 CN 121856551 A Biomarker Combination for Predicting the Efficacy of Immunotherapy in Patients with EGFR Gene Mutation NSCLC and Its Application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a biomarker combination for predicting the efficacy of immunotherapy and its application. Background Art

[0002] Lung cancer remains one of the leading malignant tumors in terms of both incidence and mortality (Sung H, Ferlay J, Siegel RL, et al.).Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249.). GLOBOCAN data shows that in 2020, there were approximately 2.2 million new cases of lung cancer, accounting for 11.4% of all diagnosed malignant tumors, ranking second in incidence, only after breast cancer. Because most lung cancers develop insidiously and have atypical early symptoms, most diagnosed patients are already in advanced stages, resulting in poor prognosis and short survival time. The 5-year survival rate is only about 20% (Wang Q, Chen Y, Feng H, et al. Prognostic and predictive value of HURP in nonsmall cell lung cancer[J]. Oncol Rep, 2018, 39(4): 1682- 1692.;Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019[J]. CA Cancer J Clin , 2019 , 69(1).). The most common type of lung cancer is non-small cell lung cancer (NSCLC), accounting for about 85%, mainly classified as lung adenocarcinoma and lung squamous cell carcinoma (Yang D, Liu Y, Bai C, et al. Epidemiology of lung cancer and lung cancer screening programs in China and the United States[J]. Cancer Lett, 2020, 468: 82-87.).

[0003] Epidermal growth factor receptor (EGFR) mutation is one of the most common gene mutation subtypes in non-small cell lung cancer (NSCLC), accounting for about 1 / 3 of the NSCLC population (Zhang Y-L, Yuan J-Q, Wang K-F, et al.).The prevalence of EGFR mutation in patients with non-small cell lung cancer: a systematic review and meta-analysis[J]. Oncotarget, 2016, 7(48): 78985-78993.). This prevalence is higher in East Asian, female, and non-smoking patients with lung adenocarcinoma. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have brought significant survival benefits to patients with advanced NSCLC harboring EGFR mutations, greatly prolonging progression-free survival (PFS) and overall survival (OS) (Zhou C, Wu Y-L, Chen G, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomized, phase 3 study [J]. Lancet Oncol, 2011, 12(8): 735-742.). They have now become the first-line standard treatment for patients with advanced EGFR-mutant NSCLC (Rocco D, Battiloro C, Gravara LD, et al. Advanced Non-Small Cell Lung Cancer with Activating Epidermal Growth Factor Receptor Mutation: First Line Treatment and Beyond[J]. Rev Recent Clin Trials, 2019, 14(2): 120‑128.However, most of these patients will develop resistance to EGFR-TKIs 8-13 months after treatment (Piotrowska Z, Sequist L V. Epidermal Growth Factor Receptor-Mutant Lung Cancer: New Drugs, New Resistance Mechanisms, and Future Treatment Options [J]. Cancer J, 2015, 21(5): 371-377.). Approximately half of patients who develop resistance to first- and second-generation EGFR-TKIs will develop a secondary EGFR T790M resistance mutation in exon 20 of EGFR (Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib[J]. N Engl J Med, 2005, 352(8): 786-792.), and can subsequently receive third-generation EGFR TKI treatment with significant efficacy. However, drug resistance is an inevitable consequence of targeted therapy.

[0004] The advent of immune checkpoint inhibitors (ICIs) has brought significant achievements to the treatment of EGFR / ALK wild-type advanced NSCLC patients (Gadgeel S, Rodríguez-Abreu D, Speranza G, et al. Updated Analysis From KEYNOTE-189: Pembrolizumab or Placebo Plus Pemetrexed and Platinum for Previously Untreated Metastatic Nonsquamous Non-Small-Cell Lung Cancer[J]. J Clin Oncol, 2020, 38(14): 1505-1517.; Paz-Ares L, Vicente D, Tafreshi A, et al.A Randomized, Placebo-Controlled Trial of Pembrolizumab Plus Chemotherapy in Patients With Metastatic Squamous NSCLC: Protocol-Specified Final Analysis of KEYNOTE-407 [J]. J Thorac Oncol, 2020, 15(10): 1657-1669.). However, the efficacy in patients with EGFR gene mutation NSCLC is unsatisfactory. Some studies have reported that EGFR gene mutation NSCLC may have an unaffected, low-inflammatory, cold tumor microenvironment (TME), which leads to poor efficacy of anti-PD-1 inhibitor therapy. Therefore, PD-1 / PD-L1 axis blockade therapy is considered a "blunt sword" under this TME in such patient populations (Dong Z-Y, Zhang J-T, Liu S-Y, et al. EGFR mutation correlates with uninflamed phenotype and weak immunogenicity, causing impaired response to PD-1 blockade in non-small cell lung cancer[J]. Oncoimmunology, 2017, 6(11): e1356145.). Some patients with EGFR mutations may benefit from PD-1 inhibitors in later-line treatment. The Impower 150 study (Reck M, Mok TSK, Nishio M, et al. Atezolizumab plus bevacizumab and chemotherapy in non-small-cell lung cancer (IMpower150): key subgroup analyses of patients with EGFR mutations or baseline liver metastases in a randomised, openlabel phase 3 trial[J]. Lancet Respir Med, 2019, 7(5): 387-401.)) and the ORIENT 31 study (Lu S, Wu L, Jian H, et al. Sintilimab plus bevacizumab biosimilar IBI305 and chemotherapy for patients with EGFR‑mutated non‑squamous non‑small‑cell lung cancer who progressed on EGFR tyrosine‑kinase inhibitor therapy (ORIENT‑31): first interim results from a randomised , double‑blind, multicentre, phase 3 trial[J]. Lancet Oncol, 2022, 23(9): 1167‑ 1179 .), the NCT03513666 study (Jiang T , Wang P , Zhang J , et al . Toripalimab plus chemotherapy as second‑line treatment in previously EGFR‑TKI treated patients with EGFR‑mutant‑advanced NSCLC: a multicenter phase‑II trial[J] . Signal Transduct Target Ther, 2021, 6(1): 355.Combination therapy strategies with anti-PD-1 inhibitors, such as PD-1 inhibitors, have shown some efficacy in the later-line treatment of EGFR-TKI resistant NSCLC patients, but there is currently a lack of evaluation mechanisms to screen patients who respond to this treatment. Therefore, finding biomarkers to predict the efficacy of ICI treatment is particularly important.

[0005] Currently, common biomarkers for predicting the efficacy of immunotherapy include programmed cell death ligand 1 (PD-L1), tumor mutation burden (TMB), and microsatellite instability / mismatch repair (MSI-H / dMMR). Although they have been approved by the U.S. Food and Drug Administration (FDA) as companion diagnostic biomarkers for pan-solid tumor immunotherapy, their role in predicting the efficacy of NSCLC immunotherapy is controversial due to their poor predictive efficacy. First, PD-L1 expression has some predictive value in ICI monotherapy for some EGFR wild-type NSCLC patients, but its predictive value in EGFR mutant NSCLC is questionable (Mazieres J, Drilon A, Lusque A, et al. Immune checkpoint inhibitors for patients with advanced lung cancer and oncogenic driver alterations: results from the IMMUNOTARGET registry[J]. Ann Oncol, 2019, 30(8): 1321-1328.; Schoenfeld AJ, Rizvi H, Bandlamudi C, et al. Clinical and molecular correlates of PD-L1 expression in patients with lung adenocarcinomas[J]. Ann Oncol, 2020, 31(5): 599-608.).Secondly, reports indicate that in patients with EGFR-mutant NSCLC receiving ICI, TMB was not significantly associated with sustained clinical benefit (Vokes N, Alguilar EJ, Umeton R, et al. Inter-test variability in tumor mutational burden (TMB) quantification and identification of TMB thresholds[J]. Cancer Research, 2019, 79(13_Supplement): 2514-2514.). Therefore, there is an urgent need for ideal biomarkers to predict the efficacy of immunotherapy for patients with EGFR gene mutations.

[0006] Currently, CCL4 expression levels are associated with the survival of non-small cell lung cancer patients, but their specificity and sensitivity are low, limiting predictive accuracy. This invention discovers that CCL4 expression levels and PD-L1 TPS (tumor cell positivity fraction) in patient biosamples are associated with the efficacy of immunotherapy in EGFR-mutant NSCLC patients, and that the combination of these two biomarkers significantly improves predictive efficacy. Based on this, this invention was completed.

[0007] In a first aspect, the present invention provides a combination of biomarkers for predicting the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations, wherein the biomarker is a combination of CCL4 and PD-L1.

[0008] Further, when the biomarker is a combination of CCL4 and PD-L1, the expression level of CCL4 in the patient's biological sample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biological sample is greater than or equal to 50%, the immunotherapy efficacy in NSCLC patients with EGFR gene mutations is good.

[0009] Further, the patient's biological sample is selected from the primary tumor tissue of NSCLC patients with EGFR gene mutations.

[0010] In a second aspect, the present invention provides the use of the biomarker as described in the first aspect of the present invention in the preparation of a reagent for predicting the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations; wherein the biomarker is a combination of CCL4 and PD-L1.

[0011] Further, when the biomarker is a combination of CCL4 and PD-L1, the expression level of CCL4 in the patient's biological sample is greater than or equal to 0.When the expression level of PD-L1 in the patient's biological sample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biological sample is greater than or equal to 50%, the immunotherapy efficacy of NSCLC patients with EGFR gene mutation is good.

[0012] Further, the patient's biological sample is selected from the primary tumor tissue of the patient with EGFR gene mutation.

[0013] In a third aspect, the present invention provides a kit for predicting the efficacy of immunotherapy in NSCLC patients with EGFR gene mutation, the kit containing a reagent for detecting the expression level of the biomarker described in the first aspect; the biomarker is a combination of CCL4 and PD-L1.

[0014] Further, when the biomarker is a combination of CCL4 and PD-L1, the expression level of CCL4 in the patient's biological sample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biological sample is greater than or equal to 50%, the immunotherapy efficacy of NSCLC patients with EGFR gene mutation is good.

[0015] Further, the patient's biological sample is selected from the primary tumor tissue of NSCLC patients with EGFR gene mutations.

[0016] Further, the kit is an ELISA detection kit and / or a colloidal gold detection kit.

[0017] Even further, the diagnostic methods of the kit include direct methods, indirect methods, double-antibody sandwich methods, and / or competitive methods.

[0018] Beneficial Effects The biomarkers of this invention are easy to sample and report results quickly. The biomarkers proposed in this invention have high accuracy: AUC = 0.771 for CCL4, sensitivity 0.64, specificity 0.67 ([95% CI: 0.62–0.93]; p < 0.05); AUC = 0.720 for PD-L1, sensitivity 0.60, specificity 0.82 ([95% CI: 0.55–0.89]; p < 0.05); AUC = 0.833 for the combined diagnosis of CCL4 and PD-L1, sensitivity 0.69, specificity 0.91 ([95% CI: 0.70–0.96]; p < 0.05).05), the diagnostic efficacy of combined CCL4 and PD-L1 is higher than that of CCL4 or PD-L1 alone; the above biomarkers have high diagnostic efficacy and predict the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations, providing guidance for clinical practice. Figure Descriptions

[0019] Figure 1 shows the relationship between CCL4 expression and the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations in the screening cohort.

[0020] Note: A represents progression-free survival; B represents the proportion of disease progression.

[0021] Figure 2 shows the relationship between PD-L1 expression and the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations in the screening cohort.

[0022] Note: A represents progression-free survival; B represents the proportion of disease progression.

[0023] Figure 3 shows the ROC curve of the combined prediction of the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations using CCL4 and PD-L1 in the screening cohort.

[0024] Figure 4 shows the relationship between CCL4 expression and the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations in the validation cohort.

[0025] Note: A represents progression-free survival; B represents the proportion of disease progression.

[0026] Figure 5 shows the ROC curve for CCL4 expression in the validation cohort.

[0027] Figure 6 shows the relationship between PD-L1 expression and the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations in the validation cohort.

[0028] Note: A represents progression-free survival; B represents the proportion of disease progression.

[0029] Figure 7 shows the ROC curve for PD-L1 expression in the validation cohort.

[0030] Figure 8 shows the ROC curve for predicting the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations using the combination of CCL4 and PD-L1 in the validation cohort.

[0031] Figure 9 shows the ROC curves for single biomarkers and combinations of biomarkers. Detailed Embodiments

[0032] The specific embodiments of the present invention will be further described below. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0033] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0034] TPS (Tumor cell Proportion Score) The Chinese term for TPS is Tumor Cell Positive Percentage Score. It is defined as the percentage of tumor cells stained with PD-L1 at any intensity out of all tumor cells. TPS = Number of PD-L1 positive tumor cells at any intensity / Total number of tumor cells * 100%. (Instructions 4 / 11, page 6, CN 121856551 A

[0035] ) CCL4,Also known as macrophage inflammatory protein (MIP-1β), it belongs to the CC chemokine family and is a protein encoded by the CCL4 gene in the human body. The CCL4 gene is located in the 17q11-q21 region of chromosome 17, with a molecular weight of approximately 8-10 kDa. CCL4 can be secreted by monocytes, B cells, T cells, NK cells, dendritic cells, neutrophils, fibroblasts, endothelial cells, and epithelial cells.

[0036] Example 1: Inclusion and Exclusion Criteria and Patient Information 1. Inclusion criteria The patient was diagnosed with NSCLC by histopathology or cytology; according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging criteria, the patient was diagnosed with unresectable stage III or stage IV NSCLC; the patient was diagnosed with EGFR-sensitive mutations (19DEL or 21L858R) by ARMSPCR or Next Generation Sequencing (NGS) and was resistant to first-line EGFR-TKI therapy, and subsequently received anti-PD-1 therapy; the patient's ECOG score was 0-2; the patient's efficacy was evaluated according to the Response Evaluation Criteria Solid Tumors (RECIST) V1.1, and at least one target lesion was available for efficacy evaluation.

[0037] Immunotherapy: PD-1 / PD-L1 inhibitor monotherapy or combination chemotherapy / anti-angiogenic drugs were used for treatment.

[0038] 2. Exclusion criteria The patient has a second primary malignant tumor; the patient has concurrent vital organ dysfunction; and the patient has concurrent active autoimmune disease.

[0039] 3. Data AcquisitionPatients sign informed consent forms to protect their privacy, and the clinical research ethics committee reviews and approves the data.

[0040] All relevant clinicopathological data of the patient are extracted from the system, including gender, age, smoking history, pathological type, TNM stage, ECOG score, EGFR mutation type, specific gene mutation status (KRAS), ICI treatment method, number of lines of ICI treatment, imaging efficacy evaluation results, etc., and follow-up data and survival information are supplemented by telephone follow-up.

[0041] The start date of ICI treatment was defined as the start time; progression-free survival (PFS) was defined as the time interval from the start date of ICI treatment to disease progression or loss to follow-up; the objective response rate (ORR) was the sum of the proportions of patients achieving complete response (CR) and partial response (PR); the disease control rate (DCR) was the sum of the proportions of patients achieving CR, PR, and stable disease (SD); the ≥6-month treatment response rate was the proportion of patients whose disease progressed more than 6 months after the start of ICI treatment. Patients without a progression event at the follow-up endpoint were defined as censored.

[0042] In summary, a total of 57 samples met the criteria, including 17 in the screening cohort and 40 in the validation cohort.

[0043] Example 2: Predicting the efficacy of immunotherapy in patients with EGFR gene mutations in a screening cohort A. Test methods 1. ImmunohistochemistryFormalin was used to fix paraffin-embedded tissue to prepare paraffin sections of tumor tissue.

[0044] PD-L1 immunohistochemistry was performed using an automated staining machine; CCL4 immunohistochemistry was performed using manual staining, with the following specific steps: After obtaining the paraffin tissue sections, they were placed in an oven and then immersed in xylene solution, anhydrous ethanol, 95% ethanol, 90% ethanol and 80% ethanol in sequence, and then immersed in water for repair.

[0045] Antigen repair: Antigen repair was performed using sodium citrate antigen repair solution via thermal repair. Sodium citrate antigen repair solution was added, heated to boiling and then cooled. After heating, the sections were washed with TBST.

[0046] After adding endogenous peroxidase inhibitor, the sections were washed with TBST and blocked. After blocking, the residual solution was removed.

[0047] Primary antibody incubation: CCL4 antibody was added to the sections, and after overnight incubation, the sections were warmed and washed with TBST.

[0048] Secondary antibody incubation: Add reaction enhancement solution, incubate, and wash with TBST; add goat anti-rabbit secondary antibody, incubate, and wash with TBST.

[0049] DAB staining: Prepare DAB chromogenic reagent working solution according to the DAB chromogenic reagent kit instructions, add DAB chromogenic reagent, and rinse after reaction; counterstain cell nuclei with hematoxylin and then perform differentiation treatment.

[0050] Dehydration and clearing: Soak in 70% ethanol, 80% ethanol, 90% ethanol, anhydrous ethanol, and xylene solutions respectively, and mount after soaking.

[0051] Observe the expression of CCL4 and PD-L1 under a microscope.

[0052] 2. IHC score The expression of CCL4 and PD-L1 was observed under a microscope, and images were taken and saved.

[0053] The expression of CCL4 was read by Image-Pro Plus 6.0 software using the mean integrated optical density (mean IOD) of the DAB chromogenic area of ​​NSCLC tumor tissue. The calculation formula is: mean IOD = IOD / area of ​​the tumor section.

[0054] The appropriate cutoff value was obtained using x-tile 3.6.1 software to define CCL4 expression as high or low expression.

[0055] The expression of PD-L1 was determined by layering based on the tumor proportion scores (TPS).

[0056] Pathological slides with unclear clinicopathological conditions of the included cases were independently reviewed and scored. The results were determined by multiple experts through consultation based on the actual situation.

[0057] 3. EGFR mutation detectionEGFR mutation detection utilizes the ARMS method, namely the amplification refractory mutation system (ARMS) technology, to detect EGFR gene mutation types in histological or cytological specimens from tumor patients.

[0058] The DNA of tumor cells in paraffin-embedded tissue sections was quantitatively extracted using the QIAamp DNA kit, and the EGFR mutation types in tumor tissues were detected using ARMS-PCR technology. The ADx-ARMS kit was used to detect 29 mutation types, with internal and external quality control samples, positive controls, and negative controls included.

[0059] 4. Statistical methods The relationship between CCL4 expression levels and clinicopathological characteristics of the included patients was analyzed using the chi-square test / Fisher's exact test. The correlation between CCL4 expression (mean IOD) and PD-L1 expression was analyzed using the Pearson correlation test. The comparison of disease control rates and response rates ≥6 months after ICI treatment between subgroups was also analyzed using the chi-square test / Fisher's exact test. Kplan-meier survival analysis (Log-rank) was used to analyze the relationship between CCL4, PD-L1, and expression levels and PFS after ICI treatment. Cox regression analysis was performed, combining patient survival data and various factors that may affect the efficacy of ICI treatment. Factors with p-values ​​<0.05 in the univariate Cox regression analysis results were included in the multivariate Cox regression analysis. A p-value <0.05 was defined as statistically significant between groups. Statistical analysis and visualization were performed using SPSS 23.0 and GraphPad Prism9.

[0060] B. Test results Instruction manual, page 6 / 11, 8 CN 121856551 A 1. Patient's clinicopathological features and diagnostic and treatment history A total of 17 patients with stage III unresectable or stage IV EGFR-TKI-resistant NSCLC who received anti-PD-1 therapy were included. The clinicopathological characteristics and diagnostic and treatment history of the patients are detailed in Table 1.

[0061] Table 1. Clinicopathological characteristics of 17 patients 2. CCL4 expression levelCCL4 expression was measured using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA), which read the mean integrated optical density (mean IOD) of the DAB chromogenic region of NSCLC tumor tissue. The formula was mean IOD = IOD / area of ​​the tumor section.

[0062] An appropriate cutoff value (mean IOD = 0.0575636) was obtained using x-tile 3.6.1 software to define CCL4 expression as high or low.

[0063] That is, when the CCL4 expression level in the patient's biological sample is greater than or equal to 0.0575636, the immunotherapy efficacy of EGFR gene-mutant NSCLC patients is good; when the CCL4 expression level in the patient's biological sample is less than 0.0575636, the immunotherapy efficacy of EGFR-positive NSCLC patients is poor.

[0064] Figure 1 shows the relationship between CCL4 expression in the screening cohort and the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations. Figure 1A shows that when the CCL4 expression level in the patient's biosample was greater than or equal to 0.0575636, the patients had a longer PFS (mPFS: 5.8 months vs 2.7 months, HR=0.06, 95%CI: 0.1-0.52, p<0.05); and Figure 1B shows that when the CCL4 expression level in the patient's biosample was greater than or equal to 0.0575636, the proportion of patients experiencing disease progression was lower (PD: 10% vs 85.70%, p=0.02).

[0065] 3. PD-L1 TPSPD-L1 expression was stratified according to tumor proportion scores (TPS): TPS < 50% was defined as low expression, i.e., the negative group, and TPS ≥ 50% was defined as high expression, i.e., the positive group.

[0066] That is, when the TPS in the patient's biological sample is greater than or equal to 50%, the immunotherapy efficacy of NSCLC patients with EGFR gene mutation is good; when the TPS in the patient's biological sample is less than 50%, the immunotherapy efficacy of NSCLC patients with EGFR gene mutation is poor.

[0067] As shown in Figure 2, the relationship between PD-L1 expression in the screening cohort and the immunotherapy efficacy of NSCLC patients with EGFR gene mutation is shown. Figure 2A shows that when the TPS in the patient's biosample is greater than or equal to 50%, the patient has a longer PFS (mPFS: 6.4 vs 3.8 months, HR=0.06, 95%CI: 0.01-0.52, p<0.05); and Figure 2B shows that when the TPS in the patient's biosample is greater than or equal to 50%, the proportion of patients experiencing disease progression is lower (PD: 0.00% vs 58.30%, p=0.036).

[0068] 4. CCL4 combined with PD-L1 When the CCL4 expression level in the patient's biosample is greater than or equal to 0.0575636 and the TPS in the patient's biosample is greater than or equal to 50%, the immunotherapy efficacy is good in NSCLC patients with EGFR gene mutations; patients have a longer PFS.

[0069] As shown in Figure 3, the ROC curve for the combined diagnosis of CCL4 and PD-L1 is shown, AUC=0.907, sensitivity 0.667, specificity 1.000 ([95% CI: 0.696–1.000]; p<0.05).

[0070] Example 3: Validation of the efficacy of immunotherapy in predicting EGFR gene mutation patients in a cohort. A. Test methods 1. ImmunohistochemistryFormalin was used to fix paraffin-embedded tissue to prepare paraffin sections of tumor tissue.

[0071] PD-L1 immunohistochemistry was performed using an automated staining machine; CCL4 immunohistochemistry was performed using manual staining, with the following specific steps: After obtaining paraffin tissue sections, they were placed in an oven, and the sections were sequentially immersed in xylene solution, anhydrous ethanol, 95% ethanol, 90% ethanol and 80% ethanol, and then immersed in water for retrieval.

[0072] Antigen retrieval: Antigen retrieval was performed using a hot retrieval method with sodium citrate antigen retrieval solution. Sodium citrate antigen retrieval solution was added, heated to boiling and then cooled, and then washed with TBST after heating.

[0073] After adding endogenous peroxidase inhibitor, the sections were washed with TBST for blocking, and the residual solution was removed after blocking.

[0074] Primary antibody incubation: CCL4 antibody was added to the sections, and after overnight incubation, the sections were warmed and washed with TBST.

[0075] Secondary antibody incubation: Add reaction enhancement solution, incubate, and wash with TBST; add goat anti-rabbit secondary antibody, incubate, and wash with TBST.

[0076] DAB staining: Prepare DAB staining working solution according to the DAB staining kit instructions, add DAB staining agent, and rinse after reaction; counterstain cell nuclei with hematoxylin and then perform differentiation treatment.

[0077] Dehydration and clearing: Soak in 70% ethanol, 80% ethanol, 90% ethanol, anhydrous ethanol and xylene solutions respectively, and mount after soaking.

[0078] Observe the expression of CCL4 and PD-L1 under a microscope.

[0079] 2. IHC score The expression of CCL4 and PD-L1 was observed under a microscope, and images were taken and saved.

[0080] The expression of CCL4 was read by Image-Pro Plus 6.0 software using the mean integrated optical density (mean IOD) of the DAB chromogenic area of ​​NSCLC tumor tissue. The calculation formula is: mean IOD = IOD / area of ​​the tumor section.

[0081] The appropriate cutoff value was obtained using x-tile 3.6.1 software to define CCL4 expression as high or low.

[0082] The expression of PD-L1 was classified according to the tumor proportion scores (TPS): TPS < 50% was defined as low expression; TPS ≥ 50% was defined as high expression.

[0083] The pathological slides of cases with unclear clinicopathological conditions were independently reviewed and scored. The results were determined by multiple experts through consultation based on the actual situation.

[0084] 3. EGFR mutation detectionEGFR mutation detection utilizes the ARMS method, namely the amplification refractory mutation system (ARMS) technology, to detect EGFR gene mutation types in histological or cytological specimens from tumor patients.

[0085] The DNA of tumor cells in paraffin-embedded tissue sections was quantitatively extracted using the QIAamp DNA kit, and the EGFR mutation types in tumor tissues were detected using ARMS-PCR technology. The ADx-ARMS kit was used to detect 29 mutation types, with internal and external quality control samples, positive controls, and negative controls included.

[0086] 4. Statistical methods The relationship between CCL4 expression levels and clinicopathological characteristics of the included patients was analyzed using the chi-square test / Fisher's exact test. The correlation between CCL4 expression (mean IOD) and PD-L1 expression was analyzed using the Pearson correlation test. The comparison of disease control rates and response rates ≥6 months after ICI treatment between subgroups was also analyzed using the chi-square test / Fisher's exact test. Kplan-meier survival analysis (Log-rank) was used to analyze the relationship between CCL4, PD-L1, and expression levels and PFS after ICI treatment. Cox regression analysis was performed, combining patient survival data and various factors that may affect the efficacy of ICI treatment. Factors with p-values ​​<0.05 in the univariate Cox regression analysis results were included in the multivariate Cox regression analysis. A p-value <0.05 was defined as statistically significant between groups. Statistical analysis and visualization were performed using SPSS 23.0 and GraphPad Prism9.

[0087] B. Test results 1. Patient's clinicopathological features and diagnostic and treatment history A total of 40 patients with stage III unresectable or stage IV EGFR-TKI-resistant NSCLC who received anti-PD-1 therapy were included. The clinicopathological characteristics and diagnostic and treatment history of the patients are detailed in Table 2.

[0088] Table 2. Clinicopathological characteristics of 40 patients. (Page 9 / 11, CN 121856551 A 2.) CCL4 expression levelCCL4 expression was measured using Image-Pro Plus 6.0 software (Media Cybernetics, Silver, manual pages 10 / 11, CN 121856551 A Spring, MD, USA), which read the mean integrated optical density (mean IOD) of the DAB chromogenic region of NSCLC tumor tissue. The formula was mean IOD = IOD / area of ​​the tumor section.

[0089] An appropriate cutoff value (mean IOD = 0.062588) was obtained using x-tile 3.6.1 software to define CCL4 expression as high or low.

[0090] That is, when the CCL4 expression level in the patient's biological sample is greater than or equal to 0.062588, the immunotherapy efficacy of NSCLC patients with EGFR gene mutation is good; when the CCL4 expression level in the patient's biological sample is less than 0.062588, the immunotherapy efficacy of NSCLC patients with EGFR gene mutation is poor.

[0091] As shown in Figure 4, the relationship between CCL4 expression and the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations was verified. Figure 4A shows that when the CCL4 expression level in the patient's biosample was greater than or equal to 0.062588, the patients had a longer PFS (mPFS: 6.9 months vs 3.5 months, HR=0.39, 95%CI: 0.16-0.98, p=0.036); and Figure 4B shows that when the CCL4 expression level in the patient's biosample was greater than or equal to 0.062588, the proportion of patients with disease progression was lower (PD: 22.20% vs 53.80%, p=0.045).

[0092] As shown in Figure 5, the ROC curve of the verification cohort CCL4 is shown. AUC=0.771, sensitivity 0.64, specificity 0.67 ([95% CI: 0.62–0.93]; p<0.05).

[0093] 3. PD-L1 TPSPD-L1 expression was stratified according to tumor proportion scores (TPS): TPS < 50% was defined as low expression (negative group); TPS ≥ 50% was defined as high expression (positive group).

[0094] That is, when the TPS in the patient's biological sample is greater than or equal to 50%, the immunotherapy efficacy of EGFR gene-mutant NSCLC patients is good; when the TPS in the patient's biological sample is less than 50%, the immunotherapy efficacy of EGFR gene-mutant NSCLC patients is poor.

[0095] As shown in Figure 6, to verify the relationship between PD-L1 expression in the cohort and the immunotherapy efficacy of EGFR gene-mutant NSCLC patients. Figure 6A shows that when the TPS in the patient's biosample is greater than or equal to 50%, the patient has a longer PFS (mPFS: 7.9 vs 3.9 months, HR=0.37, 95%CI: 0.15–0.90, p=0.022); and Figure 6B shows that when the TPS in the patient's biosample is greater than or equal to 50%, the proportion of patients experiencing disease progression is lower (PD: 10.50% vs 52.38%, p=0.005).

[0096] As shown in Figure 7, the ROC curve of PD-L1 is shown, AUC=0.720, sensitivity 0.60, specificity 0.82, ([95% CI: 0.55–0.89]; p<0.05).

[0097] 4. CCL4 combined with PD-L1When the CCL4 expression level in the patient's biosample is greater than or equal to 0.062588 and the TPS in the patient's biosample is greater than or equal to 50%, the immunotherapy efficacy of EGFR gene-mutant NSCLC patients is good, and the patients have a longer PFS.

[0098] As shown in Figure 8, the ROC curve of the combined diagnosis of CCL4 and PD-L1 is shown, AUC=0.833, sensitivity 0.69, specificity 0.91 ([95% CI: 0.70–0.96]; p<0.05).

[0099] As shown in Figure 9, the efficacy of the combined diagnosis of CCL4 and PD-L1 is higher than that of the diagnosis using CCL4 or PD-L1 alone. Instruction Manual 11 / 11 Page 13 CN 121856551 A Figure 1 Figure 2 Instruction Manual Appendix 1 / 5 Page 14 CN 121856551 A Figure 3 Figure 4 Instruction Manual Appendix 2 / 5 Page 15 CN 121856551 A Figure 5 Figure 6 Instruction Manual Appendix 3 / 5 Page 16 CN 121856551 A Figure 7 Figure 8 Instruction Manual Appendix 4 / 5 Page 17 CN 121856551 A Figure 9 Instruction Manual Appendix 5 / 5 Page 18 CN 121856551 A Abstract The present invention belongs to the field of biotechnology, and specifically relates to a panel of biomarkers for predicting the efficacy of immunotherapy in non-small cell lung cancer (NSCLC) patients with EGFR gene mutations and applications thereof. The biomarkers claimed in the present invention feature convenient sample collection and rapid result reporting, and exhibit high diagnostic accuracy: for CCL4, the area under the curve (AUC) is 0.771, with a sensitivity of 0.64, a specificity of 0.67 ([95% CI: 0.62–0.93]; p<0.05); for PD-L1, the AUC is 0.720, with a sensitivity of 0.60, a specificity of 0.82 ([95% CI: 0.55–0.89]; p<0.05). In the screening cohort, for the combined diagnosis of CCL4 and PD-L1 based on receiver operating characteristic (ROC) curve analysis, the AUC is 0.907, with a sensitivity of 0.667 and a specificity of 1.000 ([95% CI: 0.696–1.000]; p<0.05); in the validation cohort, the combined diagnosis of CCL4 and PD-L1 yields an AUC of 0.833, with a sensitivity of 0.69 and a specificity of 0.91 ([95% CI: 0.70–0.96]; p<0.05). These results demonstrate that the diagnostic performance of the combined CCL4 and PD-L1 assay outperforms that of CCL4 or PD-L1 used alone. The aforementioned biomarkers exhibit excellent diagnostic performance, enabling accurate prediction of immunotherapy efficacy in NSCLC patients with EGFR mutations, and providing clinical guidance for treatment decision-making.

Claims

1. A combination of biomarkers for predicting the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations, said biomarkers being a combination of CCL4 and PD-L1.

2. As described in claim 1, when the biomarker is a combination of CCL4 and PD-L1, the expression level of CCL4 in the patient's biosample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biosample is greater than or equal to 50%, the immunotherapy efficacy of NSCLC patients with EGFR gene mutations is good.

3. The biomarker as described in claim 2, wherein the patient's biological sample is selected from the primary tumor tissue of an NSCLC patient with an EGFR gene mutation.

4. The use of the biomarker as described in claim 1 in the preparation of a reagent for predicting the efficacy of immunotherapy in NSCLC patients with EGFR gene mutations; wherein the biomarker is a combination of CCL4 and PD-L1.

5. In the application described in claim 4, when the biomarker is a combination of CCL4 and PD-L1, the expression level of CCL4 in the patient's biosample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biosample is greater than or equal to 50%, the immunotherapy efficacy is good for NSCLC patients with EGFR gene mutations.

6. The application as described in claim 5, wherein the patient's biological sample is selected from the primary tumor tissue of an NSCLC patient with an EGFR gene mutation.

7. A kit for predicting the efficacy of immunotherapy in patients with EGFR gene-mutant NSCLC, the kit comprising a reagent for detecting the expression level of the biomarker of claim 1; wherein the biomarker is a combination of CCL4 and PD-L1.

8. The kit of claim 7, wherein when the biomarker is a combination of CCL4 and PD-L1, the expression level of CCL4 in the patient's biosample is greater than or equal to 0.062588, and the TPS of PD-L1 in the patient's biosample is greater than or equal to 50%, the immunotherapy efficacy of EGFR gene-mutant NSCLC patients is good.

9. The kit of claim 8, wherein the patient's biological sample is selected from the primary tumor tissue of an NSCLC patient with an EGFR gene mutation.

10. The kit according to claim 7, wherein the kit is an ELISA detection kit or a colloidal gold detection kit; the diagnostic methods of the kit include direct methods, indirect methods, double-antibody sandwich methods, and competitive methods.

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