Application of human endogenous retrovirus in predicting non-small cell lung cancer EGFR-TKI drug targeted therapy drug resistance

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of human endogenous retrovirus in predicting non-small cell lung cancer EGFR-TKI drug targeted therapy drug resistance. The invention finds that HERV-K (HML-2) can be used as a molecular marker for targeted treatment of drug resistance of non-small cell lung cancer EGFR-TKI for the first time. After an NSCLC patient is treated by EGFR-TKI, the expression difference of HERV-K in serum is highly related to the residual tiny focus MRD. The MRD can generate a large amount of HERV-K, the HERV-K is secreted and released into blood, and MRD tissues existing in a body after targeted therapy are indicated by detecting the content of HERV-K in serum of a patient. Expression of the HERV-K is in significant positive correlation with recurrence of the NSCLC, and the HERV-K can be used for distinguishing the response degree of the NSCLC to targeted therapy. The molecular marker has good accuracy, sensitivity and specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of human endogenous retrovirus in predicting drug resistance to EGFR-TKI targeted therapy of non-small cell lung cancer. Background Art

[0002] Lung cancer is diagnosed through a physical examination, imaging tests (such as chest X-rays, computed tomography (CT), and magnetic resonance imaging (MRI), bronchoscopy to examine the lungs, tissue samples (biopsy) for histopathological examination and identification of the specific subtype (NSCLC or SCLC), and molecular testing to identify specific genetic mutations or biomarkers to guide optimal treatment. Treatment for lung cancer, on the other hand, depends on the type of cancer, how far it has spread, and the patient's medical history. Treatment options include surgery, radiotherapy (radiation), chemotherapy, targeted therapy, and immunotherapy. Surgery is often used in the early stages of lung cancer if the tumor has not spread to other parts of the body. Chemotherapy and radiotherapy can help shrink the tumor. Systemic therapies, such as chemotherapy, targeted therapy, and immunotherapy, play a vital role in the treatment of metastatic lung cancer. Chemotherapy is generally the first-line treatment for most patients worldwide and involves the use of drugs that circulate throughout the body to kill cancer cells. Combination chemotherapy regimens are often used, with the choice of drug depending on factors such as the histological type of the cancer and the patient's general health. Targeted therapy, which aims to block the signaling pathways that drive cancer cell growth, is an important option for patients whose tumors harbor specific genetic mutations or biomarkers. Immunotherapy, particularly immune checkpoint inhibitors, has revolutionized the treatment of metastatic lung cancer. These drugs help stimulate the immune system to recognize and attack cancer cells. Local treatments, such as radiation therapy and surgery, can be used to control specific metastatic sites or alleviate symptoms caused by tumor growth.

[0003] Abnormal activation of epidermal growth factor receptors (EGFR) can promote cell proliferation and metastasis by activating signaling pathways such as MAPK, PI3K-AKT, and STAT3 / 5. EGFR is a common oncogenic driver gene in NSCLC, particularly in nonsmoking Asian women with lung adenocarcinoma, with a mutation rate as high as 67%. Therefore, targeted therapy with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has become the standard treatment for advanced NSCLC harboring EGFR mutations. However, clinical findings indicate that inevitable primary or secondary drug resistance ultimately leads to disease progression. Regardless of initial treatment efficacy, nearly all patients inevitably develop acquired resistance. In particular, the mechanisms of resistance to third-generation EGFR-TKIs are more complex, and there is no standard treatment option for those who develop resistance, posing a significant challenge to achieving long-term benefits from first-line therapy. Therefore, identifying biomarkers for early prediction of responders and exploring optimized treatment options to prolong or reverse secondary drug resistance have become research priorities both domestically and internationally.

[0004] Numerous clinical studies have shown that minimal residual disease (MRD) in patients with non-small cell lung cancer (NSCLC) remains undetectable with current clinical imaging diagnostic methods after neoadjuvant therapy and post-operative systemic adjuvant therapy. MRD, which shares or resembles the original tumor cells' phenotype and genetic characteristics, has the potential to cause tumor recurrence. MRD is a major factor in post-treatment relapse in NSCLC. MRD differs significantly from circulating tumor cells in their drug resistance characteristics. Circulating tumor cells lack reversible resistance to drug therapy and remain dormant in the patient's body for extended periods. Circulating tumor cells are not the key driver of relapse, whereas the large number of reversibly resistant MRD cells is a significant contributor to relapse. Under continued treatment pressure, MRD can evade cytotoxicity from drugs or the patient's own immune system. Following treatment cessation or driven by other oncogenic factors, MRD-retained tumor cells can repopulate and migrate, ultimately leading to relapse. Therefore, detecting residual MRD after targeted therapy and identifying molecular markers of MRD can assess patient response to treatment and are crucial for optimizing treatment strategies.

[0005] Human endogenous retroviruses (HERVs) are sequences present in large numbers in the human genome, remnants of ancient retroviral infections. These sequences are fixed and accumulate mutations or deletions over time, accounting for approximately 8% to 10% of the human genome. HERVs have influenced human evolution and physiology by contributing unique coding and non-coding sequences to the genome. In healthy individuals, HERVs are involved in immune responses, syncytiotrophoblast formation, and cell fate determination. Accumulating evidence suggests that HERVs are crucial to human health and an understudied aspect of cancer.

[0006] Human endogenous retrovirus K (HERVK-K; HML-2) is the most recently integrated and best-conserved human endogenous retrovirus family. The term "K" derives from its use of lysine tRNA to promote reverse transcription. HML-2 (Human MMTV-Like-2) is the most active, youngest, and most extensively studied subgroup of HERV-K. Other HERV-K subgroups (HML-1, HML-3–10) were integrated into the human genome much earlier (tens of millions of years ago), and most were inactivated before the divergence of humans from Old World monkeys. Their sequences are highly degenerate, with most lacking coding capacity (remaining only LTRs or partial gene fragments). Very few functional proteins are expressed, and they exist primarily as "molecular fossils." Therefore, HERV-K is often used to refer to the most active HML-2 subgroup. The HERV-K genome primarily consists of four open reading frames (ORFs): gag, pro, pol, and env, flanked by long terminal repeats (LTRs). The gag gene encodes the structural components of the matrix (MA), capsid (CA), and nucleocapsid (NC); the pol gene encodes integrase, reverse transcriptase, and RNAse; the pro gene encodes a protease; and the env gene encodes the env glycoprotein, which is composed of a surface subunit (SU) and a transmembrane (TM) subunit. Existing studies have not comprehensively evaluated the potential application of HERV-K as a novel biomarker for the diagnosis of tumor recurrence. Therefore, the discovery and application of HERV-K as a biomarker for tumor recurrence and progression, as well as its practical application as a biomarker for EGFR-TKI resistance in patients with non-small cell lung cancer, are currently lacking. Summary of the Invention

[0007] Existing clinical technical methods for detecting MRD have the following shortcomings:

[0008] 1. Imaging (including positron emission tomography): It cannot promptly and accurately reflect the presence of MRD, cannot detect potential lesions, cannot reflect the effect of targeted therapy on patients at an early stage, and has a certain lag.

[0009] 2. Tissue biopsy: Some patients have low tissue quantity or quality, which affects the assessment of their condition. Complications may also occur, and this method cannot distinguish low-risk patients with long-term tumor-free survival.

[0010] 3. Liquid biopsy: This method uses second-generation sequencing or deep sequencing of tumor-specific markers to further analyze the progression of a patient's tumor by detecting cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomes in a patient's blood, cerebrospinal fluid, or urine. This method is technically expensive, and the consistency between plasma ctDNA test results and actual lesion tissue has not yet been fully established. cfDNA from different sources can also introduce confounding factors, making test results complex and uncertain.

[0011] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides the use of human endogenous retrovirus in predicting drug resistance to EGFR-TKI targeted therapy of non-small cell lung cancer.

[0013] In some embodiments of the present invention, the human endogenous retrovirus comprises human endogenous retrovirus HERV-K.

[0014] In some embodiments of the present invention, the human endogenous retrovirus HERV-K is of the HML-2 subgroup, the NCBI Gene ID of HERV-K (HML-2) is: 64006; the GenBank accession number is: AF074086.2, also known as ERVK-6 (ERVK-6 endogenous retrovirus group K member 6, envelope [Homo sapiens (human)]).

[0015] In some embodiments of the present invention, the EGFR-TKI drug includes gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, and / or vometinib.

[0016] The second aspect of the present invention provides the use of a reagent for detecting human endogenous retroviruses in the preparation of a product for predicting resistance to EGFR-TKI targeted therapy in non-small cell lung cancer.

[0017] In some embodiments of the present invention, the reagent includes a reagent for detecting human endogenous retrovirus at the protein level or gene level.

[0018] In some embodiments of the present invention, the reagent for detecting human endogenous retroviruses at the protein level is selected from one or more detection methods of the following groups: chemiluminescence, immunofluorescence, protein chip, protein spectrum, immunohistochemistry, plaque tracing based on labeling technology, protein blotting, and enzyme-linked immunosorbent assay.

[0019] In some embodiments of the present invention, the reagent for detecting human endogenous retroviruses at the gene level is selected from the group consisting of reagents of one or more detection methods: high-throughput RNA sequencing, RNA-in situ hybridization, digital PCR, and fluorescent quantitative PCR.

[0020] In some embodiments of the present invention, the product includes a detection kit, a detection chip, or a detection test strip.

[0021] In some embodiments of the present invention, the test samples of the product include body fluids and tissue samples.

[0022] In some embodiments of the present invention, the body fluid includes blood, lymph, and cerebrospinal fluid.

[0023] In some embodiments of the present invention, the tissue sample includes lung tissue or tumor tissue.

[0024] The third aspect of the present invention provides a method for constructing a model for predicting drug resistance to EGFR-TKI targeted therapy of non-small cell lung cancer, comprising the following steps:

[0025] Obtaining the human endogenous retrovirus test results of the patient to be tested and constructing a model;

[0026] In some embodiments of the present invention, the model construction algorithm includes at least one of logistic regression, linear discriminant analysis, support vector machine, random forest, and recursive partitioning tree.

[0027] A fourth aspect of the present invention provides a system for predicting resistance to EGFR-TKI targeted therapy of non-small cell lung cancer, the system comprising a computing device for judging resistance to EGFR-TKI targeted therapy of non-small cell lung cancer based on the detection results of human endogenous retroviruses.

[0028] In some embodiments of the present invention, the system further comprises one or more of 1) to 3):

[0029] 1) a test result collection device, which may also be referred to as a test result input device, specifically, one or more of a mouse, a keyboard, a touch screen display, one or more buttons, one or more switches, one or more triggers, etc.;

[0030] 2) a diagnostic result output device, which may also be referred to as a diagnostic result display device, and may specifically be one or more of a liquid crystal display (LCD), a light emitting diode (LED) display, a plasma display, a projection display, a touch screen display, and the like;

[0031] 3) A diagnostic result sending device, which can send the result of distinguishing whether the subject is a high-risk or low-risk group to an information communication terminal device that can be viewed by the patient or medical staff.

[0032] The beneficial effects of the present invention are:

[0033] The present invention discovered for the first time that HERV-K (HML-2) can be used as a molecular marker for resistance to EGFR-TKI targeted therapy in non-small cell lung cancer. After EGFR-TKI treatment, the difference in HERV-K expression in the serum of patients with non-small cell lung cancer is highly correlated with the residual microlesions MRD. MRD can produce a large amount of HERV-K, which is secreted and released into the blood. By detecting the level of HERV-K in the patient's serum, the presence of MRD tissue in the patient's body after targeted therapy can be indicated. HERV-K expression is significantly positively correlated with the recurrence of NSCLC and can be used to distinguish the degree of response of NSCLC to targeted therapy points. This molecular marker has good accuracy, sensitivity and specificity in clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0035] Figure 1 Results were generated for in vivo models of NSCLC and minimal residual disease after targeted NSCLC therapy.

[0036] Figure 2 The figures show the statistical results of HERV-K positive cells in NSCLC and minimal residual lesion tissues after NSCLC targeted therapy in mouse models.

[0037] Figure 3 To screen the concentration results of HERV-K in paired serum of NSCLC patients before and after targeted therapy.

[0038] Figure 4 To screen the effect of HERV-K levels on survival prognosis in patients with NSCLC after targeted therapy.

[0039] Figure 5 To validate the concentration of HERV-K in the serum of patients with NSCLC who are sensitive and resistant to targeted therapy.

[0040] Figure 6ROC curve results for validating HERV-K as a marker of resistance to targeted therapy. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0042] Example 1 Detection of HERV-K expression after osimertinib treatment

[0043] In this example, an in vivo mouse model was constructed to preliminarily determine whether HERV-K has the potential to serve as a drug resistance marker.

[0044] 1. Construction of mouse model

[0045] Ten female BALB / c nude mice were purchased from Beijing Weitonglihua Co., Ltd. All mice were housed under specific pathogen-free conditions and used according to protocols approved by the Institutional Animal Welfare Care and Use Committee of Tsinghua University.

[0046] PC-9 non-small cell lung cancer cells were resuspended in sterile PBS at a density of 1 × 10 7 The cells were then injected subcutaneously into the left lower abdomen of the mice in a volume of 0.1 mL.

[0047] The EGFR-TKI targeted drug osimertinib was purchased from Selleck (Osimertinib, S7297). The powder was dissolved in DMSO, and the stock solution (20 mg / mL) was aliquoted. The dissolved stock solution was diluted to 1 mg / mL with PBS buffer before subcutaneous injection.

[0048] Ten mice were randomly divided into two groups, with 5 mice in each group, namely PC-9+placebo group and PC-9+osimertinib group.

[0049] Starting from the first day after subcutaneous injection of cells, the tumor condition was monitored every 3 days. On the 7th day, when the tumor volume reached about 100 mm 3Each mouse in the PC-9 + placebo group received 0.1 mL of PBS solution orally daily for 11 consecutive days; each mouse in the PC-9 + osimertinib group received 0.1 mL of osimertinib working solution orally daily for 11 consecutive days. Tumor volume was measured daily until the end of the experiment. After 11 days of continuous gavage treatment, the mice were sacrificed, and responding tumor tissue and minimal residual lesions after osimertinib treatment were removed for tissue fixation and HERV-K immunohistochemical staining. Immunohistochemical results were statistically analyzed by staining intensity.

[0050] 2. Experimental results

[0051] The experimental results are as follows Figure 1 、 2 After osimertinib treatment, the tumor volume of mice was significantly reduced, and the proportion of HERV-K positive cells increased significantly, with approximately 70% of the cells being HERV-K positive.

[0052] Example 2 Detection of HERV-K expression before and after targeted therapy in NSCLC patients

[0053] Based on the World Health Organization's diagnostic criteria for NSCLC, blood specimens were collected from 20 patients with NSCLC treated at Guangdong Provincial People's Hospital, with approval from the Ethics Committee of Guangdong Provincial People's Hospital and signed informed consent from the patients or their families. These specimens served as a screening set.

[0054] The patient selection criteria were:

[0055] 1. Patients with untreated advanced or unresectable non-small cell lung cancer confirmed by pathology and at least one measurable lesion on CT or MRI according to RECIST version 1.1 (measurable lesions are defined as tumor lesions with the longest diameter >10 mm and metastatic lymph node short diameter >15 mm with a scan thickness not exceeding 5.0 mm).

[0056] 2. All patients have complete follow-up data after surgery.

[0057] 3. EGFR targeted drugs are the first-line treatment.

[0058] 4. The patient has no history of active immune or autoimmune diseases, or a known history of allogeneic organ transplantation or allogeneic hematopoietic stem cell transplantation, and has no disease progression or other malignant tumors requiring active treatment.

[0059] The clinical information of the patients is shown in Table 1.

[0060] Table 1

[0061]

[0062] 5 mL of blood was collected from each patient before and after targeted therapy, and approximately 1 mL of serum was obtained by centrifugation. The serum HERV-K level was measured using a HERV-K ELISA kit. The HERV-K ELISA kit was purchased from CUSABIO (Cat. No. CSB-EL007812HU). The kit has a detection range of 0.156 ng / mL to 10 ng / mL, a sensitivity of 0.039 ng / mL, an intra-assay precision of <8%, and an inter-assay precision of <10%. The target of this kit is Uniprot No. Q69384.

[0063] After drawing the best four-parameter fitting curve using the standard, the OD value detected experimentally was substituted into the equation to calculate the concentration of HERV-K in the sample.

[0064] The experimental results are as follows Figure 3 As shown, the HERV-K content in patients increased significantly after targeted therapy.

[0065] Furthermore, the serum HERV-K concentration of patients after targeted therapy was classified into high and low concentrations, where HERV-K ≥ 20 mg / mL was high concentration (patient sample n = 14) and HERV-K < 20 mg / mL was low concentration (patient sample n = 6). Survival curves were drawn based on the patients' progression-free survival and statistically analyzed using the Gehan-Breslow-Wilcoxon test.

[0066] The results are as follows Figure 4 As shown in the results, the analysis found that after EGFR-TKI targeted therapy, the concentration of HERV-K in the patient's serum was significantly negatively correlated with the patient's prognosis and survival, indicating that patients with high concentrations of HERV-K points after targeted therapy have more tiny residual lesions in their bodies, resulting in faster disease progression in patients.

[0067] Example 3 Validation of HERV-K as a marker of tolerance to targeted therapy

[0068] Based on the World Health Organization's diagnostic criteria for NSCLC, and with approval from the Ethics Committee of Guangdong Provincial People's Hospital and written informed consent from the patients or their families, 5 mL of blood was collected from 30 patients with targeted therapy sensitivity and 30 patients with targeted therapy resistance. Approximately 1 mL of serum was obtained by centrifugation and assayed for HERV-K levels using a HERV-K ELISA kit. This sample served as a validation set, independent of the screening set in Example 2.

[0069] The clinical information of the patients is shown in Table 2.

[0070] Table 2

[0071]

[0072] The HERV-K ELISA kit was purchased from CUSABIO (Cat. No. CSB-EL007812HU). The detection range of the kit was 0.156 ng / mL-10 ng / mL, the sensitivity was 0.039 ng / mL, the intra-assay precision was CV% <8%, and the inter-assay precision was CV% <10%.

[0073] After drawing the best four-parameter fitting curve using the standard, the OD value detected experimentally was substituted into the equation to calculate the concentration of HERV-K in the sample.

[0074] The experimental results are as follows Figure 5 As shown, the level of HERV-K in the serum of patients resistant to targeted therapy was significantly increased.

[0075] Furthermore, 30 patients with lung cancer resistant to targeted therapy were used as the positive group, and 30 patients with lung cancer sensitive to targeted therapy were used as the negative group. The receiver operating characteristic curve (ROC) analysis was performed using SPSS 18.0 software. Figure 6 As shown, the results showed that the diagnostic efficacy of HERV-K molecules in serum for resistance to EGFR-TKI targeted therapy of non-small cell lung cancer can reach 0.9444 (95% CI 0.8744~0.989).

[0076] In summary, HERV-K has good specificity and sensitivity in diagnosing resistance to EGFR-TKI targeted therapy in non-small cell lung cancer and can be used as a predictive marker.

[0077] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of human endogenous retroviruses in predicting resistance to EGFR-TKI targeted therapy in non-small cell lung cancer; The human endogenous retrovirus includes the human endogenous retrovirus HERV-K HML-2 subgroup.

2. The use according to claim 1, characterized in that: The EGFR-TKI drugs include gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, and / or vometinib.

3. Application of reagents for detecting human endogenous retroviruses in the preparation of products for predicting resistance to EGFR-TKI targeted therapy in non-small cell lung cancer.

4. The use according to claim 3, characterized in that: The reagents include reagents for detecting human endogenous retroviruses at the protein level or gene level.

5. The use according to claim 4, characterized in that: The reagent for detecting human endogenous retrovirus at the protein level is selected from one or more detection methods of the following group: chemiluminescence, immunofluorescence, protein chip, protein spectrum, immunohistochemistry, plaque tracing based on labeling technology, Western blotting, and enzyme-linked immunosorbent assay; The reagent for detecting human endogenous retroviruses at the gene level is selected from the group consisting of reagents of one or more detection methods: high-throughput RNA sequencing, RNA-in situ hybridization, digital PCR, and fluorescent quantitative PCR.

6. The use according to claim 3, characterized in that: The products include detection kits, detection chips or detection test strips.

7. The use according to claim 3, characterized in that: The test samples of the product include body fluids and tissue samples.

8. A method for constructing a model for predicting resistance to EGFR-TKI targeted therapy in non-small cell lung cancer, comprising the following steps: Obtaining the human endogenous retrovirus test results of the patient to be tested and constructing a model; Preferably, the model construction algorithm includes at least one of logistic regression, linear discriminant analysis, support vector machine, random forest, and recursive partitioning tree.

9. A system for predicting resistance to EGFR-TKI targeted therapy in non-small cell lung cancer, characterized by: The system includes a computing device for judging drug resistance to EGFR-TKI targeted therapy of non-small cell lung cancer according to the detection results of human endogenous retrovirus.

10. The system according to claim 9, characterized in that: The system further includes one or more of 1) to 3): 1) Test result collection device; 2) diagnostic result output device; 3) Diagnosis result sending device.

Images