Protein markers, detection reagents or detection tools for early identification of patients who respond to induction chemotherapy for locally advanced nasopharyngeal carcinoma

Abstract

The present disclosure provides a protein marker, detection reagent or detection tool for early identification of local regional advanced nasopharyngeal carcinoma induction chemotherapy responders. Since PON1 protein, IGFBP3 protein, v‑kappa‑3 protein and DDX55 protein are significantly differentially expressed in the plasma of healthy individuals and untreated local regional advanced nasopharyngeal carcinoma patients, the plasma of induction chemotherapy responders and ineffective patients before treatment, the plasma of induction chemotherapy responders before and after treatment, and the plasma samples of induction chemotherapy responders and ineffective patients after treatment, PON1 protein, IGFBP3 protein, v‑kappa‑3 protein and DDX55 protein can be used to identify potential responders of induction chemotherapy for local regional advanced nasopharyngeal carcinoma before treatment, showing excellent test benefits. The method is simple and easy to implement, easy for patients to accept, has unified execution standards and follows objective data, and can further optimize the clinical application of induction chemotherapy for local regional advanced nasopharyngeal carcinoma, especially innovative treatment in key areas such as oncology.

Description

Technical Field

[0001] The present disclosure relates to the field of molecular biotechnology, and in particular to a protein marker, a detection reagent or a detection tool for early identification of patients who respond to induction chemotherapy for locally advanced nasopharyngeal carcinoma. Background Art

[0002] Studies have found that pre-identifying responders to induction chemotherapy before treatment is of great significance for the personalized treatment of locally advanced nasopharyngeal carcinoma. Currently, there is no effective blood test-based method to pre-identify responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma. Regarding commonly used clinical indicators, such as Epstein-Barr virus (EBV), it can only be used for the diagnosis and prognosis of nasopharyngeal carcinoma, while the value of the response evaluation criteria in solid tumors (RECIST) is limited to the determination of the efficacy after treatment.

[0003] Therefore, there is an urgent need to establish a molecular marker-driven laboratory standard to assist clinicians in formulating precise induction chemotherapy regimens. Summary of the invention

[0004] In view of this, the purpose of the present disclosure is to provide a protein marker, a detection reagent or a detection tool for early identification of responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma.

[0005] Based on the above-mentioned purpose, the first aspect of the present disclosure provides the use of protein markers in the early diagnosis of locally advanced nasopharyngeal carcinoma; or, the use of protein markers in the early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma; or, the use of protein markers in confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma; or, the use of protein markers in evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma; wherein the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0006] Based on the same purpose, the second aspect of the present disclosure provides the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for early diagnosis of locally advanced nasopharyngeal carcinoma; or, the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma; or, the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma; or, the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma; wherein the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0007] Based on the same purpose, the third aspect of the present disclosure provides a reagent for early diagnosis of locally advanced nasopharyngeal carcinoma, a reagent for early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, a reagent for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma, or a reagent for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma, characterized in that it includes an antibody that specifically binds to a protein marker; the protein marker includes at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0008] Based on the same purpose, the fourth aspect of the present disclosure provides a kit for early diagnosis of locally advanced nasopharyngeal carcinoma, a kit for early identification of potential responders of induction chemotherapy for locally advanced nasopharyngeal carcinoma, a kit for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma, or a kit for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma, characterized in that it includes antibodies that specifically bind to protein markers; the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0009] From the above, it can be seen that the protein marker, detection reagent or detection tool provided by the present disclosure for early identification of local regional advanced nasopharyngeal carcinoma induction chemotherapy responders, since PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein are significantly differentially expressed in the plasma of healthy individuals and untreated local regional advanced nasopharyngeal carcinoma patients, the plasma of induction chemotherapy responders and ineffective patients before treatment, the plasma of induction chemotherapy responders before and after treatment, and the plasma samples of induction chemotherapy responders and ineffective patients after treatment, so PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein can be used to identify potential responders of induction chemotherapy for local regional advanced nasopharyngeal carcinoma before treatment, showing excellent test benefits. The method is simple and easy to implement, easy for patients to accept, has a unified implementation standard and follows objective data, which can further optimize the clinical application of induction chemotherapy for local regional advanced nasopharyngeal carcinoma, especially innovative treatment in key fields such as oncology. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the present disclosure or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0011] Figure 1A graph showing the total expression of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein in the plasma of healthy individuals (HC), in the plasma of induction chemotherapy responders before treatment (pre-EFF), in the plasma of induction chemotherapy non-responders before treatment (pre-NEFF), in the plasma of induction chemotherapy responders after treatment (post-EFF) and in the plasma of induction chemotherapy non-responders after treatment (post-NEFF) provided in the embodiments of the present disclosure;

[0012] Figure 2 ROC curve diagram for distinguishing healthy individuals from patients with localized advanced nasopharyngeal carcinoma using PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein provided in the embodiments of the present disclosure;

[0013] Figure 3 The ROC curve diagram for the diagnosis of patients with local advanced nasopharyngeal carcinoma using the combined modeling of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein provided in the embodiments of the present disclosure;

[0014] Figure 4 ROC curve diagram of the PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein provided in the embodiments of the present disclosure for distinguishing potential responders from non-responders in plasma samples of patients with localized advanced nasopharyngeal carcinoma before induction chemotherapy;

[0015] Figure 5 ROC curve diagram for early identification of potential responders of induction chemotherapy by modeling the combination of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein provided in the embodiments of the present disclosure;

[0016] Figure 6 ROC curves of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein provided in the embodiments of the present disclosure for distinguishing the responders of induction chemotherapy before and after receiving induction chemotherapy;

[0017] Figure 7 ROC curve diagram for modeling the combination of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein provided in the embodiments of the present disclosure to distinguish the responders of induction chemotherapy before and after receiving induction chemotherapy;

[0018] Figure 8 ROC curve diagram of the PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein provided in the embodiments of the present disclosure for distinguishing between effective and ineffective patients in plasma samples of patients with local advanced nasopharyngeal carcinoma after induction chemotherapy;

[0019] Fig. 9 The ROC curve diagram of the combined modeling of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein provided in the embodiments of the present disclosure to distinguish between effective and ineffective patients in plasma samples of patients with locally advanced nasopharyngeal carcinoma after induction chemotherapy. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0021] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present disclosure should have the usual meanings understood by people with ordinary skills in the field to which the present disclosure belongs. The words "first", "second" and similar words used in the embodiments of the present disclosure do not indicate any order, quantity or importance, but are only used to distinguish different components.

[0022] Nasopharyngeal carcinoma is a highly invasive and highly metastatic head and neck malignant disease. In 2020, the World Cancer Report published by IARC showed that there were approximately 133,354 new cases of NPC and 80,008 NPC-related deaths worldwide. my country is a country with a high incidence of NPC in the world. The corresponding numbers of the above cases in 2020 were 62,444 and 34,810, accounting for 46.83% and 42.72% of the global total, respectively. It is reported that more than 70% of patients are confirmed to be locally advanced at the time of initial diagnosis. Among them, approximately 30%-40% of locally advanced NPC have treatment failures, and tumor recurrence or distant metastasis reduces the 5-year survival rate of these patients to 20%. Therefore, improving the clinical treatment of locally advanced NPC is crucial to controlling the progression of the disease and reducing mortality.

[0023] In recent years, clinical trials have confirmed that adding induction chemotherapy before radiotherapy for locally advanced nasopharyngeal carcinoma can improve patients' survival outcomes. As induction chemotherapy has been included in the NCCN guidelines for the treatment of locally advanced nasopharyngeal carcinoma, clinical work has found significant differences in the short-term efficacy of induction chemotherapy between individuals. It has been reported that some patients who received induction chemotherapy still face tumor recurrence or distant metastasis, indicating that they have undergone unnecessary treatment. In addition, the toxicity associated with chemotherapy often delays or interrupts subsequent treatment, exposing patients to the risk of disease progression and tumor resistance. In view of this, pre-identification of responders to induction chemotherapy before treatment will be of great significance for the personalized treatment of locally advanced nasopharyngeal carcinoma.

[0024] Currently, there is no effective blood test method to pre-identify patients with locally advanced nasopharyngeal carcinoma who respond to induction chemotherapy. Commonly used clinical indicators, such as Epstein-Barr virus (EBV), can only be used for the diagnosis and prognosis of nasopharyngeal carcinoma, while the value of the response evaluation criteria in solid tumors (RECIST) is limited to the determination of the efficacy after treatment.

[0025] Proteins are involved in a series of important human physiological functions, such as immunity, material exchange, metabolism, and signal pathway regulation. During the development of cancer, protein synthesis and degradation in tumor tissues are enhanced, further affecting the expression and composition of circulating proteins. This abnormal change will produce dynamic changes during the treatment process accompanied by apoptosis or proliferation of tumor cells. Therefore, the application of proteomics technology to develop peripheral circulation-based molecular markers will help improve the clinical treatment of locally advanced nasopharyngeal carcinoma and discover new therapeutic targets.

[0026] In order to solve the above problems, the present disclosure provides the use of protein markers in the early diagnosis of localized advanced nasopharyngeal carcinoma; the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, DDX55 protein

[0027] The present disclosure also provides the use of protein markers in early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma; the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0028] The present disclosure also provides the use of protein markers in confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma; the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0029] The present disclosure also provides the use of protein markers in evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma; the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0030] Among them, PON1 protein refers to paraoxonolipase 1, IGFBP3 protein refers to insulin-like growth factor binding protein 3, v-kappa-3 protein refers to rheumatoid factor D5 light chain protein, and DDX55 protein refers to epididymal secretory sperm binding protein.

[0031] In the present disclosure, in the process of using PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein to identify those who respond to induction chemotherapy for locally advanced nasopharyngeal carcinoma, the first threshold, second threshold, third threshold and fourth threshold of expression abundance can be set in advance, and then identification can be performed based on the set thresholds.

[0032] Specifically, in the plasma samples of healthy individuals and all untreated patients with locally advanced nasopharyngeal carcinoma, when the expression abundance of PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein is lower than the first threshold, the individuals can be identified as patients with locally advanced nasopharyngeal carcinoma, otherwise they are identified as healthy individuals; based on this, a kit or reagent for early diagnosis of locally advanced nasopharyngeal carcinoma can be constructed, which includes antibodies that specifically bind to protein markers.

[0033] In the plasma samples of patients identified as having locally advanced nasopharyngeal carcinoma before receiving induction chemotherapy, when the expression abundance of PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein is higher than the second threshold, they can be identified as potential responders to induction chemotherapy; otherwise, they are identified as non-responsive to induction chemotherapy; based on this, a kit or reagent for early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma can be constructed, which includes antibodies that specifically bind to protein markers.

[0034] When the expression abundance of PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein in plasma samples before and after induction chemotherapy of a person identified as a potential responder to induction chemotherapy is lower than the third threshold, the person can be confirmed as a responder after induction chemotherapy; otherwise, the person can be identified as a potential responder who did not receive induction chemotherapy; based on this, a kit or reagent for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma can be constructed, which includes antibodies that specifically bind to protein markers.

[0035] In the plasma samples of all patients with locally advanced nasopharyngeal carcinoma after receiving induction chemotherapy, when the expression abundance of PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein is higher than the fourth threshold, they can be identified as those who have responded to induction chemotherapy, otherwise they are identified as those who have not responded to induction chemotherapy; based on this, a kit or reagent for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma can be constructed, which includes antibodies that specifically bind to protein markers.

[0036] The present disclosure also provides the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for early diagnosis of locally advanced nasopharyngeal carcinoma; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0037] In some embodiments, the antibody may include at least one of an anti-PON1 protein antibody, an anti-IGFBP3 protein antibody, an anti-v-kappa-3 protein antibody, and an anti-DDX55 protein antibody.

[0038] Furthermore, the anti-PON1 protein antibody, the anti-IGFBP3 protein antibody, the anti-v-kappa-3 protein antibody, and the anti-DDX55 protein antibody can be monoclonal antibodies or polyclonal antibodies, respectively.

[0039] In some embodiments, the antibody may be linked to a detectable marker, such as an enzyme, a prosthetic group, a fluorescent substance, a luminescent substance, a bioluminescent substance, a colored substance or a radioactive isotope, etc., without limitation.

[0040] In some embodiments, the diagnostic tool may include a chip, a test strip, or a kit.

[0041] In some embodiments, the kit may be an ELISA kit, an immunofluorescence kit or an immunocolloidal gold kit, without limitation.

[0042] The present disclosure also provides the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for early identification of potential responders of induction chemotherapy for localized advanced nasopharyngeal carcinoma; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein. The antibodies may include at least one of anti-PON1 protein antibodies, anti-IGFBP3 protein antibodies, anti-v-kappa-3 protein antibodies, and anti-DDX55 protein antibodies; the diagnostic tools may include chips, test strips, or test kits.

[0043] The present disclosure also provides the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for confirming the effectiveness of induction chemotherapy for localized advanced nasopharyngeal carcinoma; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein. The antibodies may include at least one of anti-PON1 protein antibodies, anti-IGFBP3 protein antibodies, anti-v-kappa-3 protein antibodies, and anti-DDX55 protein antibodies; the diagnostic tools may include chips, test strips, or kits.

[0044] The present disclosure also provides the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for evaluating the efficacy of induction chemotherapy for localized advanced nasopharyngeal carcinoma; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein. The antibodies may include at least one of anti-PON1 protein antibodies, anti-IGFBP3 protein antibodies, anti-v-kappa-3 protein antibodies, and anti-DDX55 protein antibodies; the diagnostic tools may include chips, test strips, or kits.

[0045] The present disclosure also provides a kit for early diagnosis of locally advanced nasopharyngeal carcinoma, comprising antibodies that specifically bind to protein markers; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0046] In some embodiments, the kit may further contain a buffer and a color developing agent.

[0047] The present disclosure also provides reagents for early diagnosis of locally advanced nasopharyngeal carcinoma, including antibodies that specifically bind to protein markers; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0048] In some embodiments, the reagent may be a specific primer or probe, or may be a pharmaceutical composition comprising the primer or probe, wherein the probe is also connected with a detectable marker, such as a fluorescent group; preferably, the fluorescent group may be selected from at least one of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688, without specific limitation.

[0049] The present disclosure also provides a kit for early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, comprising antibodies that specifically bind to protein markers; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0050] The present disclosure also provides reagents for early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, including antibodies that specifically bind to protein markers; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0051] The present disclosure also provides a kit for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma, comprising antibodies that specifically bind to protein markers; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0052] The present disclosure also provides reagents for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma, including antibodies that specifically bind to protein markers; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0053] The present disclosure also provides a kit for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma, comprising antibodies that specifically bind to protein markers; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0054] The present disclosure also provides reagents for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma, including antibodies that specifically bind to protein markers; the protein markers may include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0055] It is understandable that since PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein are significantly differentially expressed in the plasma of healthy individuals and untreated patients with locally advanced nasopharyngeal carcinoma, the plasma before treatment of responders and non-responders of induction chemotherapy, the plasma before and after treatment of responders of induction chemotherapy, and the plasma samples after treatment of responders and non-responders of induction chemotherapy, PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein can be used to identify potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma before treatment, showing excellent detection efficiency.

[0056] The protein markers, detection reagents or detection tools provided by the present disclosure for identifying patients who respond to induction chemotherapy for locally advanced nasopharyngeal carcinoma are described in detail below in conjunction with specific embodiments.

[0057] It should be noted that, in this embodiment, peripheral blood of all healthy individuals and peripheral blood of all patients with local-regional advanced nasopharyngeal carcinoma before receiving induction chemotherapy are first collected; then peripheral blood of all patients with local-regional advanced nasopharyngeal carcinoma after receiving induction chemotherapy is collected, and the effectiveness of induction chemotherapy of all patients with local-regional advanced nasopharyngeal carcinoma is detected by imaging means, and based on the imaging means detection results of all patients with local-regional advanced nasopharyngeal carcinoma, all patients with local-regional advanced nasopharyngeal carcinoma are divided into an induction chemotherapy effective group and an induction chemotherapy ineffective group, and further, the peripheral blood of all patients with local-regional advanced nasopharyngeal carcinoma after receiving induction chemotherapy is divided into blood samples after treatment of effective patients and blood samples after treatment of ineffective patients; and based on the grouping results, the peripheral blood of all patients with local-regional advanced nasopharyngeal carcinoma before receiving induction chemotherapy can be divided into blood samples before treatment of effective treatment and blood samples after treatment of ineffective treatment.

[0058] Based on the expression abundance of each plasma protein in all blood samples, four specific proteins for locally advanced nasopharyngeal carcinoma are determined; then, the accuracy of the four specific proteins in early diagnosis of locally advanced nasopharyngeal carcinoma patients can be verified based on the blood samples of all healthy individuals and the blood samples of all locally advanced nasopharyngeal carcinoma patients before receiving induction therapy.

[0059] Based on the blood samples of all patients with locoregionally advanced nasopharyngeal carcinoma before receiving induction therapy, the four specific proteins can be validated to accurately identify potential responders to induction chemotherapy at an early stage.

[0060] Based on the blood samples of potential responders of locally advanced nasopharyngeal carcinoma before and after receiving induction chemotherapy, the close correlation between the four specific proteins and the effective treatment of induction chemotherapy can be verified.

[0061] Based on the blood samples of all patients with locoregionally advanced nasopharyngeal carcinoma after receiving induction therapy, it can be verified whether the four specific proteins can distinguish between effective and ineffective patients after treatment.

[0062] This embodiment uses the patient's peripheral blood as a sample for molecular marker screening, which is non-invasive, safe, easy for patients to accept, has a unified implementation standard, and follows objective experimental data.

[0063] All cases included in this example were newly diagnosed patients with locally advanced nasopharyngeal carcinoma who had not received any anti-tumor treatment, and these patients received the same induction chemotherapy regimen and treatment cycle, ensuring the specificity of the induction chemotherapy efficacy of the molecular markers finally obtained.

[0064] The molecular markers identified by high-throughput proteomics technology in this example are helpful to further optimize the clinical application of induction chemotherapy for locally advanced nasopharyngeal carcinoma, and provide an experimental basis for discovering new chemotherapy targets and individualized treatment of nasopharyngeal carcinoma.

[0065] 1. Screening and identification of protein markers associated with the benefits of induction chemotherapy

[0066] (1) Sample collection

[0067] Peripheral blood samples were collected from 50 healthy individuals in the fasting state in the morning, and paired peripheral blood samples were collected from 64 patients with localized advanced nasopharyngeal carcinoma who did not receive any anti-tumor treatment and were diagnosed according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual before and after induction chemotherapy, for a total of 178 samples. All patients only received and performed the same induction chemotherapy regimen (docetaxel + cisplatin). The present invention complies with the ethical guidelines of the Declaration of Helsinki, and all participants have signed informed consent.

[0068] (2) Sample grouping

[0069] The efficacy of induction chemotherapy was determined according to the solid tumor efficacy evaluation criteria (version 1.1). Patients with complete remission and partial remission of the total volume of the primary tumor were defined as responders, and patients with stable and progressive disease were defined as ineffective patients. Based on this standard, 64 patients with local regional advanced nasopharyngeal carcinoma were divided into 34 responders and 30 ineffective patients. Therefore, all samples were divided into a healthy control group (HC), a local regional advanced nasopharyngeal carcinoma group (LA-NPC), a responder pre-treatment group (pre-EFF), an ineffective pre-treatment group (pre-NEFF), a responder post-treatment group (post-EFF), and an ineffective post-treatment group (post-NEFF).

[0070] (3) Plasma preparation

[0071] Peripheral blood was centrifuged at 1300 g for 10 min at 4 °C under anticoagulant conditions, and the supernatant was transferred to Eppendorf tubes and stored at -80 °C.

[0072] (4) Sample pretreatment

[0073] Proteins were separated from the plasma pool using a Human 14 / Mouse 3 multi-affinity removal system column, and the fractions were desalted and concentrated using a 5 kDa ultrafiltration tube. The samples were boiled and centrifuged after adding buffer, and then quantified using the BCA method.

[0074] According to the FASP procedure, the protein was ultrafiltered with UA buffer (8M urea, 150mM Tris-HCl pH8.0) to remove detergent and low molecular weight components, and then 100μl iodoacetamide was added and incubated for 30 minutes in the dark. The filter was washed 3 times with 100μl UA buffer and 2 times with 100μl 25mM NH4HCO3 buffer. The resulting protein suspension was digested overnight with 4μg trypsin in 40μl 25mM NH4HCO3 buffer. The peptides were collected as the filtrate, desalted by C18 cartridge, concentrated by vacuum centrifugation, and reconstituted with 40μl 0.1% (v / v) formic acid, and the peptide content was estimated by UV spectral density at 280nm.

[0075] The peptides were fractionated into 10 fractions using a high pH reverse phase peptide fractionation kit. Each fraction was concentrated by vacuum centrifugation and reconstituted in 15 μL (0.1% (v / v)) formic acid. The resulting peptides were desalted and reconstituted again using a C18 cartridge and 40 μL 0.1% (v / v) formic acid. Finally, iRT was added to correct the relative retention time difference between the iRT standard peptide and the sample peptide at a volume ratio of 1:3.

[0076] (5) Data Dependent Acquisition (DDA)

[0077] The analysis of all components of the generated DDA library was performed using a Q Exactive HF X mass spectrometer connected to an Easy nLC 1200 chromatography system. 1.5 μg of peptides were first loaded onto an EASYSprayTM C18 Trap column and then separated on an EASY SprayTM C18 LC analytical column with a 120 min gradient from buffer B (0.1% formic acid and 84% acetonitrile). The mass spectrometer scan range was 300-1800 m / z, and the MS1 scan resolution was 60000 200 m / z, automatic gain control target 3e6, maximum 25MS, and dynamic exclusion 30.0 seconds. A full MS-SIM scan was performed after 20 ddMS2 scans. The MS2 scan resolution was 15000, the automatic gain control target was 5e4, the maximum IT was 25ms, and the normalized collision energy was 30eV.

[0078] (6) Data-Independent Acquisition (DIA)

[0079] Each DIA cycle consisted of a full MS-SIM scan, 30 DIA scans covering the mass range of 350-1800 m / z, with the following parameters: SIM full scan resolution at 200 m / z was 120,000, AGC 3e6, 50 ms max. The DIA scan resolution was set to 15000. The automatic gain control target was 3e6, and the normalized collision energy was 30 eV. The flow rate was 250 nl / min and the run time was 120 min under a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid). QC samples were injected in DIA mode after every 6 injections throughout the MS experiment to monitor MS performance.

[0080] (7) Mass spectrometry data analysis

[0081] The DDA library data was searched against the FASTA sequence database using SpectronautTM 14.4.200727.47784 software. The database was the Uniprot_human database (http: / / www.uniprot.org.), and the iRT peptide sequence (LGGNEQVTRYILAGVENSKGTFIIDPGGVIRGTFIIDPAAVIRGAGSSEPVTGLDAKTPVISGGPYEYRVEATFGVDESNAKTPVITGAPYEYRDGLDAASYYAPVRADVTPADFSEWSKLFLQFGAQGSPFLK) was added. Parameter settings: the digestion enzyme was trypsin, the maximum missing enzyme was 2, the fixed modification was aminomethyl (C), and the dynamic modification was oxidation modification (M) and acetyl modification (protein n item). The protein identification confidence of all reported data was 99%, and the false discovery rate (FDR = N (positive library) * 2 / (N (positive library) + N (target)) was ≤ 1%. The raw files and DDA search results were imported into Spectronaut Pulsar X TM_12.0.20491.4 (Biognosys) to build a spectral library. The raw data of DIA were processed and analyzed using SpectronautTM 14.4.200727.47784 to search the spectral library constructed above. For the setting of software parameters, the retention time prediction type was set to dynamic iRT, interference correction on MS2 level calibration was enabled, and cross-run normalization was enabled. The cutoff value of Q was set to 0.01 to filter all results, which is equivalent to FDR < 1%.

[0082] (8) Bioinformatics analysis

[0083] ① Screening of differentially expressed proteins

[0084] Proteins that showed an increase or decrease in relative abundance in the inter-group comparison were defined as differentially expressed with a fold difference greater than or equal to 1.2 or less than or equal to 0.833 and a value < 0.05.

[0085] ②Protein function enrichment analysis

[0086] NCBI BLAST+ and InterProScan were used to search for homologous sequences of differentially expressed proteins, and GO and KEGG enrichment analysis was performed using the clusterProfiler package.

[0087] ③Analysis of the correlation between protein and clinical characteristics

[0088] Weighted protein co-expression networks were constructed using the weighted gene co-expression network analysis (WGCNA) package in R (version 1.69). A power of β = 5 was chosen as a soft threshold to ensure scale-free topology. Clinical characteristics such as patient age, sex, and tumor T stage, N stage, clinical stage, and induction chemotherapy outcome were used for correlation analysis.

[0089] ④Analysis of the correlation between protein and benefits of induction chemotherapy

[0090] The fuzzy C-means algorithm was used to perform soft clustering analysis using the Mfuzz software package (http: / / mfuzz.sysbiolab.eu / ) based on R language to reveal the changes in the plasma proteome of responders before and after induction chemotherapy.

[0091] ⑤Chart drawing and ROC analysis

[0092] The ggplot2 package in R language was used to draw graphs, and the pROC package was used for ROC curve analysis.

[0093] (9) Statistical analysis

[0094] All statistical tests were performed using the R version 4.1.1 software package. The Shapiro-Wilk normality test was used to confirm whether the data followed a normal distribution. For statistical comparisons, one-way analysis of variance or student t test was used for parametric data, chi-square test was used for constituent ratios, and mean ± standard deviation was used for continuous variables. P < 0.05 for two-tailed tests was considered significant.

[0095] (10) Technical principles

[0096] The principle of data-independent acquisition (DIA) technology is to first use data-dependent acquisition (DDA) mass spectrometry technology to establish a protein spectral library, and then apply DIA to collect mass spectrometry data to achieve protein qualitative and quantitative analysis. Compared with traditional DDA technology, such as the two mainstream methods of labeled quantification (iTRAQ / TMT, SILAC) and label-free quantification (label free), the DIA mass spectrometry scanning range is divided into several windows, and then all parent ions in each window are selected, fragmented and detected in turn at high speed and cycle. DIA can perform high-speed primary MS scanning on all peptide ions in the scanning interval, and then perform secondary MS / MS analysis. This technology does not require the specification of target peptides, and can obtain all fragmentation information of all parent ions in the sample without bias and omission, with high data utilization.

[0097] 2. Experimental results

[0098] By comparing the differences in plasma protein profiles between healthy controls and patients with locally advanced nasopharyngeal carcinoma, between responders and non-responders of induction chemotherapy before treatment, between responders before and after treatment, and between responders and non-responders after treatment, it was found that all four proteins were significantly differentially expressed and were associated with clinical characteristics and benefits of induction chemotherapy, respectively. They can serve as molecular markers for early identification of responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma.

[0099] In order to evaluate the efficacy of the four proteins as candidate molecular markers for early identification of responders to induction chemotherapy, the present disclosure uses ROC analysis for verification. The area under the curve (AUC) is an important parameter indicator for evaluating the accuracy of ROC analysis. In addition, AUC values ​​between 0.5-0.7, between 0.7-0.9, and above 0.9 represent general accuracy, good accuracy, and high accuracy, respectively.

[0100] (1) Using DIA high-throughput technology, we detected significant differences in the expression of PON1 protein in the plasma protein profiles between the healthy control group and the group with locally advanced NPC, between the effective group before treatment and the ineffective group before treatment, between the effective group before treatment and the effective group after treatment, and between the effective group after treatment and the ineffective group after treatment.

[0101] Depend on Figure 1It can be seen that compared with the PON1 protein content in the plasma of healthy people, the PON1 protein content in the plasma of patients with locally advanced nasopharyngeal carcinoma was significantly downregulated by 0.83 times, P value <0.05; in the plasma of effective patients before treatment, it was significantly downregulated by 0.91 times, P value <0.05; in the plasma of ineffective patients before treatment, it was significantly downregulated by 0.74 times, P value <0.05; in the plasma of effective patients after treatment, it was significantly downregulated by 0.53 times, P value <0.05; in the plasma of ineffective patients after treatment, it was significantly downregulated by 0.44 times, P value <0.05.

[0102] Depend on Figure 2 It can be seen that the AUC value of PON1 protein in distinguishing patients with locally advanced nasopharyngeal carcinoma in healthy individuals is 0.823, indicating that PON1 protein has good diagnostic accuracy and is a protein specifically expressed in locally advanced nasopharyngeal carcinoma. Figure 4 The results showed that the AUC value of PON1 protein in identifying potential responders to induction chemotherapy in samples of locally advanced nasopharyngeal carcinoma before receiving induction chemotherapy was 0.743, indicating that PON1 protein has good predictive performance and can be used as a molecular marker for early identification of potential responders to induction chemotherapy in locally advanced nasopharyngeal carcinoma. Figure 6 The results showed that the AUC value of PON1 protein in distinguishing potential responders who did not receive induction chemotherapy from responders who had received induction chemotherapy in plasma samples of patients with locally advanced nasopharyngeal carcinoma who responded to induction chemotherapy before and after induction chemotherapy was 0.955, indicating that PON1 has a high predictive performance, further confirming its reliability as a molecular marker for early identification of responders to induction chemotherapy. Figure 8 The results showed that the AUC value of PON1 protein in distinguishing between responders and non-responders of induction chemotherapy in plasma samples of patients with locally advanced nasopharyngeal carcinoma after induction chemotherapy was 0.711, indicating that it can effectively distinguish between responders and non-responders of induction chemotherapy after treatment, and can be used as a marker for evaluating the efficacy of induction chemotherapy in locally advanced nasopharyngeal carcinoma.

[0103] In the diagnosis of locally advanced nasopharyngeal carcinoma, when the critical value of PON1 protein expression abundance was set at 9995.60, the sensitivity was 0.812 and the specificity was 0.700. When diagnosing locally advanced nasopharyngeal carcinoma, in the plasma samples of responders and responders to induction chemotherapy before treatment, if the PON1 protein expression abundance was lower than 9995.60, they were identified as patients with locally advanced nasopharyngeal carcinoma, otherwise they were identified as healthy individuals, and the false positive rate was 22.4%.

[0104] In the process of early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, when the critical value of PON1 protein expression abundance was set at 7890.79, the sensitivity was 0.600 and the specificity was 0.853; when early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma was performed, if the expression abundance of PON1 protein in the plasma samples of responders before and after induction chemotherapy was higher than 7890.79, they were identified as potential responders to induction chemotherapy, otherwise they were identified as non-responders to induction chemotherapy, and the false positive rate was 21.7%.

[0105] In the process of confirming whether the potential responders identified early are indeed suitable for induction chemotherapy, when the critical value of PON1 protein expression abundance is set to 6744.48, the sensitivity is 0.912 and the specificity is 0.941. When confirming whether the potential responders identified early have received induction chemotherapy, when the PON1 protein expression abundance in the plasma sample is lower than 6744.48, they are confirmed as responders who have received induction chemotherapy, and it is verified that the potential responders identified early are indeed suitable for induction chemotherapy. Otherwise, they are identified as potential responders who have not received induction chemotherapy, and the false positive rate is 6.1%.

[0106] In the process of distinguishing between responders and nonresponders after induction chemotherapy, when the critical value of PON1 protein expression abundance was set to 5085.02, the sensitivity was 0.800 and the specificity was 0.616. When distinguishing between responders and nonresponders after induction chemotherapy, if the PON1 protein expression abundance in the plasma samples of responders and nonresponders after induction chemotherapy was higher than 5085.02, they were further confirmed as responders to induction chemotherapy, otherwise they were identified as nonresponders to induction chemotherapy, with a false positive rate of 31.4%.

[0107] Therefore, PON1 protein can be used as a molecular marker for early identification of patients with locally advanced nasopharyngeal carcinoma who respond to induction chemotherapy.

[0108] (2) Using DIA high-throughput technology, we detected significant differences in the expression of IGFBP3 protein in the plasma protein profiles between the healthy control group and the group with locally advanced NPC, between the effective group before treatment and the ineffective group before treatment, between the effective group before treatment and the effective group after treatment, and between the effective group after treatment and the ineffective group after treatment.

[0109] Depend on Figure 1It can be seen that compared with the IGFBP3 protein content in the plasma of healthy people, the IGFBP3 protein level in the plasma of patients with locally advanced nasopharyngeal carcinoma was significantly downregulated by 0.83 times, P value <0.05; in the plasma of effective patients before treatment, it was significantly downregulated by 0.92 times, P value <0.05; in the plasma of ineffective patients before treatment, it was significantly downregulated by 0.73 times, P value <0.05; in the plasma of effective patients after treatment, it was significantly downregulated by 0.56 times, P value <0.05; in the plasma of ineffective patients after treatment, it was significantly upregulated by 1.43 times, P value <0.05.

[0110] Depend on Figure 2 It can be seen that the AUC value of IGFBP3 protein in distinguishing patients with locally advanced nasopharyngeal carcinoma in healthy individuals is 0.664, indicating that IGFBP3 protein has good diagnostic accuracy and is a protein specifically expressed in locally advanced nasopharyngeal carcinoma. Figure 4 The results showed that the AUC value of IGFBP3 protein in identifying potential responders of induction chemotherapy in samples of locally advanced nasopharyngeal carcinoma before receiving induction chemotherapy was 0.719, indicating that IGFBP3 protein has good predictive performance and can be used as a molecular marker for early identification of potential responders of induction chemotherapy in locally advanced nasopharyngeal carcinoma. Figure 6 The results showed that the AUC value of IGFBP3 protein in distinguishing potential responders who did not receive induction chemotherapy from responders who had received induction chemotherapy in plasma samples of patients with locally advanced nasopharyngeal carcinoma who responded to induction chemotherapy before and after induction chemotherapy was 0.821, indicating that IGFBP3 has a high predictive performance, further confirming its reliability as a molecular marker for early identification of responders to induction chemotherapy. Figure 8 The results showed that the AUC value of IGFBP3 protein in distinguishing between responders and non-responders of induction chemotherapy in plasma samples of patients with locally advanced nasopharyngeal carcinoma after induction chemotherapy was 0.914, indicating that it can effectively distinguish between responders and non-responders of induction chemotherapy after treatment, and can be used as a marker for evaluating the efficacy of induction chemotherapy in locally advanced nasopharyngeal carcinoma.

[0111] In the diagnosis of locally advanced nasopharyngeal carcinoma, when the critical value of IGFBP3 protein expression abundance was set at 647.93, the sensitivity was 0.609 and the specificity was 0.720. When diagnosing locally advanced nasopharyngeal carcinoma, in the plasma samples of responders and responders to induction chemotherapy before treatment, if the IGFBP3 protein expression abundance was lower than 647.93, they were identified as patients with locally advanced nasopharyngeal carcinoma, otherwise they were identified as healthy individuals, and the false positive rate was 26.4%.

[0112] In the process of early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, when the critical value of IGFBP3 protein expression abundance was set at 568.45, the sensitivity was 0.633 and the specificity was 0.765; when early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma was performed, if the expression abundance of IGFBP3 protein in the plasma samples of responders before and after induction chemotherapy was higher than 568.45, they were identified as potential responders to induction chemotherapy, otherwise they were identified as non-responders to induction chemotherapy, and the false positive rate was 29.6%.

[0113] In the process of confirming whether the potential responders identified early are indeed suitable for induction chemotherapy, when the critical value of IGFBP3 protein expression abundance is set to 464.66, the sensitivity is 0.765 and the specificity is 0.912. When confirming whether the potential responders identified early have received induction chemotherapy, when the IGFBP3 protein expression abundance in the plasma sample is lower than 464.66, they are confirmed as responders who have received induction chemotherapy, and it is verified that the potential responders identified early are indeed suitable for induction chemotherapy. Otherwise, they are identified as potential responders who have not received induction chemotherapy, and the false positive rate is 10.3%.

[0114] In the process of distinguishing between responders and nonresponders after induction chemotherapy, when the critical value of IGFBP3 protein expression abundance was set to 466.53, the sensitivity was 0.967 and the specificity was 0.765. When distinguishing between responders and nonresponders after induction chemotherapy, if the IGFBP3 protein expression abundance in the plasma samples of responders and nonresponders after induction chemotherapy was higher than 466.53, they were further confirmed as responders to induction chemotherapy, otherwise they were identified as nonresponders to induction chemotherapy, with a false positive rate of 21.6%.

[0115] Therefore, IGFBP3 protein can be used as a molecular marker for early identification of patients with locally advanced nasopharyngeal carcinoma who respond to induction chemotherapy.

[0116] (3) Using DIA high-throughput technology, we detected significant differences in the expression of v-kappa-3 protein in the plasma protein profiles between the healthy control group and the group with locally advanced NPC, between the responders before treatment and the ineffective patients before treatment, between the responders before treatment and the responders after treatment, and between the responders after treatment and the ineffective patients after treatment.

[0117] Depend on Figure 1It can be seen that compared with the v-kappa-3 protein content in the plasma of healthy people, the v-kappa-3 protein content in the plasma of patients with locally advanced nasopharyngeal carcinoma was significantly upregulated by 2.12 times, P value <0.05; in the plasma of responders before treatment, it was significantly upregulated by 1.49 times, P value <0.05; in the plasma of ineffective patients before treatment, it was significantly upregulated by 2.84 times, P value <0.05; in the plasma of responders after treatment, it was significantly downregulated by 0.88 times, P value <0.05; in the plasma of ineffective patients after treatment, it was significantly upregulated by 2.15 times, P value <0.05.

[0118] Depend on Figure 2 It can be seen that the AUC value of v-kappa-3 protein in distinguishing patients with locally advanced nasopharyngeal carcinoma in healthy individuals is 0.810, indicating that v-kappa-3 protein has good diagnostic accuracy and is a protein specifically expressed in locally advanced nasopharyngeal carcinoma. Figure 4 The results showed that the AUC value of v-kappa-3 protein in identifying potential responders to induction chemotherapy in samples of locally advanced nasopharyngeal carcinoma before receiving induction chemotherapy was 0.852, indicating that v-kappa-3 protein has good predictive performance and can be used as a molecular marker for early identification of potential responders to induction chemotherapy in locally advanced nasopharyngeal carcinoma. Figure 6 The results showed that the AUC value of v-kappa-3 protein in distinguishing potential responders who did not receive induction chemotherapy from responders who had received induction chemotherapy in plasma samples of responders to induction chemotherapy in locally advanced nasopharyngeal carcinoma before and after induction chemotherapy was 0.726, indicating that v-kappa-3 has a high predictive performance, further confirming its reliability as a molecular marker for early identification of responders to induction chemotherapy. Figure 8 The results showed that the AUC value of v-kappa-3 protein in distinguishing between responders and non-responders of induction chemotherapy in plasma samples of patients with locally advanced nasopharyngeal carcinoma after induction chemotherapy was 0.872, indicating that it can effectively distinguish between responders and non-responders of induction chemotherapy after treatment, and can be used as a marker for evaluating the efficacy of induction chemotherapy in locally advanced nasopharyngeal carcinoma.

[0119] In the diagnosis of locally advanced nasopharyngeal carcinoma, when the critical value of v-kappa-3 protein expression abundance was set at 1747.81, the sensitivity was 0.750 and the specificity was 0.720. When diagnosing locally advanced nasopharyngeal carcinoma, in the plasma samples of responders and responders to induction chemotherapy before treatment, if the v-kappa-3 protein expression abundance was lower than 1747.81, they were identified as patients with locally advanced nasopharyngeal carcinoma, otherwise they were identified as healthy individuals, and the false positive rate was 22.6%.

[0120] In the process of early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, when the critical value of v-kappa-3 protein expression abundance was set at 1993.58, the sensitivity was 1.000 and the specificity was 0.618; when early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma was performed, if the expression abundance of v-kappa-3 protein in the plasma samples of responders before and after induction chemotherapy was higher than 1993.58, they were identified as potential responders to induction chemotherapy, otherwise they were identified as non-responders to induction chemotherapy, and the false positive rate was 30.2%.

[0121] In the process of confirming whether the potential responders identified early are indeed suitable for induction chemotherapy, when the critical value of v-kappa-3 protein expression abundance is set to 1848.28, the sensitivity is 0.853 and the specificity is 0.500. When confirming whether the potential responders identified early have received induction chemotherapy, when the v-kappa-3 protein expression abundance in the plasma sample is lower than 1848.28, they are confirmed as responders who have received induction chemotherapy, and it is verified that the potential responders identified early are indeed suitable for induction chemotherapy. Otherwise, they are identified as potential responders who have not received induction chemotherapy, and the false positive rate is 37.0%.

[0122] In the process of distinguishing between responders and nonresponders after induction chemotherapy, when the critical value of v-kappa-3 protein expression abundance was set to 1571.38, the sensitivity was 0.867 and the specificity was 0.765. When distinguishing between responders and nonresponders after induction chemotherapy, if the v-kappa-3 protein expression abundance in the plasma samples of responders and nonresponders after induction chemotherapy was higher than 1571.38, they were further confirmed as responders to induction chemotherapy, otherwise they were identified as nonresponders to induction chemotherapy, with a false positive rate of 23.5%.

[0123] Therefore, v-kappa-3 protein can be used as a molecular marker for early identification of patients with locally advanced nasopharyngeal carcinoma who respond to induction chemotherapy.

[0124] (4) Using DIA high-throughput technology, we detected significant differences in the expression of DDX55 protein in the plasma protein profiles between the healthy control group and the group with locally advanced NPC, between the effective group before treatment and the ineffective group before treatment, between the effective group before treatment and the effective group after treatment, and between the effective group after treatment and the ineffective group after treatment.

[0125] Depend on Figure 1It can be seen that compared with the DDX55 protein content in the plasma of healthy people, the DDX55 protein content in the plasma of patients with locally advanced nasopharyngeal carcinoma was significantly downregulated by 0.76 times, P value <0.05; it was significantly downregulated by 0.34 times in the plasma of effective patients before treatment, P value <0.05; it was significantly upregulated by 1.23 times in the plasma of ineffective patients before treatment, P value <0.05; it was significantly downregulated by 0.89 times in the plasma of effective patients after treatment, P value <0.05; it was significantly upregulated by 1.12 times in the plasma of ineffective patients after treatment, P value <0.05.

[0126] Depend on Figure 2 It can be seen that the AUC value of DDX55 protein in distinguishing patients with locally advanced nasopharyngeal carcinoma in healthy individuals is 0.645, indicating that DDX55 protein has good diagnostic accuracy and is a protein specifically expressed in locally advanced nasopharyngeal carcinoma. Figure 4 The results showed that the AUC value of DDX55 protein in identifying potential responders of induction chemotherapy in samples of locally advanced nasopharyngeal carcinoma before receiving induction chemotherapy was 0.835, indicating that DDX55 protein has good predictive performance and can be used as a molecular marker for early identification of potential responders of induction chemotherapy in locally advanced nasopharyngeal carcinoma. Figure 6 The results showed that the AUC value of DDX55 protein in distinguishing potential responders who did not receive induction chemotherapy from responders who had received induction chemotherapy in plasma samples of patients with locally advanced nasopharyngeal carcinoma who responded to induction chemotherapy before and after induction chemotherapy was 0.926, indicating that DDX55 has a high predictive performance, further confirming its reliability as a molecular marker for early identification of responders to induction chemotherapy. Figure 8 The results showed that the AUC value of DDX55 protein in distinguishing between responders and non-responders of induction chemotherapy in plasma samples of patients with locally advanced nasopharyngeal carcinoma after induction chemotherapy was 0.700, indicating that it can effectively distinguish between responders and non-responders of induction chemotherapy after treatment, and can be used as a marker for evaluating the efficacy of induction chemotherapy in locally advanced nasopharyngeal carcinoma.

[0127] In the diagnosis of locally advanced nasopharyngeal carcinoma, when the critical value of DDX55 protein expression abundance was set at 1994.08, the sensitivity was 0.703 and the specificity was 0.600. When diagnosing locally advanced nasopharyngeal carcinoma, in the plasma samples of responders and responders to induction chemotherapy before treatment, if the DDX55 protein expression abundance was lower than 1994.08, they were identified as patients with locally advanced nasopharyngeal carcinoma, otherwise they were identified as healthy individuals, and the false positive rate was 30.8%.

[0128] In the process of early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, when the critical value of DDX55 protein expression abundance was set at 1524.40, the sensitivity was 0.733 and the specificity was 0.824; when early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma was performed, if the expression abundance of DDX55 protein in the plasma samples of responders before and after induction chemotherapy was higher than 1524.40, they were identified as potential responders to induction chemotherapy, otherwise they were identified as non-responders to induction chemotherapy, and the false positive rate was 21.4%.

[0129] In the process of confirming whether the potential responders identified early are indeed suitable for induction chemotherapy, when the critical value of DDX55 protein expression abundance is set to 1481.18, the sensitivity is 0.912 and the specificity is 0.824. When confirming whether the potential responders identified early have received induction chemotherapy, when the DDX55 protein expression abundance in the plasma sample is lower than 1481.18, they are confirmed as responders who have received induction chemotherapy, and it is verified that the potential responders identified early are indeed suitable for induction chemotherapy. Otherwise, they are identified as potential responders who have not received induction chemotherapy, and the false positive rate is 16.2%.

[0130] In the process of distinguishing between responders and nonresponders after induction chemotherapy, when the critical value of DDX55 protein expression abundance was set to 2951.23, the sensitivity was 0.600 and the specificity was 0.765. When distinguishing between responders and nonresponders after induction chemotherapy, if the DDX55 protein expression abundance in the plasma samples of responders and nonresponders after induction chemotherapy was higher than 2951.23, they were further confirmed as responders to induction chemotherapy, otherwise they were identified as nonresponders to induction chemotherapy, with a false positive rate of 30.8%.

[0131] Therefore, DDX55 protein can be used as a molecular marker for early identification of responders to induction chemotherapy in patients with locally advanced nasopharyngeal carcinoma.

[0132] (4) Application of a combination of PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein in the preparation of a kit for early diagnosis of localized advanced nasopharyngeal carcinoma

[0133] Logistic regression analysis was used to integrate PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein to distinguish healthy individuals from patients with locally advanced nasopharyngeal carcinoma; that is, a combined model of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein was used to diagnose locally advanced nasopharyngeal carcinoma at an early stage.

[0134] The logistic regression analysis equation is:

[0135] logistics1=5.9851+-6e-04*PON1+-0.0019*IGFBP3+0.0012*v-kappa-3+-4e-04*DDX55;

[0136] The results are as follows Figure 3 As shown, the AUC value of the four-protein combination modeling to distinguish healthy individuals from patients with locally advanced NPC was 0.922, and the high diagnostic accuracy demonstrated the NPC specificity of the combined protein.

[0137] In the diagnosis of locally advanced nasopharyngeal carcinoma, when the critical value of the logistic regression analysis result of the combined protein was set to -0.844, the sensitivity was 0.953 and the specificity was 0.700. When diagnosing locally advanced nasopharyngeal carcinoma, if the analysis result was lower than -0.844, it was identified as locally advanced nasopharyngeal carcinoma, otherwise it was identified as a healthy individual, and the false positive rate was 19.7%.

[0138] In view of the above results, PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein can be used as molecular markers for the combined diagnosis of locoregionally advanced nasopharyngeal carcinoma.

[0139] (5) Application of a combination of PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein in the preparation of a kit for early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma

[0140] Logistic regression analysis was used to integrate PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein to distinguish between responders and non-responders of induction chemotherapy before treatment; that is, a combined modeling of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein was used to early identify potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma.

[0141] The logistic regression analysis equation is:

[0142] logistics2=-2.5104+-1e-04*PON1+-0.0027*IGFBP3+9e-04*v-kappa-3+0.001*DDX55;

[0143] The results are as follows Figure 5 As shown, the AUC value of the four-protein combination modeling in distinguishing between responders and non-responders of induction chemotherapy before treatment was 0.948. The high prediction accuracy illustrates the specificity of the induction chemotherapy efficacy of the combination of proteins.

[0144] In the early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, when the critical value of the logistic regression analysis result of the combined protein was set to -0.531, the sensitivity was 0.967 and the specificity was 0.853. When performing early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, if the analysis result was lower than -0.531, they were identified as potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, otherwise they were identified as ineffective patients with induction chemotherapy for locally advanced nasopharyngeal carcinoma, and the false positive rate was 14.7%.

[0145] In view of the above results, PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein can be used as molecular markers for early identification of potential responders to induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma.

[0146] (6) Use of a combination of PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein in the preparation of a kit for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma

[0147] Logistic regression analysis was performed to integrate PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein to distinguish the responders to induction chemotherapy before and after induction chemotherapy; and combined modeling of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein was used to verify that the potential responders identified early were indeed suitable for induction chemotherapy, that is, to confirm whether the molecular markers were specific for the benefits of induction chemotherapy.

[0148] The logistic regression analysis equation is:

[0149] logistics3=6.4787+-9e-04*PON1+-0.0054*IGFBP3+1e-04*v-kappa-3+0.0016*DDX55;

[0150] The results are as follows Figure 7 As shown, the AUC value of the four-protein combination modeling to distinguish between those who respond to induction chemotherapy before and after induction chemotherapy was 0.971. The high efficacy confirmation indicates that the combination of proteins is specific to the benefit of induction chemotherapy.

[0151] In the process of confirming whether the potential responders identified early are indeed suitable for induction chemotherapy, the critical value of the logistic regression analysis results of the combined protein was set to -1.127, with a sensitivity of 1.000 and a specificity of 0.941. When confirming whether the potential responders identified early received induction chemotherapy, if the analysis result is lower than -1.127, they are confirmed as responders who have received induction chemotherapy, otherwise they are identified as potential responders who have not received induction chemotherapy, with a false positive rate of 5.6%.

[0152] In view of the above results, PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein can be used as molecular markers to confirm the effectiveness of induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma.

[0153] (7) Application of a combination of PON1 protein, IGFBP3 protein, v-kappa-3 protein or DDX55 protein in the preparation of a kit for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma

[0154] Logistic regression analysis was used to integrate PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein to distinguish between responders and non-responders after induction chemotherapy; that is, the combined modeling of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein was used to evaluate the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma.

[0155] The logistic regression analysis equation is:

[0156] logistics4=0.2101+-9e-04*PON1+0.0043*IGFBP3+9e-04*v-kappa-3+-3e-04*DDX55;

[0157] The results are as follows Fig. 9 As shown, the AUC value of the four-protein combination modeling to distinguish between responders and ineffective patients after induction chemotherapy was 0.941. The high efficacy evaluation performance indicates the specificity of the combination of proteins for the efficacy of induction chemotherapy.

[0158] In the process of distinguishing responders from non-responders after induction chemotherapy, the critical value of the logistic regression analysis results of the combined protein was set to 0.643, with a sensitivity of 0.800 and a specificity of 0.971. When confirming whether the potential responders identified early received induction chemotherapy, if the analysis result was higher than 0.643, they were confirmed as responders of induction chemotherapy, otherwise they were identified as non-responders of induction chemotherapy, with a false positive rate of 4.0%.

[0159] In view of the above results, the combination of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein can be used as molecular markers to evaluate the efficacy of induction chemotherapy in locoregionally advanced nasopharyngeal carcinoma.

[0160] In summary, since PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein were significantly differentially expressed in the plasma of healthy individuals and untreated patients with local advanced nasopharyngeal carcinoma, the plasma of responders and non-responders before treatment, the plasma of responders before and after treatment, and the plasma samples of responders and non-responders after treatment, PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein can be used to identify potential responders of induction chemotherapy for local advanced nasopharyngeal carcinoma before treatment, showing excellent test efficiency. This method is simple, easy to accept by patients, has unified implementation standards and follows objective data, which can further optimize the clinical application of induction chemotherapy for local advanced nasopharyngeal carcinoma, especially innovative treatment in key areas such as oncology.

[0161] It should be noted that the embodiments of the present disclosure may be further described in the following manner:

[0162] The present disclosure provides the use of protein markers in early diagnosis of locoregionally advanced nasopharyngeal carcinoma;

[0163] Or, the application of protein markers in early identification of potential responders to induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma;

[0164] or, the use of protein markers in confirming the effectiveness of induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma;

[0165] or, the application of protein markers in evaluating the efficacy of induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma;

[0166] Wherein, the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein.

[0167] The present disclosure provides the use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for early diagnosis of localized advanced nasopharyngeal carcinoma;

[0168] Or, use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for early identification of potential responders to induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma;

[0169] or, use of an antibody that specifically binds to a protein marker in the preparation of a reagent or diagnostic tool for confirming the effectiveness of induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma;

[0170] or, use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for evaluating the efficacy of induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma;

[0171] Wherein, the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein and DDX55 protein.

[0172] Optionally, the antibody includes at least one of an anti-PON1 protein antibody, an anti-IGFBP3 protein antibody, an anti-v-kappa-3 protein antibody, and an anti-DDX55 protein antibody.

[0173] Optionally, the antibody comprises a monoclonal antibody or a polyclonal antibody.

[0174] Optionally, the antibody is linked to a detectable label.

[0175] Optionally, the detectable marker comprises at least one of an enzyme, a prosthetic group, a fluorescent substance, a luminescent substance, a bioluminescent substance, a colored substance, and a radioactive isotope.

[0176] Optionally, the diagnostic tool comprises a chip, a test strip or a test kit.

[0177] The present disclosure also provides reagents for early diagnosis of locally advanced nasopharyngeal carcinoma, reagents for early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, reagents for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma, or reagents for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma, characterized in that they include antibodies that specifically bind to protein markers; the protein markers include at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0178] Optionally, the reagent is a specific primer or probe, or a pharmaceutical composition comprising the primer or probe.

[0179] The present disclosure also provides a kit for early diagnosis of locally advanced nasopharyngeal carcinoma, a kit for early identification of potential responders to induction chemotherapy for locally advanced nasopharyngeal carcinoma, a kit for confirming the effectiveness of induction chemotherapy for locally advanced nasopharyngeal carcinoma, or a kit for evaluating the efficacy of induction chemotherapy for locally advanced nasopharyngeal carcinoma, characterized in that it comprises an antibody that specifically binds to a protein marker; the protein marker comprises at least one of PON1 protein, IGFBP3 protein, v-kappa-3 protein, and DDX55 protein.

[0180] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0181] The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. The use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for early diagnosis of localized advanced nasopharyngeal carcinoma; Or, use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for early identification of potential responders to induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma; or, use of an antibody that specifically binds to a protein marker in the preparation of a reagent or diagnostic tool for confirming the effectiveness of induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma; or, use of antibodies that specifically bind to protein markers in the preparation of reagents or diagnostic tools for evaluating the efficacy of induction chemotherapy for locoregionally advanced nasopharyngeal carcinoma; in, The protein markers include at least one of PON1 protein, v-kappa-3 protein, and DDX55 protein.

2. The use according to claim 1, characterized in that: The antibody comprises at least one of an anti-PON1 protein antibody, an anti-v-kappa-3 protein antibody, and an anti-DDX55 protein antibody.

3. The use according to claim 1, characterized in that: The antibody includes a monoclonal antibody or a polyclonal antibody.

4. The use according to claim 1, characterized in that: The antibody is linked to a detectable label.

5. The use according to claim 4, characterized in that: The detectable marker comprises at least one of an enzyme, a prosthetic group, a fluorescent substance, a luminescent substance, a bioluminescent substance, a colored substance, and a radioactive isotope.

6. The use according to claim 1, characterized in that: The diagnostic tool includes a chip, a test strip or a test kit.

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