A method for screening biomarkers for primary resistance to cetuximab, biomarkers screened by this method, and their uses

Abstract

The present application relates to a method for screening biomarkers for primary resistance to cetuximab in head and neck squamous cell carcinoma, the biomarkers screened by this method, and their uses. By establishing a patient-derived xenograft (PDX) model of tumor tissues from patients with head and neck squamous cell carcinoma, accurate clinical information of the patients is obtained to construct a PDX model platform for head and neck squamous cell carcinoma. In the constructed PDX model platform, referring to the clinical phase II research mode, the corresponding PDX models of the patients are selected for inclusion in the PDX model clinical alternative trial, and cetuximab drug treatment is carried out. With the help of whole exome sequencing and transcriptome sequencing, the biomarkers for primary resistance to cetuximab are screened. Through experimental verification, the biomarkers for primary resistance to cetuximab in head and neck squamous cell carcinoma provided by the present application are very reliable biomarkers for predicting the efficacy of cetuximab in patients with head and neck squamous cell carcinoma.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, and particularly relates to a method for screening biomarkers for primary resistance to cetuximab in head and neck squamous cell carcinoma, the biomarkers screened by this method, and their uses. Background Art

[0002] Head and neck cancer is a highly prevalent malignant tumor in the cancer spectrum in China and even globally, and is one of the most serious cancers affecting people's life and health and quality of life. According to global cancer statistics, there are more than 550,000 newly diagnosed head and neck cancers worldwide each year. Its global incidence ranks third among all tumors in the body, and the lethality rate ranks sixth. Among head and neck cancers, more than 90% are head and neck squamous cell carcinomas, referred to as head and neck squamous cell carcinoma (HNSCC). The head and neck has a complex anatomy with dense organs, and most of them are important organs. Therefore, the occurrence of HNSCC not only causes great damage to the patient's appearance and basic physiological functions (such as chewing, swallowing, breathing, etc.), sensory functions (taste, smell, hearing), and language functions, but also seriously affects the patient's quality of life. Therefore, how to kill tumor cells in the head and neck with dense organs while protecting the basic physiological functions of the organs and maintaining the patient's quality of life to the greatest extent is a huge problem faced by clinicians and scientific researchers together.

[0003] The primary sites of HNSCC are numerous and the pathological types are diverse. Its complexity ranks first among all tumors in the body, seriously threatening people's life and health. Although medical devices and treatment technologies have advanced by leaps and bounds in recent years, the five-year survival rate of HNSCC patients in China has not improved significantly. At present, surgical treatment is still the only radical treatment strategy for HNSCC. In order to completely remove tumor cells as comprehensively as possible, extensive resection is required. However, due to the special anatomical location of the head and neck, radical resection of the tumor seriously affects the patient's quality of life and causes a heavy family and social burden. In addition, the conventional treatment plan for HNSCC is mainly radiotherapy and chemotherapy. However, multiple clinical studies have shown that only a small number of patients can benefit from radiotherapy and chemotherapy. More than 65% of HNSCC patients will experience recurrence and / or metastasis, and the survival rate of most HNSCC patients with recurrence and / or metastasis is less than one year.

[0004] Epidermal growth factor receptor (EGFR) is highly expressed in more than 90% of HNSCC patients. Inhibiting EGFR is the only targeted treatment strategy for HNSCC. Cetuximab is a chimeric IgG1 monoclonal antibody that blocks EGFR. As the only targeted drug recommended by the NCCN clinical guidelines for the first-line treatment of HNSCC, it is used in combination with radiotherapy to treat locally advanced HNSCC, or in combination with platinum-based chemotherapy to treat recurrent and / or metastatic HNSCC. The mechanism of action of cetuximab involves competing with endogenous EGFR ligands for binding to the accessible extracellular domain, and then blocking the receptor-dependent signal transduction pathway, thereby playing roles such as inhibiting tumor cell growth, inhibiting angiogenesis, and inhibiting tumor metastasis.

[0005] Although the combined treatment with cetuximab has improved the clinical prognosis of head and neck squamous cell carcinoma, the primary resistance and secondary resistance that occur during the treatment increase the tumor recurrence rate and limit the clinical efficacy of cetuximab. It is worth noting that the therapeutic efficacy of cetuximab in the treatment of head and neck squamous cell carcinoma is relatively low. The objective response rate of the single-agent treatment group is 13%, and that of the combined chemotherapy group is 36%. Despite continuous maintenance of cetuximab treatment, the time from the initial use until treatment failure in patients with head and neck squamous cell carcinoma using the EXTREME treatment regimen is only about 5 months. More importantly, the literature reports that only about 10-20% of advanced cancer patients can achieve tumor growth inhibition after using drugs to block the EGFR pathway, and most patients who initially respond to cetuximab will eventually show secondary resistance.

[0006] The active exploration of pharmacodynamic biomarkers for targeted drugs has always been a hot topic in cancer research and is the key to achieving precise cancer treatment. In recent years, through large-scale population cohorts and various new omics technologies, more and more studies have confirmed that tumor heterogeneity is the main reason for the significant differences in therapeutic efficacy and the emergence of drug resistance during cancer treatment. Tumor heterogeneity includes inter-tumor heterogeneity and intra-tumor heterogeneity. Among them, inter-tumor heterogeneity is mainly manifested as the primary drug resistance and sensitivity among different patients at the population level. On this basis, in recent years, a large number of studies have been conducted on the primary resistance of cetuximab; however, so far, the clinical indications for the use of cetuximab are still very scarce in most tumors including head and neck squamous cell carcinoma, and only in colorectal cancer is it clear that KRAS gene mutations can mediate the primary resistance of cetuximab.

[0007] In colorectal cancer, 1,022 tumor samples from patients treated with cetuximab combined with chemotherapy were collected from 11 centers in 7 European countries from 2001 to 2008, and the gene mutations of KRAS, BRAF, NRAS, and PIK3CA were detected. The results showed that patients with KRAS mutations had a lower response rate to cetuximab compared with wild-type patients (6.7% vs 35.8%), and a shorter median progression-free survival (12 weeks vs 24 weeks). In addition, multiple prospective clinical studies in colorectal cancer have shown that the combined use of cetuximab and radiotherapy only benefits patients with wild-type KRAS colorectal cancer, and there is no obvious clinical efficacy for patients with KRAS mutations. Therefore, the detection of the KRAS gene status helps to determine the efficacy of cetuximab in colorectal cancer patients. KRAS mutation is the first biomarker that can predict the resistance of colorectal cancer patients to the targeted therapy drug cetuximab, providing a theoretical basis for the determination of individualized treatment plans. In 2009, the NCCN updated the colorectal cancer guidelines and recommended that patients with wild-type KRAS advanced metastatic colorectal cancer choose cetuximab combined with chemotherapy as the first-line treatment.

[0008] In addition to metastatic colorectal cancer, the recommended indication for cetuximab is mainly the first-line treatment of recurrent / metastatic head and neck squamous cell carcinoma. However, compared with the relatively high mutation frequency of KRAS in colorectal cancer (more than 40% in the Chinese population), the mutation frequency of KRAS in head and neck squamous cell carcinoma is extremely low (less than 2% in the Chinese population). Therefore, KRAS mutation cannot be used as a primary drug resistance marker in head and neck squamous cell carcinoma. In head and neck cancer, due to the lag of basic and clinical research, there is no large-scale cohort study focusing on the primary drug resistance of cetuximab to reveal the pharmacodynamic biomarkers of cetuximab. In 2017, Friederike et al. screened 59 patients with head and neck squamous cell carcinoma who received palliative treatment between 2010 and 2016 and found that cetuximab resistance was associated with the EGFR-K521 polymorphism. In 2020, Nellie et al. confirmed in vitro and in vivo that the tyrosine residue Y821 of AXL mediated cetuximab resistance by activating the c-ABL kinase in head and neck squamous cell carcinoma. In 2020, researchers from the Institut Curie in France detected PIK3CA and H / N / KRAS gene mutations, as well as PTEN and EGFR immunohistochemical analysis, in tumor specimens of 118 patients with head and neck squamous cell carcinoma who were treated with cetuximab between 2006 and 2015. The results showed that the therapeutic effect of cetuximab combined with chemotherapy was not related to the PIK3CA and KRAS / HRAS gene mutations in patients with head and neck squamous cell carcinoma, and the high expression of epidermal growth factor receptor EGFR could not predict the sensitivity of cetuximab. In view of this, so far, there is no predictive biomarker in head and neck squamous cell carcinoma that can guide patients in cetuximab treatment.

[0009] In head and neck squamous cell carcinoma, retrospective studies on the primary resistance biomarkers of cetuximab that have been conducted so far mainly use patients' tumor tissues. Such retrospective studies mainly focus on the analysis of several genes that have been proven to be related to cetuximab resistance in colorectal cancer. By detecting the mutations or expression levels of target molecules, it is then evaluated whether these molecules can also serve as pharmacodynamic prediction biomarkers in head and neck squamous cell carcinoma. However, years of research have confirmed that this approach is difficult to discover primary resistance biomarkers of cetuximab in head and neck squamous cell carcinoma. On the one hand, due to the differences in the pathogenesis and genetic background of different tumors, genes with high-frequency mutations in colorectal cancer are very likely to have no obvious mutations in head and neck squamous cell carcinoma, and genes with significantly high expression in colorectal cancer are very likely to have no changes or no expression in head and neck squamous cell carcinoma. On the other hand, focusing on the changes of individual genes while ignoring the mutations and gene expression at the overall tumor level will also lead to low screening efficiency of primary resistance prediction biomarkers and difficulty in discovering accurate pharmacodynamic biomarkers. In addition, most of these studies use clinical samples for retrospective analysis. However, due to ethical restrictions, cetuximab is rarely used to treat patients as a single drug. It is often combined with various chemotherapy drugs and radiotherapy, and patients may have experienced various other drug treatments before cetuximab treatment. The primary resistance biomarkers analyzed based on these tumor samples often lack specificity and accuracy and are difficult to apply in clinical practice.

[0010] There is currently no report on relatively stable biomarkers for primary resistance of cetuximab in head and neck squamous cell carcinoma that have been mined and verified through preclinical alternative trials using xenograft tumor models. If genes with abnormal mutations or expressions in primary resistance of cetuximab can be screened out as biomarkers and corresponding detection kits can be developed, it will surely be a powerful promotion for the stratified treatment and precision medication of head and neck squamous cell carcinoma patients in China. Summary of the Invention

[0011] In view of this, the present invention takes tumor heterogeneity as the starting point, standardizes sample collection under the clinical research mode, and establishes a xenograft tumor model (patient-derived xenograft, PDX) derived from patients' tumor tissues to obtain accurate clinical information of patients and form a PDX model platform for head and neck squamous cell carcinoma. In the PDX model platform, referring to the clinical phase II research mode, the corresponding PDX models of patients are selected for inclusion to carry out preclinical alternative trials of the PDX model, and cetuximab drug treatment is carried out. With the help of whole exome sequencing and transcriptome sequencing, primary resistance biomarkers of cetuximab are mined and verified.

[0012] To address the following deficiencies of the existing technology, namely, in head and neck squamous cell carcinoma, single-agent cetuximab treatment cannot be carried out to discover the primary drug-resistant biomarkers of cetuximab; histological examination before drug administration causes pain to patients; if whole-genome and transcriptome sequencing are not performed, the efficacy of cetuximab cannot be predicted early. The present invention provides a method for screening primary drug-resistant biomarkers of cetuximab in head and neck squamous cell carcinoma using a PDX model clinical alternative trial. Based on this method, the present invention provides 8 primary drug-resistant biomarkers of cetuximab in head and neck squamous cell carcinoma.

[0013] Therefore, on the one hand, the present invention provides a method for screening primary drug-resistant biomarkers of cetuximab in head and neck squamous cell carcinoma using a patient-derived xenograft tumor model (abbreviated as PDX model) clinical alternative trial, comprising the following steps:

[0014] (1) Constructing a head and neck squamous cell carcinoma PDX model in immunodeficient mice using tumor tissues from head and neck squamous cell carcinoma patients;

[0015] (2) Obtaining the clinical information of the patients corresponding to the head and neck squamous cell carcinoma PDX model, including past medical history, clinical treatment situation, and medication situation;

[0016] (3) Construction of the first cohort of head and neck squamous cell carcinoma PDX models (also referred to as the PDX model discovery cohort hereinafter): Relying on the established head and neck squamous cell carcinoma PDX model biobank, according to the clinical information of the corresponding patients, randomly screen a number of head and neck squamous cell carcinoma PDX models for inclusion, and inoculate each live-preserved PDX xenograft tumor sample subcutaneously into nude mice for subsequent cetuximab clinical alternative trials;

[0017] (4) Conducting a cetuximab PDX model clinical alternative trial: When the tumor volume of the PDX model reaches a certain value, such as 100 - 200 mm 3 after that, each PDX case is randomly divided into a control group and a single-agent cetuximab treatment group, and drug treatment is started. The PDX models are intraperitoneally injected with PBS or cetuximab respectively, and the tumor volume and body weight of the mice are measured weekly;

[0018] (5) Evaluation of the efficacy of cetuximab in the cetuximab PDX model clinical alternative trial: To evaluate the drug response to cetuximab, the drug reaction is evaluated by the volume change of the xenograft tumor before and after drug administration in the PDX model. The specific criteria are as follows: 1) Complete tumor regression (mCR), the tumor volume is reduced by at least 40%; 2) Partial tumor regression (mPR), the tumor volume is reduced by 20% - 40%; 3) Disease progression (mPD), the tumor volume is increased by at least 30%; 4) Disease stability (mSD), the tumor volume change is between a 30% increase and a 20% decrease; the mPD model is determined as the primary drug-resistant model; the mCR model is defined as the sensitive model;

[0019] (6) Screening for primary drug resistance candidate pharmacodynamic markers through model clinical alternative trials: Perform whole-exome sequencing and transcriptome sequencing on the primary drug resistance model and the sensitive model respectively. Based on the differential gene mutation profiles and / or differential gene expression profiles between the primary drug resistance model and the sensitive model, combined with the results of gene function and signaling pathway analysis, preliminarily screen out the primary drug resistance candidate pharmacodynamic markers;

[0020] (7) Conduct independent PDX model clinical alternative trials (hereinafter also referred to as the PDX model validation cohort): Relying on the head and neck squamous cell carcinoma PDX model biobank, according to the clinical information of the corresponding patients, randomly screen several head and neck squamous cell carcinoma PDX models as the second cohort for inclusion. The selected second cohort does not overlap with the first cohort. Treat the included PDX models with cetuximab monotherapy in the same manner as in step (4). After several weeks of medication, for example, 3 weeks later, collect samples of the transplanted tumors and preserve them in vivo. By evaluating the efficacy of cetuximab, distinguish the primary drug resistance model and the sensitive model;

[0021] (8) Verifying pharmacodynamic markers using independent PDX model clinical alternative trials: Using the samples in the independent PDX model clinical alternative trials, adopt the real-time fluorescence quantitative PCR (qRT-PCR) method to verify the candidate variant genes and expressed genes. Screen out the genes with consistent gene mutations or expression trends in the first and second cohorts. Plot the receiver operating characteristic curve (ROC) for the selected pharmacodynamic predictive biomarkers, and perform analysis using SPSS 21.0 statistical software, and calculate the area under the curve AUC. Select the index with AUC > 0.80 to finally determine the head and neck squamous cell carcinoma cetuximab primary drug resistance biomarkers found in the first and second cohorts.

[0022] In a specific embodiment, the primary resistance biomarkers of cetuximab for head and neck squamous cell carcinoma screened by the method are selected from the following: transcription factor SIX2 (Sine Oculis Homeobox Homolog 2), basic transcription element binding protein KLF9 (Kruppel Like Factor 9), poly(ADP-ribose) polymerase PARP3 (Poly(ADP-Ribose)Polymerase Family Member 3), member PODXL2 (PodocalyxinLike 2) of the cell surface transmembrane protein CD34 family, cyclin-dependent kinase CDK1 (Cyclin Dependent Kinase 1), ANKH (ANKH Inorganic Pyrophosphate Transport Regulator), membrane iron transport auxiliary protein HEPHL1 (Hephaestin Like 1), and neurotransmitter transporter SLC6A (Solute carrier family6).

[0023] In the present application, high-expression events of the transcription factor SIX2 (Sine Oculis Homeobox Homolog 2), basic transcription element binding protein KLF9 (Kruppel Like Factor 9), poly(ADP-ribose) polymerase PARP3 (Poly(ADP-Ribose)Polymerase Family Member 3), and member PODXL2 (Podocalyxin Like 2) of the cell surface transmembrane protein CD34 family, and gene amplification events of cyclin-dependent kinase CDK1 (Cyclin DependentKinase 1), ANKH (ANKH Inorganic Pyrophosphate TransportRegulator), membrane iron transport auxiliary protein HEPHL1 (Hephaestin Like 1), and neurotransmitter transporter SLC6A (Solute carrier family 6) can be used as biomarkers for primary resistance to cetuximab.

[0024] The present invention also provides the use of the biomarkers SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHL1, or SLC6A in the preparation of reagents for predicting the efficacy of cetuximab in clinical applications for patients with head and neck squamous cell carcinoma.

[0025] The present invention further provides a pharmacodynamic prediction kit for biomarkers of primary resistance to cetuximab, which at least includes gene expression detection probes and / or copy number detection probes. Among them, the gene expression detection probes include gene expression detection probes for one or more of SIX2, KLF9, PARP3, and PODXL2, and the copy number detection probes include copy number detection probes for one or more of CDK1, ANKH, HEPHL1, and SLC6A.

[0026] In a specific embodiment, the kit further includes a DNA extraction reagent, an RNA extraction reagent, a PCR reverse transcription reagent, a primer-probe mixture, a Taqman copy number variation detection premix reagent, a qRT-PCR detection premix reagent, a positive control product containing plasmids corresponding to the above 8 gene sequences, and RNase-free water.

[0027] In a specific embodiment, the 5' end of the gene expression detection probes and copy number detection probes is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0028] In this application, the DNA extraction reagent, RNA extraction reagent, PCR reverse transcription reagent, copy number variation detection premix reagent, and gene expression detection premix reagent are directly selected from existing commercially available products.

[0029] In a specific embodiment, the detection probe sequences of CDK1, ANKH, HEPHL1, SLC6A, SIX2, KLF9, PARP3, and PODXL2 include but are not limited to those shown below:

[0030]

[0031] The present invention also provides the application of high expression of SIX2, KLF9, PARP3, or PODXL2 and gene amplification of CDK1, ANKH, HEPHL1, or SLC6A as biomarkers of primary resistance to cetuximab in head and neck squamous cell carcinoma.

[0032] The present invention also provides the application of a kit containing one or more of the detection reagents for SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHL1, and SLC6A in the preparation of a prediction preparation for primary resistance to cetuximab in patients with head and neck squamous cell carcinoma.

[0033] Beneficial effects

[0034] The present invention conducts relevant research based on a mouse xenograft PDX model derived from a patient's tumor tissue. According to the test requirements, suitable PDX models can be enrolled to carry out clinical alternative tests, conduct PDX model pharmacodynamic tests and sample acquisition, and single-drug administration and sample collection according to the test requirements can be achieved. This greatly saves the time and economic costs of conducting clinical research and also saves the cycle of ethical approval during the drug collection process for a large number of clinical patients. Generally speaking, using the PDX model for clinical alternative tests to screen for pharmacodynamic markers is a simple and efficient screening method.

[0035] The biomarkers for primary resistance to cetuximab in head and neck squamous cell carcinoma provided by the present invention have consistent predictive effects in the PDX model discovery cohort and the PDX model validation cohort, and both show extremely significant high expression or copy number amplification. These molecules are very reliable markers for predicting the efficacy of cetuximab in head and neck squamous cell carcinoma patients, providing a new approach for clinical patient stratification treatment and precision medication.

[0036] The kit provided by the present invention makes the clinical prediction of primary resistance to cetuximab in head and neck squamous cell carcinoma patients simple, fast, and the prediction results are reliable. Description of the Drawings

[0037] Figure 1 : Distribution of the drug response of PDX models to cetuximab in the cetuximab clinical alternative test (PDX model discovery cohort) covering 49 PDX models provided in Example 1 of the present invention.

[0038] Figure 2 : Comparison of the PDX model and clinical efficacy of a cetuximab-sensitive patient provided in Example 1 of the present invention.

[0039] Figure 3 : Comparison of the PDX model and clinical efficacy of a cetuximab-insensitive patient provided in Example 1 of the present invention.

[0040] Figure 4 : Display of the sample gene variation in the PDX model discovery cohort provided in Example 2 of the present invention.

[0041] Figure 5 : Display of the sample gene differential expression in the PDX model discovery cohort provided in Example 2 of the present invention.

[0042] Figure 6 : Distribution of the drug response of PDX models to cetuximab in the cetuximab clinical alternative test (PDX model validation cohort) covering 61 PDX models provided in Example 3 of the present invention.

[0043] Figure 7:Demonstration of copy number variations of CDK1, ANKH, HEPHL1, and SLC6A in the validation cohort provided in Example 3 of the present invention.

[0044] Figure 8 :Illustration of the ability of the receiver operating characteristic curve (ROC) provided in Example 3 of the present invention to evaluate the ability of SIX2, KLF9, PARP3, and PODXL2 to distinguish between primary cetuximab-resistant and sensitive samples. Figure A shows the ability of SIX2, KLF9, PARP3, and PODXL2 to distinguish between the resistant group and the relatively sensitive group in the transcriptome sequencing results of the discovery cohort; Figure B shows the ability of SIX2, KLF9, PARP3, and PODXL2 to distinguish between the resistant group and the relatively sensitive group in the qRT-PCR results of the validation cohort. Detailed implementation manners

[0045] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited do not limit the present invention. Conditions and methods not specified in the embodiments are carried out according to the conventional or the conditions recommended by the manufacturer.

[0046] The mice used to construct the PDX model in the present invention are female BALB / c-nu nude mice, 6-8 weeks old, purchased from the Experimental Animal Business Department of the Shanghai Institute of Planned Parenthood Research (Animal License No.: SCXK (Shanghai) 2018-0006). The mice are kept in separate cages under SPF-level conditions. The room temperature is maintained at 18-25 °C, and the relative humidity is 40%-60%. Special mouse cages, bedding, feed, and drinking water are sterilized by high pressure at 121 °C for 30 minutes. The bedding is changed at least once a week.

[0047] Example 1. Construction of a head and neck squamous cell carcinoma PDX model cohort and evaluation of the efficacy of cetuximab

[0048] For the corresponding patients for constructing the PDX model of the present invention, their clinical information is recorded, including basic information (gender, age, smoking and drinking history, etc.), clinicopathological diagnosis (tumor size and location, TNM stage, HPV infection status), previous treatment history (surgery, radiotherapy and chemotherapy conditions), prognosis information such as recurrence and metastasis, etc. Tumor, adjacent cancer, and blood samples of the patients are collected. Pathological tissue morphology identification and genetic information identification are performed on the tumor samples.

[0049] After the tumor tissue is surgically removed, observe the tissue color, morphology, and texture, remove the necrotic tissue, and select the central part of the lesion for sampling. Since head and neck squamous cell carcinoma generally grows in contaminated parts such as the oral cavity and nasal mucosa, the samples need to be sterilized with 0.05% sodium hypochlorite and quickly washed with PBS containing 1% penicillin-streptomycin double antibody for 30 seconds before being inoculated and transplanted into mice. Gently scrape off the peripheral tissue of the tissue sample, and cut the tumor into 1-2 mm3 Small pieces of tissue are transplanted into the areas with rich blood supply and lymph nodes of immunodeficient mice (such as bilateral axillae) under sterile conditions to construct subcutaneous PDX models, or inoculated into the bilateral submandibular spaces of animals to construct orthotopic PDX models. To improve the inoculation success rate, Matrigel can be mixed with the patient's tumor tissue before inoculation, and 3 - 5 mice are inoculated with each patient's tissue. After 1 - 2 weeks of modeling, the growth trajectory of the PDX model is traced. When the tumor volume exceeds 800 mm 3 or the tumor volume shows no obvious increase for two weeks, the transplanted tumor is serially passaged. Generally, when the transplanted tumor is passaged more than 3 generations, the model is considered to be able to stably passage.

[0050] Based on the PDX model biobank of head and neck squamous cell carcinoma established by the present invention, according to the clinical information of the corresponding patients, 49 cases of head and neck squamous cell carcinoma PDX models are randomly selected for inclusion to construct a discovery cohort of PDX models. Each sample of the PDX transplanted tumor preserved in vivo is inoculated subcutaneously into nude mice (30 - 50 mm 3 ) for subsequent clinical alternative trials of cetuximab. In the first round of drug administration trials, the experimental group of PDX models is intraperitoneally injected with cetuximab ( Merck), at a dose of 10 mg / kg, twice a week; the control group is intraperitoneally injected with PBS, twice a week. The tumor volume and body weight of the models are continuously measured until 21 days or the tumor volume reaches 1000 - 1500 mm 3 . If adverse reactions occur in the mice within 14 days from the start of the experiment, they should be euthanized and removed from the group.

[0051] Evaluation criteria for the efficacy of cetuximab and screening of efficacy characteristic models: As shown in the following formula, the drug response (Response) is evaluated by the volume change of the transplanted tumor in the PDX model before and after drug administration:

[0052]

[0053] In the above formula, △Volt represents the change in tumor volume, V t represents the tumor volume on the t - th day of drug administration, and V initial represents the tumor volume on the 0 - th day of drug administration.

[0054] As follows, the tumor efficacy is evaluated according to △Volt:

[0055] 1. Modified complete remission (mCR): △Volt < - 40%;

[0056] 2. Modified partial remission (mPR): - 40% < △Volt < - 20%;

[0057] 3. Modified disease stability (mSD): - 20% < △Volt < 30%;

[0058] 4. Modified progression of disease (mPD): △Volt > 30%.

[0059] According to the above criteria, the mPD model was defined as a primary drug resistance model with the 21st day as the node; for other models, their tumor volume and body weight were continuously monitored until the 90th day; with the 90th day as the second node, the mCR model was defined as a sensitive model; models that relapsed within 90 days and did not meet the mCR criteria entered the acquired drug resistance study. When the tumor volume of the relapsed model reached 100 - 200 mm 3 , cetuximab was intraperitoneally injected at a dose of 10 mg / kg, twice a week. After 3 weeks of treatment, the drug response was evaluated, and the mPD model was defined as a secondary drug resistance model. Models that relapsed during the first round of treatment but showed mPR or mCR during the drug administration in the acquired drug resistance study were defined as having a reversible drug-tolerant persister (DTP) state. After the experiment ended, the mice were sacrificed and samples were retained for subsequent sequencing and verification work.

[0060] The results were as Figure 1 shown. In the pharmacodynamic experiment covering the discovery cohort of 49 PDX models, the progression of tumor disease (mPD) after drug administration was defined as primary drug resistance (n = 21), accounting for 42.86%; complete remission of the tumor (mCR) without relapse within 90 days was defined as sensitive (n = 9), accounting for 18.37%; tumor control (mCR + mPR + mSD) after drug administration and relapse within 90 days required re-administration, and the progression of tumor disease (mPD) after re-administration was defined as secondary drug resistance (n = 8), accounting for 16.33%; models with mPR or mCR of the tumor after re-administration were defined as having a reversible drug-tolerant persister state (n = 3), accounting for 6.12%; another 4 groups could not be differentiated pharmacodynamically due to large within-group differences, accounting for 8.16%; and 4 cases stopped drug administration due to various factors such as death in nude mouse experiments, poor nude mouse condition, and severe side effects, accounting for 8.16%. In this PDX model treatment cohort, the complete remission rate of 18.37% was consistent with the response rate of 13% in the clinical application of cetuximab, indicating that this PDX model treatment cohort could accurately simulate clinical treatment and had extremely high value for studying drug resistance mechanisms and exploring intervention strategies.

[0061] In the present invention, the pharmacodynamic effect of the PDX model on cetuximab was consistent with that of cetuximab in clinical use by patients. As Figure 2 shown, for a patient sensitive to cetuximab, the corresponding PDX model was also sensitive to cetuximab; as Figure 3 shown, for a patient relatively insensitive to cetuximab, the corresponding PDX model was also insensitive to cetuximab.

[0062] Example 2. Screening of primary resistance biomarkers for cetuximab

[0063] All samples collected in the discovery cohort of the PDX model of the present invention were obtained from mice, snap-frozen in liquid nitrogen, and sent to a sequencing company for whole-exome sequencing and transcriptome sequencing.

[0064] Whole-exome sequencing: Whole-exome sequencing of the snap-frozen patient tumors and PDX samples was performed using the Illumina Novaseq6000 platform. The sequencing depth of the patient tumor samples was 200X, and that of the PDX samples was 100X. For PDX samples, murine genes needed to be removed. The human and mouse hybrid genomes (hs37d5 and mm10) were mapped to the sequencing data through the Burrows-Wheeler Aligner (BWA), and human-derived mutations were filtered using SAMtools, Genome Analysis Toolkit (GATK-UnifiedGenotyper), and FreeBayes (Garrison and Marth).

[0065] RNA sequencing: RNA libraries were constructed using the Illumina Hiseq platform. The sequencing reads were mapped to the human and mouse genomes through Hisat2 v2.0.5, and transcripts were assembled using Cufflinks v2.2.1 to estimate the abundance of transcripts and perform differential expression analysis.

[0066] The present invention obtained a series of potential drug sensitivity-related biomarkers by analyzing the mutated genes, gene copy numbers, and expression differences of the PDX samples in the sensitive group and the primary resistance group. The sensitive group and the primary resistance group were screened out, and gene mutations and copy number variations with significant differences between the two groups (P<0.05) were shown in Figure 4 Moreover, gene variations with significant differences between the sensitive group and the primary resistance group could be found. For example, the copy number amplification of CDK1 only occurred in the resistant PDX, suggesting that it might be a potential drug efficacy biomarker. In addition Figure 5 showed the gene expression profiles with significant differences between the sensitive model and the resistant model. Some genes were highly expressed in the resistant model compared to the sensitive model, such as MAPK15, SIX2, and PTPN18, which might be predictive biomarkers for primary resistance to cetuximab.

[0067] Example 3. Verification of primary resistance biomarkers for cetuximab

[0068] In order to verify the sensitivity and specificity of the drug efficacy biomarkers related to cetuximab resistance screened in the discovery cohort of the PDX model, the present invention further carried out an independent PDX cohort for cross-validation of the biomarkers.

[0069] According to the aforementioned PDX model inoculation and passage methods, the present invention constructs multiple PDX models with stable passage and complete histological evaluation and informatics identification, enrolls 61 independent PDX models as the PDX model validation cohort, and conducts a clinical substitution trial of cetuximab.

[0070] Among the PDX model validation cohort constructed by the present invention, 28 PDXs showed disease progression (mPD), defined as primary resistant samples, accounting for 45.90%; 6 cases had a complete response (mCR, 9.84%) and 12 cases (22.95%) showed a partial response (mPR), defined as relatively sensitive samples; 13 cases showed stable disease (mSD), accounting for 21.31% ( Figure 6 ).

[0071] The present invention collects pre-dose samples from the PDX model validation cohort to detect potential biomarkers by qRT-PCR. The results show that the amplification of CDK1, ANKH, HEPHL1, and SLC6A genes can be used as drug-sensitive biomarkers for cetuximab ( Figure 7 ).

[0072] Subsequently, the present invention selects potential biomarkers with differential expression in the PDX model discovery cohort for qRT-PCR detection, and uses the Receiver Operating Characteristic Curve (ROC) to judge the sensitivity and specificity of each biomarker to evaluate its clinical significance. The results show that the transcriptome sequencing of the discovery cohort ( Figure 8 A) or the qRT-PCR of the validation cohort ( Figure 8 B) both identify that the high expression of SIX2, KLF9, PARP3, and PODXL2 can better distinguish the resistant group and the sensitive group (AUC>0.80) ( Figure 8 ).

[0073] Example 4. Composition and usage method of a kit for detecting primary resistance to cetuximab in head and neck squamous cell carcinoma

[0074] A kit for detecting primary resistance to cetuximab in head and neck squamous cell carcinoma, which consists of a DNA extraction reagent, an RNA extraction reagent, a PCR reverse transcription reagent, a primer-probe mixture, a Taqman copy number variation detection premix reagent, a qRT-PCR detection premix reagent, a positive control product containing plasmids with the corresponding 8 gene sequences, and RNase-free water.

[0075] Among them, the DNA extraction reagent (Thermofisher, 10503027), RNA extraction reagent (Thermofisher, 12183555), PCR reverse transcription reagent (Takara, 639505), copy number variation detection premix reagent (Thermofisher, A30866) and gene expression detection premix reagent (Thermofisher, 4444556) are directly selected from existing commercially available products.

[0076] The detection probe sequences of SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHL1 and SLC6A can be designed according to the gene sequences. For example, as shown in Table 1, the 5' end of the probe sequence needs to be labeled with a fluorescent group, and the 3' end needs to be labeled with a quenching group to be suitable for TaqMan probe-based copy number variation detection and qRT-PCR method detection.

[0077] Table 1 Probe sequences

[0078]

[0079] Operation and result determination of the kit:

[0080] (1) Add about 10 - 50 ng of different sample DNA (DNA extracted from head and neck squamous cell carcinoma tumor tissue and adjacent tissue) or cDNA (RNA extracted from head and neck squamous cell carcinoma tumor tissue and adjacent tissue, reverse transcribed into cDNA) to each PCR reaction tube, and add 10 μL of qRT-PCR premix reagent and 1 μL of each forward and reverse primer probe to the tube, and make up to 20 μL with ultrapure water to prepare the system. Cover the tube cap and put it into a fluorescence quantitative PCR instrument for fluorescence PCR detection. At the same time, set up a blank control without sample DNA or RNA, and a positive control containing plasmids with the corresponding 8 amplified gene sequences mentioned above.

[0081] (2) Set the conditions for the PCR amplification reaction in the instrument as follows:

[0082] Copy number amplification program: Pre-denaturation / DNA polymerase activation, 95 °C, 10 min; Denaturation, 95 °C, 15 s; Annealing / extension, 60 °C, 60 s; 40 cycles.

[0083] Gene expression program: UNG enzyme incubation, 50 °C, 2 min; DNA polymerase activation, 95 °C, 2 min; Denaturation, 95 °C, 3 s; Annealing / extension, 60 °C, 20 s; 40 cycles.

[0084] (3) After the reaction is completed, the baseline is set to be automatically adjusted, and the detection results are analyzed according to the amplification curve graph and Ct value.

[0085] (4) Validity determination:

[0086] If there is a signal rise in the Ct value detected by RNase-free water and the Ct value < 35, the experimental result is invalid, and it is recommended to repeat the experiment; if the Ct value of the positive control plasmid > 28, the experimental result is invalid, and it is recommended to repeat the experiment.

[0087] (5) Copy number and gene expression calculation:

[0088] Use the plasmid corresponding to the target gene as a standard product for gradient dilution, and add standard product plasmids with five concentration gradients to the PCR reaction for PCR reaction.

[0089] Take the logarithm of the copy number of the standard product as the abscissa and the measured Ct value as the ordinate to draw a standard curve. When quantifying an unknown sample, according to the Ct value of the unknown sample, the copy number of the sample can be obtained from the standard curve. Log (initial concentration) has a linear relationship with the number of cycles. A standard curve can be made through a standard product with a known initial copy number, that is, the linear relationship existing in this amplification reaction is obtained. From the Ct value of the sample, the amount of template contained in the sample can be calculated.

[0090] According to the standard curve, using the Ct value measured from the sample, calculate the corresponding gene copy number or gene absolute expression value in the tumor tissue and adjacent tissue of the cancer. Compare the copy number of the target gene in the tumor tissue and adjacent tissue of each sample. If the copy number of the tumor tissue is greater than that of the adjacent tissue, it is defined as a sample with amplified copy number of the target gene, indicating that the patient may be initially resistant to cetuximab. Compare the absolute expression level of the target gene in the tumor tissue and adjacent tissue of each sample. If the expression level of the tumor tissue is greater than that of the adjacent tissue and the expression level of the target gene is significantly higher than the average level, it is defined as a sample with high expression of the target gene, indicating that the patient may be initially resistant to cetuximab.

[0091] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims. Sequence Listing <110> Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital <120> A method for screening biomarkers for primary resistance to cetuximab, biomarkers screened by this method and their uses <130> DI22-0003-XC03 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> CDK1 forward probe <400> 1 tgtctcctga tgggcaaagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> CDK1 reverse probe <400> 2 acattgaggc tcgaagggaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ANKH forward probe <400> 3 agaactagac gaggcttgcg 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ANKH reverse probe <400> 4 tggcacaaga caaacccgat 20 <210> 5 <211> 21 <212> DNA <213> Artificial sequence <220> <223> HEPHL1 forward probe <400> 5 atttctcgaa agagggccca a 21 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> HEPHL1 reverse probe <400> 6 gttccatccg tgaagcgtct 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SLC6A3 forward probe <400> 7 gtttacacct ttccgtgccg 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SLC6A3 reverse probe <400> 8 tcgctgcaca gatctacgtc 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PODXL2 forward probe <400> 9 cctctgctac cttgggacaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PODXL2 reverse probe <400> 10 cagattgctc cagtccttgc 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SIX2 forward probe <400> 11 ttccgcgagc tctacaagat 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SIX2 reverse probe <400> 12 cttctccgcc tcgatgtagt 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PARP3 forward probe <400> 13 gcatcatgcc acattctggt 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PARP3 reverse probe <400> 14 ccagctgact tgctgttctc 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> KLF9 forward probe <400> 15 tcagtgttcg aggctgtagg 20 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> KLF9 reverse probe <400> 16 aggtgcgtct agaactgagg 20

Claims

1. A method for screening primary resistant biomarkers of cetuximab in head and neck squamous cell carcinoma using a patient-derived xenograft model (PDX model) of tumor tissue for clinical alternative trials. The method comprises the following steps: (1) Construct a head and neck squamous cell carcinoma PDX model in immunodeficient mice using tumor tissue from head and neck squamous cell carcinoma patients; (2) Obtain the clinical information of the patients corresponding to the head and neck squamous cell carcinoma PDX model, including past medical history, clinical treatment situation, and medication situation; (3) Construction of the first cohort of head and neck squamous cell carcinoma PDX models: Relying on the established biobank of head and neck squamous cell carcinoma PDX models, according to the clinical information of the corresponding patients, randomly screen a number of head and neck squamous cell carcinoma PDX models for inclusion. Inoculate each cryopreserved PDX xenograft tumor sample subcutaneously into nude mice for subsequent clinical alternative trials of cetuximab; (4) Conduct a clinical alternative trial of cetuximab in the PDX model: After the tumor volume of the head and neck squamous cell carcinoma PDX model reaches a certain value, randomly divide each PDX case into a control group and a cetuximab monotherapy group, start drug treatment, and the PDX models receive intraperitoneal injection of PBS or cetuximab respectively. Measure the tumor volume and body weight of the mice weekly; (5) Evaluation of the efficacy of cetuximab in the clinical alternative trial of the PDX model: To evaluate the drug response to cetuximab, evaluate the drug reaction through the volume change of the xenograft tumor before and after drug treatment in the PDX model. The specific criteria are as follows: 1) Complete tumor regression (mCR), the tumor volume is reduced by at least 40%; 2) Partial tumor regression (mPR), the tumor volume is reduced by 20% - 40%; 3) Disease progression (mPD), the tumor volume is increased by at least 30%; 4) Disease stability (mSD), the tumor volume change is between a 30% increase and a 20% decrease; The mPD model is determined as the primary resistant model, and the mCR model is defined as the sensitive model; (6) Screen primary resistant candidate efficacy biomarkers by means of the model clinical alternative trial: Perform whole exome sequencing and transcriptome sequencing on the primary resistant model and the sensitive model respectively. According to the differential gene mutation spectrum and / or differential gene expression spectrum between the primary resistant model and the sensitive model, combined with the results of gene function and signaling pathway analysis, preliminarily screen out primary resistant candidate efficacy biomarkers; (7) Conduct an independent clinical alternative trial of the PDX model: Relying on the biobank of head and neck squamous cell carcinoma PDX models, according to the clinical information of the corresponding patients, randomly screen a number of head and neck squamous cell carcinoma PDX models again as the second cohort for inclusion. The selected second cohort does not overlap with the first cohort. In the same way as in step (4), perform cetuximab monotherapy on the included PDX models. After several weeks of medication, for example, 3 weeks later, collect samples of the xenograft tumors and cryopreserve them alive. By evaluating the efficacy of cetuximab, distinguish the primary resistant model and the sensitive model; (8)Verifying pharmacodynamic markers using independent PDX model clinical surrogate trials: Using samples from independent PDX model clinical surrogate trials, candidate variant genes and expressed genes were verified by real-time fluorescence quantitative PCR (qRT-PCR). Genes with consistent gene variations or expression trends in the first and second cohorts were screened out. The selected pharmacodynamic predictive biomarkers were plotted on a Receiver Operating Characteristic Curve (ROC), analyzed using SPSS 21.0 statistical software, and the area under the curve AUC was calculated. An index with AUC > 0.80 was selected, and finally, the primary resistance biomarkers of cetuximab in head and neck squamous cell carcinoma found in the first and second cohorts were determined.

2. The method according to claim 1, wherein, the primary resistance biomarkers of cetuximab in head and neck squamous cell carcinoma screened by the method are selected from the following: transcription factor SIX2 (Sine Oculis Homeobox Homolog 2), basic transcription element binding protein KLF9 (Kruppel Like Factor 9), poly(ADP-ribose) polymerase PARP3 (Poly(ADP-Ribose)Polymerase Family Member 3), member PODXL2 (Podocalyxin Like2) of the cell surface transmembrane protein CD34 family, cyclin-dependent kinase CDK1 (Cyclin Dependent Kinase 1), inorganic pyrophosphate transport regulator ANKH (ANKH Inorganic Pyrophosphate Transport Regulator), hephaestin-like protein HEPHL1 (Hephaestin Like 1), and neurotransmitter transporter SLC6A (Solute carrier family 6).

3. Use of biomarkers SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHL1 or SLC6A in the preparation of a reagent for predicting the efficacy of clinical application of cetuximab in patients with head and neck squamous cell carcinoma.

4. Use of a kit in the preparation of a reagent for predicting primary resistance of cetuximab in patients with head and neck squamous cell carcinoma, wherein, the kit contains one or more of the detection reagents for SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHL1, SLC6A.

5. The use according to claim 4, wherein, The kit at least includes gene expression detection probes and / or copy number detection probes. Among them, the gene expression detection probes include gene expression detection probes for one or more of SIX2, KLF9, PARP3, and PODXL2, and the copy number detection probes include copy number detection probes for one or more of CDK1, ANKH, HEPHL1, and SLC6A.

6. The application according to claim 5, wherein, the kit further includes tissue DNA extraction reagents, PCR reverse transcription reagents, primer-probe mixtures, Taqman copy number variation detection premixed reagents, qRT-PCR detection premixed reagents, positive control products, and RNase-free water.

7. The application according to claim 5, wherein, the 5' ends of the gene expression detection probes and the copy number detection probes are labeled with fluorescent groups, and the 3' ends are labeled with quenching groups.

8. The application according to claim 5, wherein, the detection probe sequences of CDK1, ANKH, HEPHL1, SLC6A, SIX2, KLF9, PARP3, and PODXL2 are:

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