CN114354936A - Method for screening cetuximab drug resistance biomarkers, biomarkers screened by method and application of biomarkers - Google Patents

Method for screening cetuximab drug resistance biomarkers, biomarkers screened by method and application of biomarkers Download PDF

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CN114354936A
CN114354936A CN202210032285.1A CN202210032285A CN114354936A CN 114354936 A CN114354936 A CN 114354936A CN 202210032285 A CN202210032285 A CN 202210032285A CN 114354936 A CN114354936 A CN 114354936A
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cetuximab
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cell carcinoma
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孙树洋
张志愿
姚艳丽
杨桂柱
王玉珏
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Abstract

The application relates to a method for screening a cetuximab single antigen-induced drug resistance biomarker for head and neck squamous cell carcinoma, a biomarker screened by the method and application of the biomarker. According to the method, accurate clinical information of a patient is obtained by establishing a mouse xenograft tumor model (PDX) derived from tumor tissues of the patient with the head and neck squamous cell carcinoma, and a PDX model platform of the head and neck squamous cell carcinoma is constructed. And in the constructed PDX model platform, referring to a clinical second-stage research mode, selecting a PDX model corresponding to a patient into a group to carry out a PDX model clinical substitution test, carrying out cetuximab drug treatment, and screening a primary drug-resistant biomarker of the cetuximab by means of whole exon sequencing and transcriptome sequencing. Experiments prove that the head and neck squamous cell carcinoma cetuximab antigen-induced drug resistance biomarker provided by the application is a marker for predicting the drug effect of head and neck squamous cell carcinoma patients on cetuximab with high reliability.

Description

Method for screening cetuximab drug resistance biomarkers, biomarkers screened by method and application of biomarkers
Technical Field
The application belongs to the field of biomedicine, and particularly relates to a method for screening a head and neck squamous cell carcinoma cetuximab single antigen-induced drug resistance biomarker, a biomarker screened by the method and application of the biomarker.
Background
Head and neck cancer is a high malignant tumor of cancer spectrum in China and even the world, and is one of the most serious cancers affecting the life health and the quality of life of people. According to global cancer statistics, there are about more than 55 ten thousand new head and neck cancers worldwide each year, the global incidence rate is third of the systemic tumor, and the mortality rate is sixth. Of the head and neck cancers, more than 90% are head and neck squamous cell carcinomas, abbreviated as head and neck squamous cell carcinomas. The head and neck are complex in anatomy, and various organs are dense and are mostly important organs. The occurrence of head and neck squamous carcinoma therefore causes great disruption not only to the patient's appearance and basic physiological functions (chewing, swallowing, breathing, etc.), sensory functions (taste, smell, hearing), and speech functions, but also seriously affects the patient's quality of life. Therefore, how to protect the basic physiological functions of organs while killing tumor cells in the multi-organ dense head and neck, and to maintain the quality of life of patients to the greatest extent is a great problem facing both clinicians and researchers.
The head and neck squamous cell carcinoma has numerous primary parts and various pathological types, the complexity of the head and neck squamous cell carcinoma is the first of tumors of the whole body, and the head and neck squamous cell carcinoma seriously threatens the life and health of people. Although medical appliances and treatment technologies are rapidly advanced in recent years, the five-year survival rate of the patients with the squamous cell carcinoma of head and neck in China is not obviously improved. At present, the operation treatment is still the only radical treatment strategy of the head and neck squamous cell carcinoma, and in order to completely remove tumor cells as far as possible, enlarged removal is needed, but in view of the special anatomical position of the head and neck, the radical removal of the tumor seriously affects the life quality of patients, and causes heavy burden of family society. Furthermore, the conventional treatment regimen for head and neck squamous cell carcinoma is dominated by chemoradiotherapy, however, several clinical studies have shown that only a small percentage of patients benefit from chemoradiotherapy, and that more than 65% of head and neck squamous cell carcinoma patients experience relapse and/or metastasis, with the survival rate for most relapsed and/or metastatic head and neck squamous cell carcinoma patients being less than one year.
The epidermal growth factor receptor EGFR is highly expressed in more than 90% of patients with head and neck squamous cell carcinoma, and the inhibition of the EGFR is the only targeted treatment strategy for the head and neck squamous cell carcinoma. Cetuximab is a monoclonal antibody that blocks the chimeric IgG1 of EGFR and is suggested as the only targeted drug for first-line treatment of head and neck squamous cell carcinoma as recommended by NCCN clinical guidelines for the treatment of locally advanced head and neck squamous cell carcinoma in combination with radiotherapy or for the treatment of relapsed and/or metastatic head and neck squamous cell carcinoma in combination with platinum-based chemotherapy. The action mechanism of cetuximab involves competition with endogenous EGFR ligands for binding to accessible extracellular domains, followed by blocking receptor-dependent signal transduction pathways, thereby exerting effects of inhibiting tumor cell growth, inhibiting angiogenesis, inhibiting tumor metastasis, and the like.
Although cetuximab combination treatment improves the clinical prognosis of head and neck squamous cell carcinoma, primary resistance and secondary resistance generated during the course of drug administration increase the tumor recurrence rate and limit the clinical efficacy of cetuximab. It is noted that the therapeutic effect of cetuximab in the treatment of head and neck squamous cell carcinoma is low, the objective remission rate of the single-drug treatment group is 13%, and the objective remission rate of the combined chemotherapy group is 36%. Despite sustained maintenance of cetuximab therapy, patients with head and neck squamous cell carcinoma who use the exteme treatment regimen also spend only around 5 months from initial use until failure of therapy. More importantly, the literature reports that approximately only 10-20% of patients with advanced cancer achieve tumor growth inhibition after blocking the EGFR pathway with drugs, and most patients who initially respond to cetuximab will eventually also exhibit secondary resistance.
The active search for the target drug efficacy biomarker is always a hotspot of tumor research, and is the key to realizing accurate cancer treatment. Through the research of large-scale crowd queues and various novel omics technologies in recent years, more and more researches have proved that: tumor heterogeneity is the main reason for the obvious difference in curative effect and drug resistance in the process of tumor treatment. Tumor heterogeneity includes intratumoral and intratumoral heterogeneity, where the intratumoral heterogeneity is primarily manifested as primary drug resistance and sensitivity to drugs between patients at varying levels in the human population. On this basis, in recent years, a great deal of research has been conducted around the primary drug resistance of cetuximab; however, clinical medication indications of cetuximab are still very poor in most tumors including head and neck squamous cell carcinoma, and it is only clear in colorectal cancer that KRAS gene mutations can mediate primary drug resistance of cetuximab.
Among colorectal cancers, 1022 tumor samples treated with cetuximab combined chemotherapy from 2001 to 2008 were collected by 11 centers from 7 european countries and examined for KRAS, BRAF, NRAS and PIK3CA gene variation. The results show that KRAS mutant patients are less effective on cetuximab (6.7% vs 35.8%) and have a shorter median progression-free survival (12 weeks vs 24 weeks) than wild-type patients. Furthermore, prospective clinical studies in multiple colorectal cancers indicate that cetuximab in combination with radiation therapy only benefited KRAS wild-type colorectal cancer patients, while there was no significant clinical benefit in KRAS mutant patients. Therefore, the KRAS gene state detection is helpful for determining the curative effect of cetuximab in colorectal cancer patients, KRAS mutation is the first biomarker capable of predicting the drug resistance of the targeted therapeutic drug cetuximab in the colorectal cancer patients, and theoretical basis is provided for determining individualized treatment schemes. Updating the colorectal cancer guideline by NCCN in 2009 suggested that first line therapy of KRAS wild-type advanced metastatic colorectal cancer patients chose cetuximab in combination with chemotherapy.
Cetuximab recommends indications other than metastatic colorectal cancer, primarily first-line treatment of recurrent/metastatic squamous cell carcinoma of the head and neck. However, compared with the higher mutation frequency of KRAS in colorectal cancer (more than 40% in Chinese population), the mutation frequency of KRAS in head and neck squamous cell carcinoma is extremely low (less than 2% in Chinese population), so 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 studies, there is no temporary large-scale cohort studies focusing on primary drug resistance of cetuximab, revealing pharmacodynamic biomarkers of cetuximab. In 2017, Friederike et al screened 59 patients with palliative head and neck squamous cell carcinoma between 2010 and 2016, and found that cetuximab resistance was associated with EGFR-K521 polymorphisms. In 2020 Nellie et al demonstrated by in vitro and in vivo studies that AXL tyrosine residue Y821 mediates resistance to cetuximab by activating head and neck squamous cell carcinoma c-ABL kinase. In 2020, tumor specimens from 118 patients with head and neck squamous carcinoma treated with cetuximab between 2006 and 2015 were examined for mutations in PIK3CA and H/N/KRAS genes and for PTEN and EGFR immunohistochemistry by a french curie institute. The results show that the therapeutic effect of cetuximab in combination with chemotherapy is independent of the mutations in PIK3CA and KRAS/HRAS genes in head and neck squamous cell carcinoma patients, and that high expression of epidermal growth factor receptor EGFR does not predict the sensitivity of cetuximab. In view of this, to date, none of the predictive biomarkers in head and neck squamous cell carcinoma are capable of guiding patients for cetuximab therapy.
In head and neck squamous cell carcinoma, the primary drug-resistant biomarker studies of cetuximab that have been carried out at present have mainly used patient tumor tissues for retrospective studies. This retrospective study focused on several analyses of genes that have been confirmed in colorectal cancer and associated with cetuximab resistance, and further evaluated whether the molecule could be used as a drug efficacy prediction marker in head and neck squamous cell carcinoma by detecting mutations or expression levels of the target molecule. However, research for many years proves that the cetuximab antigen-induced drug resistance biomarker is difficult to find in head and neck squamous cell carcinoma in the mode. On one hand, due to the difference of pathogenesis and genetic background of different tumors, genes with high frequency variation in colorectal cancer are likely not to have obvious variation in head and neck squamous cell carcinoma, and genes with obvious high expression in the colorectal cancer are likely not to have variation or expression in the head and neck squamous cell carcinoma; on the other hand, focusing on the change of individual genes, neglecting the mutation and gene expression of the whole tumor level, also causes the low screening efficiency of the primary drug resistance prediction marker, and is difficult to find the accurate drug effect marker; in addition, clinical samples are mostly used for retrospective analysis in these studies, but due to ethical limitations, cetuximab rarely treats patients with a single drug, and is often combined with multiple chemotherapeutic drugs and radiotherapy, and patients may undergo multiple other drug treatments before cetuximab treatment, and primary drug resistance markers analyzed based on these tumor samples are often insufficient in specificity and accuracy and are difficult to apply in clinical practice.
At present, no stable biomarker report for the cetuximab single antigen-induced drug resistance of the head and neck squamous cell carcinoma, which is excavated and verified by means of a clinical alternative test of a xenograft tumor model, exists, and if abnormal variation or expression genes in the cetuximab single antigen-induced drug resistance can be screened out to be used as biomarkers and corresponding detection kits are developed, the method can be used for effectively promoting the layered treatment and accurate medication of head and neck squamous cell carcinoma patients in China.
Disclosure of Invention
In view of this, the invention takes tumor heterogeneity as an entry point, takes standardized sample materials in a clinical research mode, obtains accurate clinical information of a patient by establishing a patient tumor tissue-derived xenograft tumor model (PDX), and constructs a head and neck squamous cell carcinoma PDX model platform. In a PDX model platform, referring to a clinical second-stage research mode, selecting a PDX model corresponding to a patient into a group to carry out a PDX model clinical substitution test, carrying out cetuximab drug treatment, and mining and verifying a primary drug-resistant biomarker of cetuximab by means of whole exon sequencing and transcriptome sequencing.
In order to solve the following defects of the prior art, namely, cetuximab single-drug therapy cannot be carried out in head and neck squamous cell carcinoma so as to discover a cetuximab primary drug-resistance biomarker; when histological examination is carried out before administration, pain is caused to patients; cetuximab potency cannot be predicted early without whole gene and transcriptome sequencing. The invention provides a method for screening a cetuximab single antigen-induced drug resistance biomarker of head and neck squamous cell carcinoma by using a PDX model clinical alternative test. Based on the method, the invention provides 8 biomarkers of the drug resistance of the cetuximab of the head and neck squamous cell carcinoma.
Therefore, in one aspect, the present invention provides a method for screening cetuximab single antigen-induced drug resistance biomarkers for head and neck squamous cell carcinoma by using a patient tumor tissue-derived xenograft tumor model (abbreviated as PDX model) clinical alternative test, comprising the following steps:
(1) constructing a head and neck squamous cell carcinoma PDX model in an immunodeficiency mouse by using tumor tissues of the head and neck squamous cell carcinoma patient;
(2) acquiring clinical information of a patient corresponding to the PDX model of the head and neck squamous cell carcinoma, wherein the clinical information comprises the past medical history, the clinical treatment condition and the medication condition;
(3) construction of a first cohort of head and neck squamous cell carcinoma PDX models (hereinafter also referred to as PDX model discovery cohort): based on a head and neck squamous cell carcinoma PDX model biological sample library established, randomly screening a plurality of head and neck squamous cell carcinoma PDX models into groups according to clinical information of corresponding patients, and inoculating PDX transplantation tumor samples preserved in each living body to the subcutaneous part of a nude mouse for subsequent clinical substitution tests of cetuximab;
(4) development of cetuximab PDX model clinical alternatives: when the tumor volume of the PDX model reaches a certain value, for example, 100-3Then, randomly dividing each PDX case into a control group and a cetuximab single-drug treatment group, starting drug treatment, carrying out intraperitoneal injection on the PDX model by PBS or cetuximab respectively, and measuring the tumor volume and the weight of the mouse every week;
(5) evaluating the drug effect of cetuximab in a PDX model clinical alternative test: to evaluate drug response to cetuximab, drug response was evaluated by volume change before and after drug administration of the transplanted tumor in the PDX model, with the following specific criteria: 1) complete tumor regression (mCR), at least 40% reduction in tumor volume; 2) partial regression of tumor (mPR), tumor volume reduction 20% -40%; 3) disease progression (mPD), at least 30% increase in tumor volume; 4) disease stabilization (mSD), tumor volume change between 30% increase and 20% decrease; the mPD model is determined as a primary drug resistance model; defining the mCR model as a sensitive model;
(6) screening primary drug resistance candidate pharmacodynamic markers by means of model clinical alternative tests: respectively carrying out whole exome sequencing and transcriptome sequencing on the primary drug resistance model and the sensitive model, and preliminarily screening out primary drug resistance candidate pharmacodynamic markers according to a difference gene variation spectrum and/or a difference gene expression spectrum of the primary drug resistance model and the sensitive model and combining gene functions and a signal channel analysis result;
(7) clinical alternative trials of independent PDX models (hereinafter, also referred to as PDX model validation cohorts) were performed: depending on a biological sample library of the head and neck squamous cell carcinoma PDX model, randomly screening a plurality of cases of head and neck squamous cell carcinoma PDX models as a second queue for grouping according to clinical information of corresponding patients, wherein the selected second queue is not overlapped with the first queue, carrying out cetuximab single-drug treatment on the grouped PDX model in the same way as in the step (4), carrying out sample collection and living body preservation on transplanted tumors after taking the drugs for a plurality of weeks, for example, 3 weeks, and distinguishing an original drug-resistant model and a sensitive model by evaluating the drug effect of the cetuximab;
(8) the pharmacodynamic markers were verified using an independent PDX model clinical surrogate test: the method comprises the steps of utilizing a sample in an independent PDX model clinical substitution test, adopting a real-time fluorescent quantitative PCR (qRT-PCR) method to verify candidate variation genes and expression genes, screening out genes with gene variation or expression trends consistent in a first queue and a second queue, drawing a Receiver Operating Characteristic Curve (ROC) for the selected pharmacodynamic predictive biomarkers, adopting SPSS 21.0 statistical software to analyze, calculating an area under the Curve, selecting an index with AUC >0.80, and finally determining the head and neck cancer cetuximab single antigen development drug resistance biomarkers found in the first queue and the second queue.
In specific embodiments, the cetuximab primary antigen-resistance biomarkers of head and neck squamous cell carcinoma screened by the method are selected from the following: transcription factors SIX2(Sine accumis Homeobox Homolog 2), basic transcription element binding protein KLF9(Kruppel Like Factor 9), Poly (adenosine diphosphate Ribose) PARP3(Poly (ADP-Ribose) Polymerase Family 3), Member of the cell surface transmembrane protein CD34 Family PODXL2(Podocalyxin Li2), cell cycle Dependent Kinase CDK1(Cyclin Dependent Kinase 1), Inorganic Pyrophosphate Transport regulator ANKH (ANKH Inorganic pyrophor transporter regulator), membrane iron Transport accessory protein HEPHHL 1 (Hephastin Like 1), and neurotransmitter SLC6A (solvent carrier 6).
In the present application, the high expression events of transcription factors SIX2(Sine accumlis Homeobox Homolog 2), the basic transcription element binding protein KLF9(Kruppel Like Factor 9), Poly-adenosine diphosphate Ribose Polymerase PARP3(Poly (ADP-Ribose) Polymerase Family 3) and the Member PODXL2(Podocalyxin Like2) of the cell surface transmembrane protein CD34 Family, as well as the cell cycle-Dependent kinases CDK1(Cyclin Dependent Kinase 1), the Inorganic Pyrophosphate Transport regulator ANKH Inorganic phosphate transporter regulator, the membrane iron Transport accessory protein HEPHHL 1(Hephaestin Like SLC 1) and the neurotransmitter transporter 6A (Solute car Family6) can serve as markers for the resistance to western drugs.
The invention also provides application of the biomarkers SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHHL 1 or SLC6A in preparation of a clinical cetuximab curative effect prediction reagent for patients with head and neck squamous cell carcinoma.
The invention further provides a cetuximab primary drug resistance biomarker efficacy prediction kit, which at least comprises a gene expression detection probe and/or a copy number detection probe, wherein the gene expression detection probe comprises a gene expression detection probe for one or more of SIX2, KLF9, PARP3, PODXL2, and the copy number detection probe comprises a copy number detection probe for one or more of CDK1, ANKH, HEPHL1, SLC 6A.
In a specific implementation mode, the kit further comprises a DNA extraction reagent, an RNA extraction reagent, a PCR reverse transcription reagent, a primer probe mixed solution, a Taqman copy number variation detection premixed reagent, a qRT-PCR detection premixed reagent, a plasmid containing the corresponding 8 gene sequences in a positive quality control product and RNase-free water.
In specific embodiments, the gene expression detection probe and the copy number detection probe are labeled with a fluorophore at the 5 'end and a quencher at the 3' end.
In the present 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 commercially available products.
In particular embodiments, CDK1, ANKH, HEPHL1, SLC6A, SIX2, KLF9, PARP3, PODXL2 detection probe sequences include, but are not limited to, the following:
Figure BDA0003466994710000071
the invention also provides application of SIX2, KLF9, PARP3 or PODXL2 with high expression and CDK1, ANKH, HEPHHL 1 or SLC6A gene amplification as a cetuximab drug-resistant marker in head and neck squamous cell carcinoma.
The invention also provides application of a kit containing one or more of SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHHL 1 and SLC6A detection reagents in preparation of a prediction preparation for cetuximab-induced drug resistance of patients with head and neck squamous cell carcinoma.
Advantageous effects
The invention carries out related research based on the mouse transplanted tumor PDX model derived from the tumor tissue of the patient, can be combined into a proper PDX model according to the test requirements, carries out clinical substitution tests, carries out the drug effect test of the PDX model and obtains samples, and can realize single drug administration and material obtaining according to the test requirements. Greatly saves the time and economic cost for developing clinical research and the period of ethical examination and approval in the process of taking medicine materials of a large number of clinical patients. Generally, the method for screening the pharmacodynamic markers by using the PDX model clinical alternative test is a simple and efficient screening method.
The head and neck squamous cell carcinoma cetuximab single antigen drug resistance biomarker provided by the invention has consistent prediction effect in a PDX model discovery queue and a PDX model verification queue, and both show abnormal significant high expression or copy number amplification. The molecules are very reliable markers for predicting the drug effect of the patient with head and neck squamous cell carcinoma on cetuximab, and a new way is provided for layered treatment and accurate medication of clinical patients.
The kit provided by the invention enables clinical prediction of drug resistance of head and neck squamous carcinoma patients to cetuximab antigen to be simple, convenient and quick, and the prediction result is reliable.
Drawings
FIG. 1: the drug response condition distribution of the PDX model to cetuximab in the cetuximab clinical alternative test (PDX model discovery cohort) covering 49 PDX models provided in embodiment 1 of the present invention.
FIG. 2: the PDX model for 1 cetuximab-sensitive patient provided in example 1 of the present invention was compared to clinical efficacy.
FIG. 3: the PDX model for 1 cetuximab insensitive patient provided in example 1 of the present invention was compared to clinical efficacy.
FIG. 4: the PDX model provided in embodiment 2 of the present invention shows the genetic variation of the samples in the queue.
FIG. 5: the PDX model provided by the embodiment 2 of the invention shows the sample gene differential expression in the queue.
FIG. 6: the drug response condition distribution of the PDX model to cetuximab in the cetuximab clinical alternative test (PDX model validation cohort) covering 61 PDX models provided in embodiment 3 of the present invention.
FIG. 7: the copy number variation conditions of CDK1, ANKH, HEPHHL 1 and SLC6A in the verification queue provided by the embodiment 3 of the invention are shown.
FIG. 8: graphical representation of the ability of the receiver working characteristic curve (ROC) provided in example 3 of the present invention to evaluate resistant and sensitive samples against cetuximab in SIX2, KLF9, PARP3, and PODXL 2. Wherein panel a shows the ability of SIX2, KLF9, PARP3, PODXL2 to distinguish between a drug-resistant group and a relatively sensitive group in the transcriptome sequencing results found in cohorts; panel B shows the ability of SIX2, KLF9, PARP3, PODXL2 to distinguish between drug resistant and relatively sensitive groups in the validation cohort qRT-PCR results.
Detailed Description
The present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention. Conditions, methods and the like not described in the examples were carried out according to the conventional conditions or conditions recommended by the manufacturers.
The mice used for constructing the PDX model in the invention are female BALB/c-nu nude mice, 6-8 weeks old, and purchased from the Ministry of laboratory animals of family planning science institute of Shanghai (animal license number: SCXK (Shanghai) 2018-. The mice are raised in cages under the condition of meeting the SPF level. Keeping the room temperature at 18-25 deg.C and relative humidity at 40-60%, and sterilizing special mouse cage, padding, feed and drinking water at 121 deg.C for 30 min. Dunnage is changed at least 1 time per week.
Example 1 construction of PDX model cohort for head and neck squamous cell carcinoma and evaluation of drug efficacy of cetuximab
For the corresponding patient of the PDX model constructed by the invention, the clinical information of the corresponding patient is recorded, and the clinical information comprises basic data (sex, age, smoking and drinking history and the like), clinical pathological diagnosis (tumor size and position, TNM stage, HPV infection condition), past treatment history (operation, radiotherapy and chemotherapy condition), recurrence, metastasis and other prognosis information and the like. Tumor, paracarcinoma and blood samples of the patients were collected. And carrying out pathological tissue morphological identification and genetic information identification on the tumor sample.
After the tumor tissue is removed by operation, the color, shape and texture of the tissue are observed, the necrotic tissue is removed, and the central part of the focus is selected to take the material. Because head and neck squamous cell carcinoma generally grows in the oral cavity, nasal mucosa and other contaminated parts, the sample needs to be sterilized by 0.05 percent of sodium hypochlorite and quickly cleaned by 1 percent of PBS of penicillin-streptomycin double antibody for 30 seconds before being transplanted to a mouse. Gently scraping peripheral tissues of the tissue sample, and cutting the tumor into 1-2 mm3The small blocks are used for transplanting patient tissues to a region with abundant blood supply and lymph nodes (such as bilateral axilla) of an immunodeficiency mouse under a sterile condition to construct a subcutaneous PDX model, or inoculating the small blocks to bilateral submaxillary spaces of an animal to construct an in-situ PDX model. To increase the success rate of vaccination, Matrigel gel was mixed with patient tumor tissue and vaccinated, 3-5 mice were vaccinated per patient tissue. Starting to track the growth track of the PDX model after modeling for 1-2 weeks, and when the tumor volume exceeds 800mm3Or when the tumor volume does not obviously increase for two weeks, carrying out sequence passage on the transplanted tumor, and generally considering that the model can be stably passaged when the transplanted tumor is passaged to more than 3 generations.
Based on the inventionAccording to the clinical information of corresponding patients, 49 head and neck squamous cell carcinoma PDX models are randomly screened into groups, and a PDX model discovery queue is constructed. Each of the in vivo-preserved PDX graft tumor samples was inoculated subcutaneously into nude mice (30-50 mm for each model)3) And the clinical substitution test of the cetuximab is used for follow-up clinical substitution tests. In the first round of administration, the experimental group PDX model was injected intraperitoneally with Cetuximab Cetuximab (R) ((R))
Figure BDA0003466994710000102
Merck), dose 10mg/kg, twice a week; control groups were injected intraperitoneally with PBS twice a week. The tumor volume and body weight of the model are continuously measured until 21 days or the tumor volume reaches 1000-3. If the mice develop adverse reactions within 14 days of the start of the experiment, they should be sacrificed and removed from the group.
The Cetuximab drug effect evaluation standard and the screening of a drug effect characteristic model are as follows: drug Response (Response) was assessed by volume change before and after tumor implantation in the PDX model as shown below:
Figure BDA0003466994710000101
in the above formula,. DELTA.Volt represents the change in tumor volume, VtRepresents the tumor volume on day t of administration, VinitialRepresents the tumor volume at day 0 of administration.
Tumor efficacy was assessed according to Δ Volt as follows:
1. improved complete remission (mCR): delta Volt < -40%;
2. modified partial remission (mPR): -40% <Δvolt < -20%;
3. improved disease stability (mSD): -20% <Δvolt < 30%;
4. improved disease progression (mPD): delta Volt > 30%.
According to the above standards, defining the mPD model as a primary drug resistance model by taking the day 21 as a node; other models continued to monitor their tumor volume and body weight until day 90; defining the mCR model as a sensitive model by taking the 90 th day as a second nodeMolding; models that recur within 90 days and fail to meet the mCR standard enter the study of acquired resistance. The tumor volume of the model to be repeatedly developed reaches 100-3The Cetuximab Cetuximab is injected into the abdominal cavity, the dosage is 10mg/kg, the Cetuximab Cetuximab is injected twice a week, the drug reaction condition is evaluated after 3 weeks of treatment, and the mPD model is defined as a secondary drug resistance model. The model that recurs in the first round of treatment but appears as mPR or mCR in the acquired resistance study dose is defined as a reversible drug-resistance persistence (DTP) state. And (5) sacrificing the mice after the test is finished, and reserving the samples for subsequent sequencing and verification.
As shown in fig. 1, in the pharmacodynamic test covering 49 PDX model discovery cohorts, tumor disease progression (mPD) after administration was defined as primary drug resistance (n 21) at 42.86%, tumor complete remission (mCR) with no recurrence within 90 days as sensitization (n 9) at 18.37%, tumor control (mCR + mPR + mSD) after administration and recurrence within 90 days was defined as re-administration, tumor disease progression (mPD) after re-administration was defined as secondary drug resistance (n 8) at 16.33%, the model of tumor mPR or mCR after re-administration was defined as reversible drug resistance persistence (n 3) at 6.12%, 4 groups failed to pharmacodynamic differentiation at 8.16% due to large intra-group differences, and 4 groups stopped administration due to various factors such as death in nude mice, poor nude mice status, severe adverse side reactions, etc. at 8.16%. In the PDX model treatment queue, the complete remission rate of 18.37 percent is matched with the response rate of 13 percent in clinical application of cetuximab, so that the PDX model treatment queue can accurately simulate clinical treatment and has extremely high drug resistance mechanism research and intervention strategy mining values.
The drug effect of the PDX model on the cetuximab is matched with the drug effect of the cetuximab clinically used by a patient, an example of a cetuximab sensitive patient is shown in figure 2, and the PDX model corresponding to the patient is also shown to be sensitive to the cetuximab; an example of a patient with relatively insensitive cetuximab is shown in fig. 3, which corresponds to a PDX model that also appears insensitive to cetuximab.
Example 2 cetuximab single antigen drug resistance biomarker screening
According to the invention, samples collected in a PDX model discovery queue are all obtained from a mouse, then are subjected to liquid nitrogen quick-freezing storage, and are sent to a sequencing company for whole exome sequencing and transcriptome sequencing.
Sequencing of all exons: the liquid nitrogen quick-frozen patient tumor and PDX samples are subjected to whole exon sequencing by using an Illumina Novaseq6000 platform, the sequencing depth of the patient tumor sample is 200X, and the PDX sample is 100X. For PDX samples requiring removal of murine genes, the human and mouse hybrid Genome (hs37d5 and mm10) was mapped into sequencing data by Burrows-Wheeler Aligner (BWA), and the human mutations were filtered using SAMtools, Genome Analysis Toolkit (GATK-Unified genotype) and FreeBaies (Garrison and Marth).
RNA sequencing: RNA library construction using Illumina Hiseq platform, mapping sequencing reads into human and mouse genomes via Hisat2 v2.0.5 and assembly of transcripts using Cufflinks v2.2.1, estimation of transcript abundance and differential expression analysis.
The invention obtains a series of potential drug sensitivity related biomarkers by analyzing mutant genes, gene copy numbers and expression differences of PDX samples of a sensitive group and a primary drug resistance group. The sensitive group and the primary drug-resistant group are screened, the gene mutation and copy number variation with significant difference (P <0.05) are shown in figure 4, and the gene variation with significant difference between the sensitive group and the primary drug-resistant group can be found, for example, the copy number amplification of CDK1 only appears in the drug-resistant PDX, which indicates that the gene variation is a potential drug efficacy marker. In addition, fig. 5 shows that there are significant differences in gene expression profiles between the sensitive model and the drug resistance model, and some genes exhibit high expression in the drug resistance model compared with the sensitive model, such as MAPK15, SIX2, and PTPN18, and may be predictive markers of cetuximab-induced drug resistance.
Example 3 validation of cetuximab antigen-mediated drug resistance biomarkers
In order to verify the sensitivity and specificity of the cetuximab drug-resistance related drug effect biomarkers screened from the PDX model discovery queue, the invention further carries out cross validation of the biomarkers by using the independent PDX queue.
According to the PDX model inoculation and passage method, a plurality of PDX models which are stable in passage and have complete histological evaluation and informatics identification are constructed, 61 independent PDX models are combined to serve as a PDX model verification queue, and a clinical substitution test of cetuximab is developed.
In the PDX model verification queue constructed by the invention, 28 cases of PDX disease progression (mPD) are defined as primary drug-resistant samples, accounting for 45.90%; 6 cases (9.84%) and 12 (22.95%) exhibited partial remission (mPR), defined as a relatively sensitive sample; 13 cases showed stable disease (mSD), accounting for 21.31% (fig. 6).
The invention collects the pre-drug sample of PDX model validation queue to carry out qRT-PCR detection on potential biomarkers. The results show that the amplification of CDK1, ANKH, HEPHL1, SLC6A genes can serve as cetuximab drug-sensitive biomarkers (fig. 7).
Subsequently, the method selects potential biomarkers of differential expression in a PDX model discovery queue to carry out qRT-PCR detection, and judges the sensitivity and specificity of each marker by using a Receiver Operating Curve (ROC) so as to evaluate the clinical significance of the markers. The results show that both transcriptome sequencing of cohort (fig. 8A) or qRT-PCR of validation cohort (fig. 8B) identified that high expression of SIX2, KLF9, PARP3, PODXL2 better distinguished between the drug-resistant and sensitive groups (AUC >0.80) (fig. 8).
Example 4 composition and use methods of a kit for detecting drug resistance in cetuximab-single antigen of head and neck squamous cell carcinoma
The kit for detecting the drug resistance of the cetuximab single antigen of the head and neck squamous cell carcinoma comprises a DNA extraction reagent, an RNA extraction reagent, a PCR reverse transcription reagent, a primer probe mixed solution, a Taqman copy number variation detection premixed reagent, a qRT-PCR detection premixed reagent, a plasmid containing the corresponding 8 gene sequences in a positive quality control product and RNase-free water.
Wherein the DNA extraction reagent (Thermofisiher, 10503027), the RNA extraction reagent (Thermofisiher, 12183555), the PCR reverse transcription reagent (Takara, 639505), the premixed reagent for detecting copy number variation (Thermofisiher, A30866) and the premixed reagent for detecting gene expression (Thermofisiher, 4444556) are directly selected from the existing commercial products.
The SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHL1 and SLC6A detection probe sequences can be designed according to gene sequences, for example, as shown in Table 1, the 5 'end of the probe sequences needs to be marked with a fluorescent group, and the 3' end needs to be marked with a quenching group, so that the probe sequences are suitable for TaqMan probe-based copy number variation detection and qRT-PCR method detection.
TABLE 1 Probe sequences
Figure BDA0003466994710000131
Operation and result judgment of the kit:
(1) adding about 10-50ng of DNA (DNA extracted from head neck scale cancer tumor tissue and tissues beside cancer) or cDNA (RNA extracted from head neck scale cancer tumor tissue and tissues beside cancer and reverse transcribed into cDNA) of different samples into each PCR reaction tube, adding 10 mu L of qRT-PCR mixed reagent and 1 mu L of forward and reverse primer probes into the tubes, supplementing the ultrapure water to 20 mu L, preparing a system, covering the tube cover, and placing the system into a fluorescence quantitative PCR instrument for fluorescence PCR detection. Simultaneously setting blank control without sample DNA or RNA and plasmid containing corresponding 8 amplification gene sequences in positive quality control.
(2) The conditions for PCR amplification reaction in the instrument were set as follows:
copy number amplification procedure: pre-denaturation/DNA polymerase activation at 95 ℃ for 10 min; denaturation, 95 ℃ for 15 s; annealing/stretching at 60 ℃ for 60 s; 40 cycles.
Gene expression program: incubation with UNG enzyme at 50 deg.C for 2 min; activating DNA polymerase at 95 deg.C for 2 min; denaturation, 95 ℃ for 3 s; annealing/stretching at 60 ℃ for 20 s; 40 cycles.
(3) After the reaction is finished, setting the base line as automatic adjustment, and analyzing the detection result according to the amplification curve graph and the Ct value.
(4) And (3) judging the effectiveness:
if the Ct value detected by the RNase-free water has a signal rising and is less than 35, the experimental result is invalid, and a new experiment is recommended; if the Ct value of the positive control plasmid is greater than 28, the experimental result is invalid, and re-experiment is recommended.
(5) Copy number and gene expression calculation:
and (3) taking the plasmid corresponding to the target gene as a standard substance to carry out gradient dilution, and adding five standard substance plasmids with concentration gradients in the PCR reaction to carry out the PCR reaction.
And (5) taking the logarithmic value of the copy number of the standard product as an abscissa and the measured Ct value as an ordinate to draw a standard curve. When the unknown sample is quantified, the copy number of the sample can be obtained in the standard curve according to the Ct value of the unknown sample. Log (initial concentration) and cycle number are in linear relation, a standard curve can be made through a standard substance with known initial copy number, the linear relation existing in the amplification reaction is obtained, and the template amount contained in the sample can be calculated according to the Ct value of the sample.
And calculating the corresponding gene copy number or gene absolute expression value in the tumor tissue and the tissue beside the cancer by using the Ct value measured by the sample according to the standard curve. And comparing the copy numbers of the target genes of the tumor tissue and the para-carcinoma tissue of each sample, and if the copy number of the tumor tissue is greater than that of the para-carcinoma tissue, defining the target gene copy number amplification sample as the sample, which indicates that the patient is possibly resistant to primary drug of the cetuximab. And comparing the absolute expression quantity of the target gene of the tumor tissue and the para-carcinoma tissue of each sample, and if the expression quantity of the tumor tissue is greater than that of the para-carcinoma tissue and the expression level of the target gene is obviously higher than the average level, defining the target gene as a high-expression sample, which indicates that the patient is possibly resistant to primary cetuximab.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and the present invention shall be covered thereby. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
<110> Shanghai university of traffic medical college affiliated ninth people hospital
<120> a method for screening cetuximab antigen-induced drug resistance biomarkers, biomarkers screened by the method and uses thereof
<130> DI22-0003-XC03
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<170> PatentIn version 3.5
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Claims (8)

1. A method for screening cetuximab single antigen-mediated drug resistance biomarkers for head and neck squamous cell carcinoma using a patient tumor tissue-derived xenograft tumor model (PDX model) clinical surrogate test, the method comprising the steps of:
(1) constructing a head and neck squamous cell carcinoma PDX model in an immunodeficiency mouse by using tumor tissues of the head and neck squamous cell carcinoma patient;
(2) acquiring clinical information of a patient corresponding to the head and neck squamous cell carcinoma PDX model, wherein the clinical information comprises the past medical history, clinical treatment conditions, medication conditions and the like;
(3) constructing a first queue of a head and neck squamous cell carcinoma PDX model: based on a head and neck squamous cell carcinoma PDX model biological sample library established, randomly screening a plurality of head and neck squamous cell carcinoma PDX models into groups according to clinical information of corresponding patients, and inoculating PDX transplantation tumor samples preserved in each living body to the subcutaneous part of a nude mouse for subsequent clinical substitution tests of cetuximab;
(4) development of cetuximab PDX model clinical alternatives: after the tumor volume of the head and neck squamous cell carcinoma PDX model reaches a certain value, randomly dividing each PDX case into a control group and a cetuximab single-drug treatment group, starting drug treatment, respectively receiving PBS or cetuximab intraperitoneal injection by the PDX model, and measuring the tumor volume and the body weight of the mouse every week;
(5) evaluating the drug effect of cetuximab in a PDX model clinical alternative test: to evaluate drug response to cetuximab, drug response was evaluated by volume change before and after drug administration of the transplanted tumor in the PDX model, with the following specific criteria: 1) complete tumor regression (mCR), at least 40% reduction in tumor volume; 2) partial regression of tumor (mPR), tumor volume reduction 20% -40%; 3) disease progression (mPD), at least 30% increase in tumor volume; 4) disease stabilization (mSD), tumor volume change between 30% increase and 20% decrease; the mPD model is determined as a primary drug resistance model, and the mCR model is defined as a sensitive model;
(6) screening primary drug resistance candidate pharmacodynamic markers by means of model clinical alternative tests: respectively carrying out whole exome sequencing and transcriptome sequencing on the primary drug resistance model and the sensitive model, and preliminarily screening out primary drug resistance candidate pharmacodynamic markers according to a difference gene variation spectrum and/or a difference gene expression spectrum of the primary drug resistance model and the sensitive model and combining gene functions and a signal channel analysis result;
(7) development of an independent PDX model clinical surrogate test: depending on a biological sample library of the head and neck squamous cell carcinoma PDX model, randomly screening a plurality of cases of head and neck squamous cell carcinoma PDX models as a second queue for grouping according to clinical information of corresponding patients, wherein the selected second queue is not overlapped with the first queue, carrying out cetuximab single-drug treatment on the grouped PDX model in the same way as in the step (4), carrying out sample collection and living body preservation on transplanted tumors after taking the drugs for a plurality of weeks, for example, 3 weeks, and distinguishing an original drug-resistant model and a sensitive model by evaluating the drug effect of the cetuximab;
(8) the pharmacodynamic markers were verified using an independent PDX model clinical surrogate test: the method comprises the steps of utilizing a sample in an independent PDX model clinical substitution test, adopting a real-time fluorescent quantitative PCR (qRT-PCR) method to verify candidate variation genes and expression genes, screening out genes with gene variation or expression trends consistent in a first queue and a second queue, drawing a working Characteristic Curve (ROC) of a subject for the selected pharmacodynamic predictive biomarkers, adopting SPSS 21.0 statistical software to analyze, calculating an area under the Curve, selecting an index with the AUC >0.80, and finally determining the head and neck squamous cell carcinoma cetuximab single antigen development resistant biomarkers found in the first queue and the second queue.
2. The method of claim 1, wherein the head and neck squamous carcinoma cetuximab primary drug resistance biomarkers screened by said method are selected from the following: transcription factors SIX2(Sine accumis Homeobox Homolog 2), basic transcription element binding protein KLF9(Kruppel Like Factor 9), Poly (adenosine diphosphate Ribose) PARP3(Poly (ADP-Ribose) Polymerase Family 3), Member of the cell surface transmembrane protein CD34 Family PODXL2(Podocalyxin Li 2), cell cycle Dependent Kinase CDK1(Cyclin Dependent Kinase 1), Inorganic Pyrophosphate Transport regulator ANKH Inorganic pyrophor transporter regulator), membrane iron Transport accessory protein HEPHHL 1 (Heestron Li1), and neurotransmitter transporter SLC6A (solvent carrier 6).
3. Use of the biomarkers SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHL1 or SLC6A in the preparation of a clinical prognostic agent for cetuximab efficacy in patients with head and neck squamous cell carcinoma.
4. A cetuximab primary drug resistance biomarker efficacy prediction kit comprising at least gene expression detection probes and/or copy number detection probes, wherein the gene expression detection probes comprise gene expression detection probes for one or more of SIX2, KLF9, PARP3, PODXL2, and the copy number detection probes comprise copy number detection probes for one or more of CDK1, ANKH, HEPHL1, SLC 6A.
5. The kit of claim 4, wherein the kit further comprises a tissue DNA 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 quality control substance and RNase-free water.
6. The kit according to claim 4, wherein the gene expression detection probe and the copy number detection probe are labeled with a fluorescent group at the 5 'end and a quenching group at the 3' end.
7. The kit of claim 4, wherein the CDK1, ANKH, HEPHHL 1, SLC6A, SIX2, KLF9, PARP3, PODXL2 detection probe sequences comprise:
Figure FDA0003466994700000031
8. the application of a kit in preparing a preparation for predicting drug resistance of cetuximab of patients with head and neck squamous cell carcinoma is disclosed, wherein the kit contains one or more detection reagents of SIX2, KLF9, PARP3, PODXL2, CDK1, ANKH, HEPHHL 1 and SLC 6A.
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CN115410705A (en) * 2022-08-22 2022-11-29 哈尔滨医科大学 Auxiliary evaluation method, device and equipment for resectable tumor after transformation therapy
CN115910214A (en) * 2022-10-13 2023-04-04 南京普恩瑞生物科技有限公司 Method for evaluating drug effect of anti-tumor drug by simulating clinical test by using tumor living tissue biological sample library and application thereof
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