WO2012126162A1

WO2012126162A1 - Test kit for selection of esophageal cancer treatment protocols and/or prognostic evaluation

Info

Publication number: WO2012126162A1
Application number: PCT/CN2011/002037
Authority: WO
Inventors: 王云松; 张力建; 姜文国
Original assignee: 吴江鉴治生物科技有限公司
Priority date: 2011-03-24
Filing date: 2011-12-06
Publication date: 2012-09-27
Also published as: CN102199664A; CN102199664B

Abstract

Disclosed in the invention is a test kit for selection of esophageal cancer treatment protocols and/or prognostic evaluation, which comprises primers used for testing multiple genes, and the primer sequences are shown as SEQ ID NO:1-36. By testing and identifying the expression level of a plurality of genes in this invention, the therapeutic effect of clinical treatment is evaluated.

Description

Test kit for treatment options and/or prognosis evaluation of esophageal cancer

The present invention relates to a test kit, and more particularly to a test kit for treatment selection and/or prognosis evaluation of esophageal cancer.

Background technique

Esophageal cancer is one of the most malignant tumors occurring in the upper gastrointestinal tract. It occurs from the epithelial tissue of the esophagus and can be roughly classified into adenocarcinoma and squamous cell carcinoma. It is currently believed that the occurrence of esophageal cancer may be related to smoking, alcoholism, gastroesophageal reflux disease, infection, eating habits (such as eating spicy foods), chewing tobacco, age and other unknown factors.

Although esophageal cancer has a low overall incidence worldwide, it is a high-risk malignancy in China, Iceland, Ireland, Japan, South Korea and the United Kingdom. In the United States, the incidence of esophageal cancer is low. In 2007, there were only more than 15,000 new cases in the United States, while in the United Kingdom there were nearly 8,000 new cases, making esophageal cancer the ninth most common malignant tumor in the UK. There are about 500,000 new cases of esophageal cancer every year in the world, especially in the past 20 years, the incidence of esophageal cancer has continued to rise. The incidence of esophageal cancer in China ranks first in the world, followed by certain African countries and Japan.

Esophageal cancer is a malignant tumor with poor prognosis. The tumor has already metastasized at the time of initial diagnosis. As a result, the efficacy of esophageal cancer patients is very poor, and the five-year survival rate is less than 5%. In the UK, patients with esophageal cancer have a 30% annual survival rate and a five-year survival rate of 8%. Another cause of poor prognosis in esophageal cancer is the presence of serious medical problems that prevent patients from undergoing surgery or adjuvant therapy. As a result, nearly 7,600 esophageal cancer patients died in the UK in 2007, and more than 400,000 patients worldwide died.

At present, there is no effective means to detect the prognosis of patients with esophageal cancer, and it can assist doctors to choose a reasonable and targeted treatment plan.

Summary of the invention

It is an object of the present invention to provide an accurate and rapid test kit for the selection and/or post-evaluation of treatment options for esophageal cancer.

Another object of the invention is to propose a gene chip for the treatment option selection and/or prognosis evaluation of esophageal cancer.

A third object of the present invention is to propose a novel use of a gene group in the preparation of a treatment plan selection and/or prognosis evaluation test kit for esophageal cancer. The inventive idea of the present invention is: The inventors identified a set of genes whose expression levels have significant clinical significance. After a large amount of statistical analysis in the later period, 16 genes (including two housekeeping genes) were finally identified and a test kit was prepared. The comprehensive analysis of the expression levels of these genes can well evaluate the prognosis of patients. The kit allows rapid clinical testing of postoperative or tissue biopsy patients, helping clinicians to evaluate patients' prognosis well. In addition, the test results can identify which patients are not suitable for a conventional treatment regimen, suggesting that patients choose a more appropriate individualized treatment regimen. Finally, these molecular markers may be used as potential drug targets in the future for the treatment of tumors.

In order to achieve the above object, the specific technical solution of the present invention is:

A test kit for treatment option selection and/or prognosis evaluation of esophageal cancer, the kit comprising an agent for detecting expression of a gene;

The genes include:

BMP-8, CD133, CD29, Carl, CL5, Endomus2, Nectin3, Nectin Kai PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, IL22R and VEGF-D.

The genes also include housekeeping genes: GAPDH and CK19.

The reagent is an agent for immunohistochemical detection of the gene of claim 1.

The kit has the specific composition:

(1) Hot-start Q-master mix (Abgene);

(2) a primer for detecting the gene of claim 1;

(3) Universal probes for FAM labeling.

The primer sequences are shown in SEQ ID No. 1 to 36.

Primers for detecting the expression of each of the above genes, wherein the primer sequences are shown as SEQ ID No. 1 to 36.

A gene chip for treatment selection and/or prognosis evaluation of esophageal cancer, the gene chip comprising a solid phase carrier and a probe, the probe hybridizing with a gene and/or a complement thereof; the gene include:

BMP-8, CD133, CD29, Carl, CL5, Endomus2, Nectin3, NectinK KaiK PPARGCA, WAVE-WAVE-3, BMP9, CMG IL22R and VEGF-D.

A gene group for use in the preparation of a therapeutic protocol selection and/or prognostic evaluation kit for esophageal cancer, the gene group comprising the following genes:

BMP-8, CD133, CD29, Carl, CL5, Endomus2, Nectin3, NectinK KaiK PPARGCA, WAVE-1, WAVE-3, BMP9, CMG1, IL22R and VEGF-D. A gene group for use in the preparation of a therapeutic plan selection and/or prognosis evaluation gene chip for esophageal cancer, the gene group comprising the following genes:

BMP-8, CD133, CD29, Carl, CL5, Endomus2, Nectin3, Nectin Kail, PPARGCA, WAVE-WAVE-3, BMP9, CMG1, IL22R and VEGF-D.

The naming of each of the above genes is derived from the www.NCBLLM.NIH.gov database or the nomenclature recognized in the literature, all well known to those skilled in the art.

The beneficial effects of the invention:

The kit of the invention evaluates the therapeutic effect of the clinical treatment by detecting and identifying the expression levels of a plurality of genes, thereby determining which patients have poor prognosis, which patients are not suitable for using the conventional treatment plan, and providing a supplementary reference for the doctor to select a treatment plan. . The detection method of the kit of the invention is simple, accurate, and low in cost, and is easily accepted by a large number of patients.

DRAWINGS

Figure 1 shows molecular markers in CTF8 mode that divide esophageal cancer patients into two groups. 0 was the "prognosis" group; 1 was the "prognosis of poor prognosis" group, and there was a significant statistical difference between the two groups (p < 0.00001).

Figure 2 shows the molecular markers of the CTF 8.12 model dividing esophageal cancer patients into three groups. 1 was in the "prognosis" group; 2 was in the "prognosis" group; 3 was in the "prognosis" group, and there was a significant statistical difference between the three groups (p < 0.00001).

Figure 3 shows the relationship between lymph node metastasis status and prognosis of esophageal cancer. 0 is negative for lymph node metastasis; 1 is positive for lymph node metastasis.

Figure 4 shows the relationship between TNM staging and prognosis of esophageal cancer. 2 is TNM staging Stagell; 3 is TNM staging Stagelll; 4 is TNM staging StageIV.

Figure 5 shows the relationship between tumor cell differentiation status and prognosis of esophageal cancer. 1 is highly differentiated; 2 is high school differentiation;

3 is moderately differentiated; 4 is low-medium differentiation; 5 is poorly differentiated.

Figure 6 shows the relationship between tumor embolism and prognosis of esophageal cancer. 0 is tumor embolism; 1 is no tumor embolism. Figure 7 shows the relationship between pathological type and prognosis of esophageal cancer. 1 is squamous cell carcinoma; 2 is adenocarcinoma.

Figure 8 shows that molecular markers in the CTF8 model divide patients with esophageal cancer who are negative for lymph node metastasis into two groups. 0 is the "prognosis of good" group; 1 is the "poor prognosis" group.

Figure 9 shows that molecular markers of CTF8 mode divide lymph node metastasis-positive esophageal cancer patients into two groups. 0 is the "prognosis of good" group; 1 is the "poor prognosis" group.

detailed description

Example 1 Acquisition of target gene

1. Collection, storage and recording of the organization The test tissue samples were collected by the inventors, and the discarded tissue samples (including normal tissues, paracancerous tissues, and tumor tissues) after surgical resection of esophageal cancer patients were stored in a refrigerator at -80 ° C for use.

2. Sample processing of esophageal cancer tissue and construction of cDNA library

The esophageal cancer tissues were frozen and sectioned with a cryostat cryostat (Ldca), and some sections were preserved for histological analysis. About 30 sections of the tissue were used for RNA extraction (method is as follows). After quantifying the RNA, use The same amount of RNA was used to construct a cDNA library.

3. Tissue RNA extraction and cDNA synthesis

The frozen tissue was cut into 5-10 μΐΉ thickness, some of which were used for immunohistochemical analysis (Jiang et al 2003a) _o 20-30 sections of tissue were homogenized in ice-cold RNA isolation reagent After the pulping. The concentration of RNA was detected by an ultraviolet spectrophotometer (Jiang et al 2003a;). Using 3 g of total RNA as a template and Oligo-dT as a primer, reverse transcription was performed using a RT kit (Promega), and the quality of cDNA synthesized by β-actin primer was used as an internal reference. PCR primers were designed using Beacon Designer (California, USA) and synthesized at InvitrogenTM Ltd and Sigma. The molecular biology grade agarose and DNA Ladder used in the experiments were purchased from Invitrogen, and conventional PCR reagents and quantitative PCR reagents were purchased from AbGene and Biorad, respectively.

4. Quantitative analysis of molecular markers

The detection of the expression level of the test gene in the above synthetic cDNA was carried out using a real-time quantitative PCR technique based on the AmplifuorTM principle (Nazarenko et al 1997 and Jiang et al 2003a and 2003b). First, PCR primers were designed using Beacon Designer software (version 2, Biosoft, Palo Alto, California, USA). One primer (usually an antisense primer) was terminated with a Z sequence (5 'actgaacctgaccgtaca'3, sequence and universal Z The probe sequences are complementary). Taqman ™ kit for the detection of β-actin was purchased from Perkin-Elmer ^TM.

The reaction system was as follows: Hot-start Q-master mix, specific upstream primer l Opmol, downstream primer containing Z sequence l pmol, FAM labeled probe (Intergen Inc.) l Opmol and about 50 ng RNA. The reaction was carried out on IcyclerlQTM (Bio-RadTM, Hemel Hamstead, England, UK). The specific reaction conditions were as follows: First, denaturation at 94 °C for 12 min, then 50 thermal cycles, including denaturation at 94 °C for 15 s, 55 V 40s, extending at 72 °C for 20s (Jiang et al 2003b, 2003c). In the reaction, an internal standard is used to simultaneously amplify the sample to be tested, thereby determining the transcription level of the target gene in the sample. The results are expressed in two ways: The species is the transcription level of the same amount of RNA, and the other is the relative ratio of the gene of interest to the expression level of the GAPDH gene or the CK19 gene.

5. Select molecular markers based on the expression pattern of the target gene

The target gene transcription levels of the samples were first analyzed using Minitab software (Minitab Inc., State College, PA 16801, USA).

Determining the final enrolled gene: The final selection based on the expression level of the candidate gene and whether it can distinguish the long-term disease group from the other groups. Screening conditions: Collect the clinical stage, pathological type, treatment mode, and survival time of the patient. Clinical data, if the clinical data of the patient can be well distinguished by the expression level of the candidate gene, especially to distinguish patients with different survival periods, it is determined as the final selected gene. Grouping and simple statistical analysis were performed by Excel (Microsoft Office 2007 version) software, and SPSS (SPSS Inc., Chicago, Illinois, US) software was used for further statistical analysis of patient survival.

Preparation of the kit: After determining the final selected genetic markers, specific gene markers are shown in Table 1, and test kits are prepared based on these markers. All reagents for detecting gene expression were first formulated and then automatically spotted into 96-well plates for clinical and cellular samples. After the kit is prepared in the laboratory, store it at -20 °C for later use.

6. Identification of molecular markers

After the experimental analysis, 16 genes were finally identified as molecular markers for evaluating the prognosis and efficacy of esophageal cancer patients, as shown in Table 1. BMP-8, CD133, CD29, Carl, CL5, Endomus2, Nectin3, Nectin 1, Kai 1, PPARGCA, WAVE-1, WAVE-3, BMP9, CMG 1, IL22R and VEGF-D, GAPDH housekeeping gene control and CK19 housekeeper Gene control.

Table 1 16 genetic markers as genetic markers for esophageal cancer

7, grouping of esophageal cancer molecular markers The above 16 genes were analyzed according to the patient's survival time, and were classified into "prognosis markers" and "prognosis markers". Whether the selected marker gene is up-regulated or down-regulated is regarded as abnormal expression. If there are more than 8 gene expression abnormalities, the patient is classified as "prognosis", if less than or equal to 8 genes are abnormally expressed, For the "prognosis good" group.

8. Verification of the relationship between molecular markers of esophageal cancer and prognosis

(1) Two groups of classification methods and results verification based on 16 molecular markers to evaluate patient prognosis:

As shown in Fig. 1, patients can be classified into a "prognosis group (indicated by 0)" and a "prognosis group (indicated by 1)" based on the expression levels of these molecular markers. If there are more than 8 gene expression abnormalities, the patients are classified as "prognosis", and if there are less than or equal to 8 gene expression abnormalities, they are classified as "prognosis". The marker pattern of the prognosis group was named CTF8, that is, the test result was CTF8 mode, and the average survival time of the validated patients was 39.3 months. Compared with this, the "prognosis of poor prognosis" markers showed that the average survival time of the patients was only 15.3. Months (p<0.00001). During follow-up, 78.3% of the "prognosis" groups were able to survive until the end of follow-up, compared with 12.5% in the "prognosis" group. Entering the survival time, survival status, and expression level of the marker into the SPSS software for survival analysis can be grouped and the average survival time can be obtained.

The clinical data of patients including tumor embolism, lymph node metastasis, tumor stage, TNM stage, differentiation status, histological type and CTF8 input SPSS for COX multivariate analysis can be concluded that CTF8 is an independent prognostic factor (p <0.001), the same tumor embolism can also be used as an independent prognostic factor (ρ = 0.0 Π), while other factors such as lymph node metastasis (ρ = 0.227), tumor stage (p = 0.255), TNM staging (p = 0.313), differentiation status (p=0.162), and histological type (p=0.78) were not independent prognostic factors.

(2) Three groups of classification methods and results verification based on 16 molecular markers to evaluate patient prognosis:

In order to more accurately distinguish the prognosis of patients, we used the CTF8.12 model to divide patients into three groups. Less than 8 gene expression abnormalities were "prognosis group" and 8-12 gene expression abnormalities were "prognosis group". More than 12 gene expression abnormalities are "prognosis group". As shown in Figure 2, patients can be divided into "prognosis group (represented by 1)", "prognosis group (represented by 2)" and "prognosis group" by detecting 16 molecular markers. In the middle of 3) ". According to Kaplan-Meier statistical analysis, there was a statistically significant difference in survival between the three groups (p < 0.00001). 87% of patients with "prognosis" group survived to the end of follow-up, with a median survival of 44.6 months, and 35% of patients in the "prognosis" group survived to follow-up, with a median survival of 20.1 months, and all There was no survival in the "prognosis of poor prognosis" group until the end of follow-up, and the median survival was only 6.5 months. Also based on multivariate analysis, CTF8.12 was an independent prognostic factor (p<0.00001), and the patient's clinical data included the presence of tumor embolism, lymph node metastasis, tumor stage, TNM stage, differentiation status, histological type, and CTF8 input SPSS for COX multi-factor analysis can be obtained. All other clinical and pathological factors could not be used as independent prognostic factors (lymph node metastasis p=0.314, tumor embolization p=0.074, tumor stage p=0.076, TNM stage p=0.231, differentiation status p=0.403, tissue type p= 0.891).

9. Clinical and pathological factors and their prognostic value

According to statistical analysis, the three-group classification of prognosis CTF8.12 is an independent prognostic factor (p<0.00001), whereas the prior art lymph node metastasis status (Fig. 3), tumor stage (Fig. 4), tumor cell differentiation Clinical and pathological factors such as status (Figure 5), tumor embolization (Figure 6), and histological type (Figure 7) cannot be used as independent prognostic factors to assess patient prognosis. ,

10. Verification result of the kit of the present invention

We evaluated the prognosis of patients with lymph node metastasis status and 16 molecular markers. As shown in Figure 8, 85.6% of patients with "prognosis" in the lymph node metastasis group survived to the end of follow-up, with a median survival of 43.3. Months, and all patients with negative lymph node metastasis in the "prognosis" group did not survive, with a median survival of only 4 months. The "prognosis" group with positive 70% lymph node metastasis survived to the end of follow-up with a median survival of 28.5 months. 16.7% of patients with a positive prognosis in lymph node metastasis survived to the end of follow-up, with a median survival of 18.4 months, as shown in Figure 9.

11. Conclusion

The present invention provides a novel test kit for assessing the prognosis of patients with esophageal cancer, and the present invention also provides an assessment of the likelihood of postoperative chemotherapy failure in patients with esophageal cancer.

It has always been a problem for prognosis evaluation of patients with esophageal cancer. At present, some clinical and pathological methods after surgery are mainly used to evaluate prognosis, such as lymph node metastasis status, tumor stage, and TNM stage. This study found that the presence of TNM staging and tumor embolization does have some value in assessing prognosis, and tumor cell differentiation status, lymph node metastasis status, and histological type are also valuable in assessing prognosis, but most of these factors are not statistical. The meaning of learning has little statistical significance. The 16 molecular markers we found have significant advantages over these factors (see Figure 4-8). Statistical analysis showed that the two groups and three groups of classifications had statistically significant prognostic assessment, and 16 points. Sub-markers can be used as independent prognostic factors. Example 2 How to use the kit

1. Reaction system: The reaction was carried out in a 96 low-profile PCR plate. Three parallel reactions were required for each marker gene, and two housekeeping genes were also tested as internal controls. The reaction system for each reaction (per well) is as follows: q-PCR master mix (8μ1), upstream primer (lOpmol./μΙ, Ι μΐ), downstream primer (lpmol/μΐ, Ι μΐ), general probe for FAM labeling Needle (lOpmol./μΙ, Ι μΐ), PCR grade Η ₂ Ο } μ1, sample cDNA or housekeeping gene 4μ1.

The extraction of the sample _{cDNA to} be tested is known to those skilled in the art, and the extracted cDNA is used.

After the PCR grade H ₂ O was dissolved, 4 μl was added to the reaction system.

2, Reaction conditions: The reaction was carried out in Bio-Rad Q-PCR thermo cycler, the specific reaction conditions are as follows: 94 Ό 12 minutes, then 50 cycles, 94 Ό 15 seconds in each cycle, 55 ° C 40 seconds 1T seconds. At the same time as the sample was amplified, the transcript was quantified based on the internal reference. The results are expressed in two ways: one is the transcript level based on the average total RNA amount, and the other is the relative ratio of the gene of interest to the amount of the GAPDH gene or the CK19 gene.

Claims

― Claims

A test kit for treatment selection and/or prognosis evaluation of esophageal cancer, characterized in that the kit comprises an agent for detecting expression of a gene;

The genes include:

BMP-8, CD 133, CD29, Car CL5, Endomus2, Nectin3, NectinK Kail, PPARGCA, WAVE-WAVE-3. BMP9, CMG1, IL22R and VEGF-D.

The test kit according to claim 1, wherein the gene further comprises a housekeeping gene: GAPDH and CK19.

The test kit according to claim 1 or 2, wherein the reagent is a reagent for detecting the gene of claim 1.

The kit according to claim 3, wherein the kit is composed of:

(1) Hot-start Q-master mix (Abgene);

(2) a primer for detecting the gene of claim 1;

(3) Universal probes for FAM labeling.

The kit according to claim 4, wherein the primer sequence is as shown in SEQ ID Nos. 1 to 36.

6. A test kit for treatment selection and/or prognosis evaluation of esophageal cancer, characterized in that the kit comprises a primer sequence for detecting genes including: BMP-8, CD133, CD29, Carl, CL5, Endomus2, Nectin3, Nectin Kail, PPARGCA, WAVE-WAVE-3, BMP9, CMG1, IL22R and VEGF-D.

A primer for detecting the gene of claim 1, wherein the primer sequence is as shown in SEQ ID Nos. 1 to 36. ■

8. A gene chip for treatment selection and/or prognosis evaluation of esophageal cancer, the gene chip comprising a solid phase carrier and a probe, characterized in that the probe and the following gene and/or their complementary sequences Hybridization; the genes include: BMP-8, CD133, CD29, Carl, CL5, Endomus2, Nectin3, Nectin Kail, PPARGCA, WAVE-K WAVE-3, BMP9, CMG IL22R and VEGF-D.

9. Use of a gene group for the preparation of a therapeutic protocol selection and/or prognostic evaluation kit for esophageal cancer, characterized in that the gene group comprises the following genes:

BMP-8. CD133, CD29, Carl, CL5, Endomus2, Nectin3, NectinK Kail, PPARGCA, WAVE-U WAVE-3, BMP9, CMGK IL22R and VEGF-D.

- Jn

10. Use of a gene group for the preparation of a therapeutic plan selection and/or prognostic evaluation gene chip for esophageal cancer, characterized in that the gene group comprises the following genes:

BMP-8, CD133, CD29, CarK CL5, Endomus2, Nectin3, NectinK Kail PPARGCA, WAVE-WAVE-3. BMP9, CMGK IL22R and VEGF-D.

n