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Biomarker for forecasting new auxiliary chemotherapy curative effect of breast cancer and fluorescence quantitative immune PCR kit

A biomarker and fluorescence quantitative technology, applied in the field of biomedicine, can solve the problems of NACT insensitivity, delayed local treatment, low pCR rate, etc., and achieve the effect of avoiding blindness

Inactive Publication Date: 2015-11-11
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The clinical application of NACT has a history of more than 30 years. Although great progress has been made in recent years, problems and deficiencies that need to be solved have also been discovered with the deepening of research: about 20% of breast cancer patients are not sensitive to NACT. For some patients, if NACT is not sensitive and the treatment course is too long, it may delay the timing of local treatment. It is for this reason that some doctors and patients are overly conservative in carrying out NACT, and the pCR rate is less than 10%.
[0006] However, the genome-wide chip used to screen NACT-related genes has the following disadvantages: when the chip is designed, it covers the gene probes in the whole gene range and the tumor specimen itself has a large heterogeneity, resulting in a high FDR of the chip data (FalseDiscoveryRate) value and it is difficult to identify important genes

Method used

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  • Biomarker for forecasting new auxiliary chemotherapy curative effect of breast cancer and fluorescence quantitative immune PCR kit
  • Biomarker for forecasting new auxiliary chemotherapy curative effect of breast cancer and fluorescence quantitative immune PCR kit
  • Biomarker for forecasting new auxiliary chemotherapy curative effect of breast cancer and fluorescence quantitative immune PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] 20 cases of breast cancer patients without NACT who were treated in Zhejiang Cancer Hospital were punctured to obtain breast cancer tissue samples. Patients were required to have a clear cytological diagnosis, no treatment before surgery, and complete pathological information .

[0069] After the sampling was completed, the patients were given neoadjuvant chemotherapy, and the pCR group and the NpCR group were distinguished according to the curative effect; at the same time, the breast cancer tissue samples were processed as follows:

[0070] 1. Extraction of total RNA from breast cancer tissue

[0071] (1) Total RNA extraction

[0072] 1) In a clean area with less RNase interference (all utensils are wiped with 1‰ DEPC water), the mortar needs to be sterilized at 200°C for 5 hours and cooled for 4 hours. In a mortar pre-cooled with liquid nitrogen, grind to powder with a pestle (incomplete grinding will seriously affect the yield of RNA);

[0073] 2) Add 700 μl of Q...

Embodiment 2

[0112] Fluorescence quantitative PCR analysis of embodiment 2 differentially expressed genes

[0113] For the CCL2 gene, CCR1 gene, CXCL10 gene, CXCL11 gene and IL2RG gene, primer5.0 was used to design fluorescent quantitative PCR primers, and the primer design information is shown in Table 2.

[0114] Table 2 Specific primers for fluorescent quantitative PCR

[0115]

[0116] 2. Real-Time PCR

[0117] 1) Synthesis of the first strand of cDNA

[0118] The total RNA in the tissue was used as a template to carry out reverse transcription reaction to synthesize cDNA.

[0119] Add the following reagents sequentially into a new 200 μl optical PCR eight-tube tube:

[0120]

[0121]

[0122] 2) After mixing, centrifuge slightly and carry out PCR reaction in ABIPRISM7300. The reaction conditions are set as:

[0123] Two-step PCR amplification standard procedure

[0124] Stage1: Pre-denaturation

[0125] Number of cycles: 1

[0126] 95℃30s

[0127] Stage2: PCR reaction...

Embodiment 3

[0138] Embodiment 3 Fluorescent quantitative PCR kit based on SYBRGreen fluorescent dye method

[0139] A fluorescent quantitative PCR kit for predicting the curative effect of neoadjuvant chemotherapy for breast cancer, including the primers shown in Table 2, and reagents such as Taq enzyme and dNTP.

[0140] Using the kit provided in this embodiment, the method of using the fluorescent quantitative PCR detection based on the DNA dye method is as follows:

[0141] In a new 0.2μl optical PCR reaction tube, add in sequence:

[0142]

[0143] The value of this kit lies in the detection of the expression levels of five cytokines and cytokine receptor interaction signaling pathway-related genes by simple DNA dye method through the patient's cancer tissue RNA samples, and then predicting the breast cancer by the degree of difference in the expression levels. The efficacy of NACT in cancer patients, thereby reducing unnecessary treatment. Therefore, the kit is put into producti...

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Abstract

The invention discloses a biomarker for forecasting the new auxiliary chemotherapy curative effect of breast cancer and a fluorescence quantitative immune PCR kit. The biomarker is at least one of a CCL2 gene, a CCR1 gene, a CXCL10 gene, a CXCL11 gene and an IL2RG gene. The five genes of the biomarker are efficiently expressed in a pCR group; the IL2RG gene has an extremely obvious expression difference (P is less than 0.01) in the pCR group and the NpCR group, and the other four genes have an obvious expression difference (P is less than 0.05) in the pCR group and the NpCR group; the five genes are indicated to be biomarkers for forecasting the new auxiliary chemotherapy curative effect of the breast cancer, and the blindness of new auxiliary chemotherapy is avoided.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a biomarker and a fluorescent quantitative immune PCR kit for predicting the curative effect of neoadjuvant chemotherapy for breast cancer. Background technique [0002] Breast cancer neoadjuvant chemotherapy (neoadjuvant chemotherapy, NACT), also known as preoperative chemotherapy (primary chemotherapy), refers to the treatment of non-metastatic tumors before local treatment, systemic cytotoxic drugs. This chemotherapeutic approach is not only used in advanced breast cancer but also increasingly in early pathological states. NACT has the following three effects on operable breast cancer patients: (1) The long-term survival of patients with pathologic complete remission (pCR) is significantly improved. pCR is an effective evaluation index for NACT in breast cancer. The international expert committee recommends that pCR be defined as: after neoadjuvant chemotherapy, there i...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 丁先锋王青郭江峰盛清傅枭男洪祝平
Owner ZHEJIANG SCI-TECH UNIV
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