Method for monitoring and controlling drug resistance of gastrointestinal stromal tumor patient to imatinib/sunitinib through ddPCR technology

A technology for drug resistance mutation sites and objects, applied in the medical and biological fields, can solve the problems of difficult to detect the type and sequence of cell-free DNA, low content of cell-free DNA in plasma, and lack of it.

Pending Publication Date: 2016-08-10
SHANGHAI BIOTECAN PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, due to the extremely small amount of plasma cell-free DNA in the blood and the interference of a large amount of blood-derived DNA, it is still difficult to accurately isolate and purify DNA with high-efficiency DNA enrichment methods and high-sensitivity detection methods (such as sequencing, digital PCR). and reliably detect the specific types and sequences of tumor-associated cell-free DNA
For example, it has been reported in the literature that although the average content of circulating cell-free DNA (4771ng / ml) in patients with colorectal cancer is significantly different from that in healthy people (0.85ng / ml), for 67 patients with carcinoembryonic antigen (CEA) Only 47% of the test results are meaningful for diagnosis
[0008] Therefore, although the detection technology based on free blood DNA is the focus of research and development, there is still a lack of satisfactory mutant DNA detection technology in this field that can truly meet the clinical needs of high sensitivity, high accuracy and high reliability.

Method used

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  • Method for monitoring and controlling drug resistance of gastrointestinal stromal tumor patient to imatinib/sunitinib through ddPCR technology
  • Method for monitoring and controlling drug resistance of gastrointestinal stromal tumor patient to imatinib/sunitinib through ddPCR technology
  • Method for monitoring and controlling drug resistance of gastrointestinal stromal tumor patient to imatinib/sunitinib through ddPCR technology

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Experimental program
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Embodiment 1

[0237] Example 1 A method for monitoring the secondary resistance of gastrointestinal stromal tumors to imatinib / sunitinib by using ddPCR technology to detect C-kit / PDGFRα gene mutations in serum / plasma

[0238] (1) Extract 4mL of peripheral blood from the patient and separate to obtain plasma / serum. Use the "QIAamp Circulating Nucleic Acid Kit" to extract free DNA in the plasma / serum. The extraction method is as described in the general method. High-concentration / high-purity DNA was obtained through sample dissolution, enrichment, column elution, and finally AVE elution, and finally the fragment size (<313bp) was determined by agarose gel electrophoresis to ensure the extraction quality.

[0239] (2) Reaction system preparation

[0240] a. Design of probes and primers: The present invention adopts the conventional Taqman probe method to design, and designs wild-type and mutant types respectively for the three detection sites of C-kit gene (T670I, D816V) / PDGFRα gene (D842V) T...

Embodiment 2

[0253] Example 2 A method for monitoring the secondary drug resistance of gastrointestinal stromal tumors to imatinib / sunitinib by using ddPCR technology to detect C-kit / PDGFRα gene mutations in serum / plasma

[0254] (1) Extract 4mL of peripheral blood from the patient and separate to obtain plasma / serum. Use the "QIAamp Circulating Nucleic Acid Kit" to extract free DNA in the plasma / serum. The extraction method is as described in the general method. High-concentration / high-purity DNA was obtained through sample dissolution, enrichment, column elution, and finally AVE elution, and finally the fragment size (<313bp) was determined by agarose gel electrophoresis to ensure the extraction quality.

[0255] (2) Reaction system preparation

[0256] a. Design of probes and primers: The present invention adopts the conventional Taqman probe method to design, and designs wild-type and mutant types respectively for the three detection sites of C-kit gene (T670I, D816V) / PDGFRα gene (D842...

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Abstract

The invention discloses a method for monitoring and controlling drug resistance of a gastrointestinal stromal tumor patient to imatinib / sunitinib through ddPCR technology. The method comprises: extracting DNA from peripheral plasma or serum of a gastrointestinal stromal tumor patient, detecting a mutation situation of a drug-resistant mutation site T670I, D816V, or D842V of C-kit or PDGFR[alpha] in the DNA through ddPCR technology, and judging if the patient produces drug resistance according to the mutation situation.

Description

technical field [0001] The present invention relates to the fields of medicine and biotechnology, in particular to a molecular detection technology that can be used to guide tumor treatment, especially for highly sensitive detection of C-kit gene (T670I, D816V) / PDGFRα gene (D842V) mutations and Its application in the monitoring of imatinib / sunitinib resistance in patients with gastrointestinal stromal tumors. Background technique [0002] Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the digestive tract, and their biological behaviors are complex and changeable. GIST is not sensitive to traditional radiotherapy and chemotherapy, and previous surgery is the only effective treatment for GIST. In recent years, molecular targeting research has made great progress in gastrointestinal stromal tumors. The tyrosine kinase inhibitor imatinib (imatinib) can significantly improve the clinical prognosis of patients with advanced GIST, and has mild sid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 易静许骋吴云鸣张桢珍丁飞飞楼敬伟何青卿马国栋周箴刘晓晨张存李迎雪
Owner SHANGHAI BIOTECAN PHARMA
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