Method for monitoring secondary drug resistance to imatinib (Glivec)/nilotinib through ddPCR technology

A technology for drug resistance mutation sites and sequences, 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

AI Technical Summary

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-fre...

Method used

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  • Method for monitoring secondary drug resistance to imatinib (Glivec)/nilotinib through ddPCR technology
  • Method for monitoring secondary drug resistance to imatinib (Glivec)/nilotinib through ddPCR technology
  • Method for monitoring secondary drug resistance to imatinib (Glivec)/nilotinib through ddPCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0215] Example 1 Detection of ABL gene and PDGFRα gene mutations in plasma by ddPCR technology to monitor secondary resistance to imatinib (Gleevec) / nilotinib in patients with chronic myelogenous leukemia

[0216] (1) Before starting imatinib (Gleevec) / nilotinib treatment in a patient with chronic myeloid leukemia, 4mL of peripheral blood was extracted from the patient, and the plasma was separated, and the free DNA in the plasma was analyzed by "QIAamp Circulating Nucleic Acid Kit". Extraction is performed. 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.

[0217] (2) Reaction system preparation

[0218] a. Design of probes and primers: The present invention adopts the conventional Taqman probe method to design, respectively designs wild-type and mutant probes for the target frag...

Embodiment 2

[0232] Example 2 Detection of ABL gene and PDGFRα gene mutations in plasma by ddPCR technology to monitor chronic myeloid leukemia patients' secondary resistance to imatinib (Gleevec) / nilotinib

[0233] (1) After the patient in Example 1 started imatinib (Gleevec) / nilotinib treatment for 6 months, his peripheral blood was collected by the same method as in Example 1, plasma was separated, and genomic DNA was extracted And perform PCR amplification. The test results are described in the table below:

[0234]

[0235]

[0236] Based on the analysis of the above data, it was found that PDGFRα (T674I) and ABL (T315I) of this sample did not mutate, suggesting that the patient has not yet developed secondary drug resistance to imatinib (Gleevec) / nilotinib, but Real-time monitoring should be carried out regularly (every 3-6 weeks). Once secondary drug resistance to imatinib (Gleevec) / nilotinib is found, the patient should be guided in time for further drug treatment.

Embodiment 3

[0237] Example 3 Detection of ABL gene and PDGFRα gene mutations in plasma by ddPCR technology to monitor secondary resistance to imatinib (Gleevec) / nilotinib in patients with chronic myelogenous leukemia

[0238] (1) After the patient in Example 1 started imatinib (Gleevec) / nilotinib treatment for 12 months, his peripheral blood was collected by the same method as in Example 1, plasma was separated, and genomic DNA was extracted And perform PCR amplification.

[0239] (2) Result analysis:

[0240] figure 1 A, B and figure 2 A and B are the detection result diagrams of Example 3. figure 1 (A) and figure 2 In (A), both mutant and wild-type PDGFRα(T674I) were detected. figure 1 (B) and figure 2 In (B), both mutant and wild type were detected in ABL(T315I), indicating that both PDGFRα(T674I) and ABL(T315I) were mutation-positive.

[0241] The test results are described in the table below:

[0242]

[0243] According to the analysis of the above data, it was found th...

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Abstract

The invention provides a method for monitoring secondary drug resistance to imatinib (Glivec)/nilotinib through ddPCR technology, and an application of the method to monitoring and control of imatinib (Glivec)/nilotinib in a process of treating chronic granulocytic leukemia. The method comprises: firstly, extracting DNA from peripheral plasma of a patient suffering from chronic granulocytic leukemia, then detecting a mutation situation of a drug-resistant mutation site T315I of an ABL gene or a drug-resistant mutation site T674I of a PDGFR[alpha] gene in the DNA through ddPCR technology, and finally judging if the patient produces drug resistance according to the mutation situation.

Description

technical field [0001] The 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 a method for highly sensitive detection of ABL gene or PDGFRα gene site mutation in peripheral plasma, and its role in monitoring chronic myelogenous leukemia The application of secondary resistance to imatinib (Gleevec) / nilotinib in patients. Background technique [0002] Chronic myelogenous leukemia (CML) is a malignant proliferative disease originating from hematopoietic stem cells, characterized by the formation of Philadelphia chromosome (Ph) by translocation of chromosomes 9 and 22, which forms a new BCR-ABL fusion gene, the fusion protein encoded by this gene can lead to abnormal clonal proliferation of myeloid hematopoiesis. The emergence of tyrosine kinase inhibitors (tyrosine kinase inhibitors, TKIs) is a milestone in the history of CML treatment, and now TKIs have become t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 易静许骋吴云鸣张桢珍丁飞飞楼敬伟王潍博李明峰何志杰张威浩易村犍
Owner SHANGHAI BIOTECAN PHARMA
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