Method for detecting BCR-ABL inhibitor resistance-related mutations, and data acquisition method for predicting resistance to BCR-ABL inhibitor using said method

A resistance-related and detection method technology is applied in the detection of BCR-ABL inhibitor resistance-related mutations and the data acquisition field using the detection for predicting BCR-ABL inhibitor resistance, which can solve the problem that mutations cannot be accurately performed detection, etc.

Inactive Publication Date: 2017-08-18
TOYO KOHAN CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, since cells expressing the BCR-ABL fusion gene are mixed with (normal) cells expressing the ABL gene without the above-mentioned translocation in the biological sample collected from the subject, it is therefore possible that resistance to BCR-ABL inhibitors When detecting mutations, sometimes the mutations in the ABL kinase domain on chromosome 9 derived from (normal) cells contained in biological samples are also detected, because the above-mentioned mutations in the ABL kinase domain are not Directly contributes to BCR-ABL inhibitor resistance, so there is a problem that mutations associated with BCR-ABL inhibitor resistance cannot be accurately detected

Method used

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  • Method for detecting BCR-ABL inhibitor resistance-related mutations, and data acquisition method for predicting resistance to BCR-ABL inhibitor using said method
  • Method for detecting BCR-ABL inhibitor resistance-related mutations, and data acquisition method for predicting resistance to BCR-ABL inhibitor using said method
  • Method for detecting BCR-ABL inhibitor resistance-related mutations, and data acquisition method for predicting resistance to BCR-ABL inhibitor using said method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065]

[0066] In this example, five samples summarized in the table below were prepared.

[0067] [Table 1]

[0068]

[0069]

[0070] The cell line K562 was cultured, and the cells were confirmed to be 1.25×10 using a TC20 automatic cell counter (manufactured by Bio-Rad). 6 individual / mL. Add 10 mL of culture medium to a 15 mL centrifuge tube, and centrifuge at 300×g for 5 minutes to form a precipitate. After the supernatant was carefully and completely removed, RNA was extracted using RNeasy Mini Kit (manufactured by QIAGEN). The RNA concentration was measured using NanoDrop 2000 (manufactured by Thermo Fisher Scientific).

[0071]

[0072] The cell line HL60 was cultured, and RNA was extracted using the RNeasy Mini Kit (manufactured by QIAGEN) in the same manner as in the preparation of Sample 1. The RNA concentration was measured using NanoDrop 2000 (manufactured by Thermo Fisher Scientific).

[0073]

[0074] Using 2 mg of total RNA extracted from the ce...

Embodiment 2-2

[0122] [Example 2-2] Evaluation of primer pairs

[0123] In this example, the total amount of sample 3 and sample 4 mixed was set to 10 ng, and the mixed samples at ratios of 100:0, 50:50, and 0:100 were used as templates, respectively. In addition, the hybridization reaction by PCR and a DNA chip, the subsequent fluorescence detection, etc. were performed in the same manner as in Example 1-1.

[0124] Table 7 shows the results of fluorescence intensity measurements using primer pair A and primer pair B in samples 3 and 4 that do not contain the normal cell-derived c-ABL gene, respectively. As shown in Table 7, in both cases when primer pair A and primer pair B were used, a correlation was obtained between the mixing ratio of the sample mixed as the template and the mutation ratio based on the signal intensity.

[0125] [Table 7]

[0126]

[0127]

Embodiment 2-3

[0128] [Example 2-3] Evaluation of primer pairs

[0129]In this example, 1 ng of sample 4 and 100 ng of sample 2 were added as templates. In addition, the hybridization reaction by PCR and a DNA chip, the subsequent fluorescence detection, etc. were performed in the same manner as in Example 1-1.

[0130] Table 8 shows the results of measuring the fluorescence intensity using primer pair A and primer pair B when sample 2 containing the c-ABL gene derived from normal cells is included. As shown in Table 8 below, when primer pair A is used, the mutation ratio (mutant type: 100%) of the BCR-ABL fusion gene used as a template is correlated with the detected signal intensity. On the other hand, when primer pair B was used, the ratio of mutations mixed as a template and the signal intensity of detection were reversed, and it was found that it was greatly affected by the sample 2 that did not have the BCR-ABL fusion gene. From this, it can be seen that when the primer pair A is use...

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Abstract

The invention provides a method for detecting BCR-ABL inhibitor resistance-related mutations, and a data acquisition method for predicting resistance to BCR-ABL inhibitor using said method. The objective of the present invention is to detect, with a higher degree of accuracy, mutations related to resistance to BCR-ABL inhibitors in chronic myeloid leukemia and the like. This method comprises: a step in which, using a biological sample taken from a subject, regions that include fusion sites contained in BCR-ABL fusion genes and mutation sites involved in resistance to BCR-ABL inhibitors are amplified; and a step in which, using nucleic acid amplified in the aforementioned step, the genotype of the mutation site that is involved with resistance to BCR-ABL inhibitors is determined.

Description

technical field [0001] The invention relates to a detection method for BCR-ABL inhibitor resistance-related mutations in chronic myelogenous leukemia and the like and a data acquisition method for predicting BCR-ABL inhibitor resistance using the detection method. Background technique [0002] In almost all cases of chronic myeloid leukemia (CML), a gene mutation called Ph chromosome (Philadelphia chromosome) can be confirmed. Chromosome 9 of the Ph chromosome translocates with the respective long arms of chromosome 22. Due to this translocation, the BCR (breakpoint cluster region) on chromosome 22 overlaps with the ABL gene region on chromosome 9 to generate a BCR-ABL fusion (chimeric) gene. The fusion protein BCR-ABL encoded by the fusion gene continuously increases the activity of tyrosine kinase, which is the root cause of chronic myelogenous leukemia. [0003] On the other hand, BCR-ABL inhibitors represented by imatinib are currently used as molecular targeted therap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09C12N15/11G16B20/00
CPCC12Q2600/106G16B20/00C12Q1/6886C12Q1/6853C12Q2600/156C12Q2600/158
Inventor 大场光芳阿部健一山野博文汤尻俊昭
Owner TOYO KOHAN CO LTD
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