Eml4-alk gene mutation analysis method

An analysis method and gene technology, applied in the field of EML4-ALK, can solve the problems of cost and time

Pending Publication Date: 2020-04-10
CYTOGEN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ALK gene mutations represent various types of gene fusions, and immunohistochemistry (immunohistochemistry), reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), etc. are available as methods for detecting them In the clinic, currently, as a standard companion diagnostic test for administering crizotinib, Abbott's Vysis ALK fluorescence in situ hybridization method approved by the US Food and Drug Administration is utilized, but this method exists Problems requiring a lot of cost and time when testing many patients due to difficulty and complexity

Method used

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  • Eml4-alk gene mutation analysis method
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  • Eml4-alk gene mutation analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Preparation of high-density microchips

[0048] For the high-density microchip used in the experiment, the pore-size is formed from 5.5 to 8.5 μm, and white blood cell (WBC) and red blood cell (RBC) are smaller than 5.5 μm. The chip is removed by passing it through, and the target cells with a size greater than 5.5 μm are microchips developed in a way that they can be prepared into a chip to selectively recover a specific size.

[0049] For reference, in order to confirm the cell recovery rate of high-density microchips, 10, 100, and 1000 cancer cells of cancer patients were spiked through the chip to perform the recovery of cancer cells on the observation chip. Rate of experiment. The results are shown in Table 1 below.

[0050] Table 1

[0051] Sample Number of spiked cells Number of recovered cells Cell recovery rate (%) 110990 21008686 3100085085

[0052] Calculating the cell recovery rate of the chip, the result showed a high cell recovery rate of about 8...

Embodiment 2

[0053] Example 2: Separation process of circulating cancer cells (CTC) in blood

[0054] 1. Put 250μl of antibody polymer in 5ml of blood, mix for about 3 seconds, and react for 20 minutes at room temperature.

[0055] 2. Add 5 ml of phosphate buffered saline (PBS) containing 1% fetal bovine serum (FBS).

[0056] 3. Carefully put 10 ml of the reaction solution in a 50 ml tube containing 15 ml of Ficoll solution.

[0057] 4. Centrifuge the solution at 1200g for 20 minutes to remove blood cells for the first time.

[0058] 5. In order to prevent the adsorption of unnecessary cells, a high-density microchip (HD Microporous chip, filter) is treated with 0.1% bovine serum albumin solution for 10 minutes and coated, and then washed with phosphate buffer.

[0059] 6. Put the supernatant solution of Ficoll on the filter, and gravity filter the red blood cells present in a small amount to separate high-purity circulating cancer cells in the blood for the second time. It does not perform centrif...

Embodiment 3

[0061] Example 3: Short-term culture of circulating cancer cells in isolated blood

[0062] Circulating cancer cells in the blood separated by the high-density microchip of the present invention are seeded in an ultra low attachment culture coated with a hydrogel with neutral charge and hydrophilicity. board. The above-mentioned culture plate contains a culture medium containing 11 ng / ml insulin, 22 ng / ml transferrin, 2 ng / ml EGF and 8 μm Roche kinase inhibitor. For the above-mentioned short-term culture, from the time point of culture to 14 During the day, in a cell incubator at 37℃ and 5-10% CO 2 Culture under the conditions.

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Abstract

According to an embodiment of the present invention, a qRT-PCR primer capable of detecting blood-borne circulating cancer cell-based EML4-ALK gene mutations at a higher sensitivity detection limit than existing methods is provided. According to another embodiment of the present invention, EML4-ALK gene mutations can be detected using blood-borne circulating lung cancer tumor cells even in the caseof lung cancer patients for whom ALK-FISH testing has been impossible due to the difficulty of solid biopsy collection of lung cancer tissue, and thus a lung cancer patient screening method for determining whether an anticancer drug targeted to EML4-ALK gene mutations can be applied to a lung cancer patient is provided.

Description

Technical field [0001] The present invention relates to a method for detecting EML4-ALK mutations, and in more detail, to a polymerase chain reaction (PCR) primer pair for detecting EML4-ALK mutations based on circulating cancer cells in the blood, using nested polymerase chain A method for detecting the reactive EML4-ALK mutation and a method for screening patients with non-small cell lung cancer using cancer cells derived from the blood of patients with non-small cell lung cancer as anticancer agents. Background technique [0002] Lung cancer occupies the fourth place (10.3%) among the domestic cancer patients (202053) in Korea in 2010, and is a common cancer in Koreans. However, the 5-year survival rate is 19.7%, which is a poor prognosis compared with other cancers. Lung cancer is divided into non-small cell lung cancer and small cell lung cancer according to the size and morphology of the cancer cells through the microscope. In this way, when distinguishing between non-smal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806
CPCC12Q1/6886C12Q2600/156C12Q2600/106C12Q1/6806C12Q2527/156C12Q2600/158C12Q2549/119C12Q1/6827G01N2800/7028
Inventor 全炳熙
Owner CYTOGEN CORP
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