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A uveal melanoma metastasis risk prediction kit

A technology for melanoma and uvea, applied in the field of disease molecular diagnosis kits, can solve problems such as unclear mechanism and relationship

Active Publication Date: 2022-08-02
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The relationship between EZH2 and UM is not yet clear. There are only two reports on UM cell lines, and EZH2 can silence the IFN-γ-induced MHC2TA transcription level; UM cell lines are resistant to EZH2 inhibitor EPZ-6438, and the mechanism is not yet clear. clear

Method used

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  • A uveal melanoma metastasis risk prediction kit
  • A uveal melanoma metastasis risk prediction kit
  • A uveal melanoma metastasis risk prediction kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 PCR detection and sequencing kit composition of the present invention

[0033] The kit of the invention includes amplification reagents for amplifying the p.E725K mutation site of the EZH2 gene, and reagents for Sanger sequencing.

[0034] 1. Amplification reagent

[0035] The PCR amplification reagent is used to amplify a DNA sequence where the SNP site is located, and its composition is shown in Table 1.

[0036] Table 1 PCR amplification reagents

[0037] component concentration volume PCR mix 2× 600μl primer pair 10μM 100μl pure water 2ml

[0038] The PCR mixture in Table 1 includes Taq enzyme, dNTP, magnesium ions and other components required for conventional PCR; the primer pair information is shown in Table 2.

[0039] Table 2 Primers used for gene amplification

[0040]

[0041] 2. Sequencing reagents

[0042] The reagent included the components shown in Table 3.

[0043] Table 3 Gene variant typing...

Embodiment 2

[0047] Example 2 The method of using the kit of the present invention

[0048] 1. DNA extraction

[0049] The paraffin blocks of UM patients were extracted for sectioning, 5-10 μm thick, 5-6 slices. Use a scalpel to scrape off the tumor tissue on the section, try to scrape only the target tissue part, and remove excess paraffin. The scraped tissue was collected in a 1.5 mL EP tube, 1 mL of xylene was added, and 1 mL of xylene was added to mix well. After fully dissolving the paraffin, centrifuge at 12,000 rpm for 2 min at room temperature, and discard the supernatant. Add 1 ml of absolute ethanol, shake and mix well, centrifuge at 12,000 rpm for 2 min at room temperature, and discard the supernatant. Open the cap and dry at room temperature or 37°C for 5-10 minutes to completely remove residual ethanol. Add 200 μl of lysis buffer and 20 μl of Proteinase K, mix well, and incubate at 55°C until the sample is completely lysed. Incubate in a 90°C water bath for 1 h, add 200 μl...

experiment example 1

[0069] Experimental Example 1 The relationship between UM metastasis and p.E725K mutation

[0070] 1. Selection of experimental subjects

[0071] From 2009 to 2017, 85 patients with primary UM were collected from West China Hospital of Sichuan University. Among them, 19 had UM cell metastasis within 5 years of illness, and 66 had no UM cell metastasis within 5 years of illness. The rate was 22.35%. The specific clinical case parameters are shown in Table 7.

[0072] Table 7 Clinicopathological parameters of 85 UM patients

[0073]

[0074] 2. Sequencing

[0075] The method of Example 2 was used to sequence the p.E725K mutation site of the EZH2 gene of the experimental subject.

[0076] 3. Sequencing results

[0077] The results showed that there was a p.E725K mutation site in the EZH2 gene of the experimental subjects, and the mutation rate was 41.2% (35 / 85). Among the patients with UM cell metastasis for 5 years, 13 patients carried the p.E725K mutation (metastasis). ...

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Abstract

The invention provides a new uveal melanoma metastasis risk prediction kit, which belongs to the field of disease molecular diagnosis kits. The invention expounds the relationship between the p.E725K mutation site of the EZH2 gene and the metastasis of uveal melanoma, and provides a new type of kit, which realizes grapevine by detecting the p.E725K mutation site of the EZH2 gene. Membranous melanoma metastasis risk is effectively predicted and has good application prospects.

Description

technical field [0001] The invention belongs to the field of disease molecular diagnosis kits. Background technique [0002] Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults, and its incidence ranks first in intraocular tumors. It can involve the choroid, iris and ciliary body, and choroidal melanoma is the most common. . It has a high degree of malignancy and a poor prognosis, which can not only lead to vision loss, but also cause distant metastasis to the liver and lungs, which seriously threatens the life of patients. The 10-year disease-related survival rate is only 50%. [0003] In the past three decades, the diagnosis and treatment of UM have improved significantly. The DecisionDx-UM test can classify UM patients into Class 1 (low risk of metastasis) and Class 2 (high risk of metastasis). The main treatment methods for UM are brachytherapy, laser therapy, local excision and enucleation, but the 5-year survival rate of patients has ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/156C12Q2600/118
Inventor 闫乃红肖丽容侯宸
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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