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Method for determining prognosis of renal cell carcinoma

a renal cell carcinoma and prognosis technology, applied in the field of renal cell carcinoma prognosis determination, can solve the problems of time and labor required for electrophoresis used in analysis, the reaction rate of sulfonation as the first step is very slow, and the methylated cytosine is left unaltered throughout the reaction, so as to improve the survival rate of cancer patients, the risk of recurrence in cancer patients can be determined more quickly, convenient and highly accura

Inactive Publication Date: 2018-11-29
NAT CANCER CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a way to quickly and accurately determine if a cancer patient is likely to come back or come back with a more aggressive form of cancer. This helps doctors make treatment decisions for cancer patients faster, which can improve their chances of survival.

Problems solved by technology

On the other hand, methylated cytosine is left unaltered throughout the reaction time of actually performed bisulfite treatment because the reaction rate of sulfonation as the first step is very slow.
A problem of the methods, however, is time and labor required for electrophoresis used in the analysis.
Thus, it is difficult to predict the prognosis of RCC using existing clinicopathological factors or the like.
This causes problems that the presence or absence of cells having highly methylated DNA cannot be determined, and a risk of estimating DNA methylation rates to be lower than actual values cannot be avoided.

Method used

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  • Method for determining prognosis of renal cell carcinoma
  • Method for determining prognosis of renal cell carcinoma
  • Method for determining prognosis of renal cell carcinoma

Examples

Experimental program
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Effect test

reference example 1

[Reference Example 1] Determination of CIMP Negativity or Positivity by Conventional Method

[0160]The determination of CIMP negativity or positivity by a conventional method was conducted according to the MassARRAY method described in Patent Literature 4 (Example 5). The DNA methylation levels at the CpG sites of 17 genes (FAM150A, GRM6, ZNF540, ZFP42, ZNF154, RIMS4, PCDHAC1, KHDRBS2, ASCL2, KCNQ1, PRAC, WNT3A, TRH, FAM78A, ZNF671, SLC13A5, and NKX6-2) (Tables 1 to 4) were detected by the MassARRAY method, which is one of the methods for detecting methylated DNA.

[0161]The MassARRAY method is a method which involves amplifying DNA after bisulfite treatment, transcribing the amplification product to RNA, further cleaving the RNA with RNase in a base-specific manner, and then detecting the difference in molecular weight between a methylated DNA fragment and an unmethylated DNA fragment using a mass spectrometer.

[0162]First, MassARRAY primers were designed for CpG islands containing the ...

reference example 2

[Reference Example 2] Preparation of Anion Exchange Column

[0166]In a reactor equipped with a stirrer, a mixture of 200 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 100 g of triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 100 g of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.), and 1.0 g of benzoyl peroxide (manufactured by Kishida Chemical Co., Ltd.) was added to 2,000 mL of an aqueous solution containing 3 wt % of polyvinyl alcohol (manufactured by The Nippon Synthetic Industry Co., Ltd.). The reaction mixture was heated with stirring and polymerized at 80° C. for 1 hour in the nitrogen atmosphere. Next, 100 g of ethyl methacrylate trimethylammonium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved as the hydrophilic monomer having the strong cationic group in ion exchange water. This solution was added to the reactor mentioned above. Similar...

reference example 3

[Reference Example 3] Detection of Methylated DNA by Ion Exchange Chromatography

(1) Extraction and Bisulfite Treatment of Genomic DNA

[0169]Fresh frozen tissue samples obtained from the patients were each treated with phenol-chloroform and subsequently dialyzed to extract high-molecular-weight DNA (see Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY, p. 6.14-6.15). 500 ng of the DNA was treated with bisulfite using EZ DNA Methylation-Gold™ kit (manufactured by Zymo Research Corp.).

(2) PCR

[0170]The bisulfite-treated genomic DNA obtained in the preceding step (1) was amplified by PCR. The PCR was performed using a 25 μL of a reaction solution containing 10 ng of template DNA, GeneAmp 1×PCR buffer (manufactured by Life Technologies Corp.), 200 μmol / L GeneAmp dNTP Mix (manufactured by Life Technologies Corp.), 0.75 U AmpliTaq Gold DNA Polymerase (manufactured by Life Technologies Corp.), and 0.25 μmol / L forward and reverse...

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Abstract

It is intended to provide a rapid, convenient, and highly accurate method for determining the prognosis of cancer. The present invention provides a method for determining the prognosis of a renal cell carcinoma patient, comprising: (1) treating genomic DNA prepared from a renal tissue of a subject with bisulfite; (2) amplifying the bisulfite-treated DNA by PCR; (3) subjecting the obtained PCR amplification product to ion exchange chromatography; (4) obtaining the retention time of a detection signal obtained by the chromatography; and (5) determining the renal cell carcinoma of the subject as having poor prognosis when the result of the step (4) is shorter than a retention time serving as a reference.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation of U.S. application Ser. No. 15 / 121,656, filed on Aug. 25, 2016, which is a 35 U.S.C. § 371 national stage patent application of International patent application PCT / JP2015 / 056108, filed on Mar. 2, 2015, and claims the benefit of the filing date of Japanese application nos. 2014-039417, filed on Feb. 28, 2014, and 2014-044943, filed on Mar. 7, 2014.FIELD OF THE INVENTION[0002]The present invention relates to a method for determining the prognosis of renal cell carcinoma by use of the detection of methylated DNA.BACKGROUND OF THE INVENTION[0003]In recent years, abnormal methylation of DNA has been found to be deeply involved in malignant transformation and has received attention. Abnormal DNA methylation of CpG islands in some gene promoter regions is known as a characteristic epigenetic abnormality in cancer cells. The CpG island is a region in which a two-nucleotide sequence of cytosine (C)-guani...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/6886G01N30/88C12Q1/6827G01N30/96B01D15/36
CPCG01N30/88C12Q1/6886C12Q2600/154G01N30/96G01N2030/8813C12Q2600/118B01D15/362C12Q1/6827B01D15/363C12Q2523/125C12Q2565/137
Inventor KANAI, YAEARAI, ERINEMOTO, YURIKOYOTANI, TAKUYA
Owner NAT CANCER CENT