DNA methylation and mutational analysis methods for bladder cancer surveillance

A technology of bladder cancer and methylation, applied in the fields of biochemical equipment and methods, analytical materials, biomaterial analysis, etc.

Pending Publication Date: 2019-12-27
基美健有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Typically, CpG dinucleotides in CpG islands are unmethylated in normal cells, however CpG dinucleotides found in isolated sequence segments in the coding regions of genes may be methylated in normal cells

Method used

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  • DNA methylation and mutational analysis methods for bladder cancer surveillance
  • DNA methylation and mutational analysis methods for bladder cancer surveillance
  • DNA methylation and mutational analysis methods for bladder cancer surveillance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1: Method of evaluating methylation and proof-of-concept study

[0133] Urine cell sediments from 66 patients with bladder cancer (25 patients without recurrence, 14 low-risk (LR) (Ta low grade) patients, and 27 high-risk (HR) (T1 or high grade) patients) were used for conception Validation (POC) study. 11 methylation gene markers and 1 methylation-independent (MIP) reference gene selected based on literature were used to assess recurrence. Methylation-specific quantitative PCR (MS-qPCR) analysis after bisulfate conversion was used to determine the methylation levels of these 12 genes. This study was used to evaluate the performance of individual methylation markers and the combined score for predicting the presence of recurrence. Based on this research, several methods for evaluating methylation signals have been developed and used in the refinement of a larger set of candidate markers.

[0134] The methylation fraction (MF) of each gene is estimated from the met...

Embodiment 2

[0143] Example 2: Methylome discovery study

[0144] A gene discovery study was conducted to identify other gene candidates that are differentially methylated in samples from patients with bladder cancer and patients without cancer. A total of 26 tumor tissue samples and 7 urine cells from cancer patients (14 patients previously diagnosed with low risk of recurrence (LR) and 19 patients previously diagnosed with high risk of recurrence (HR)) were used in the analysis The sediment, as well as 7 urine cell sediments and 5 tissue samples from cancer-free patients (7 healthy individuals and 5 patients who were not previously diagnosed with bladder cancer or who were previously diagnosed with no malignancy at the time of surgery). Based on the analysis of the Cancer Genome Atlas (TCGA), the methylome assessment targets approximately 6 million nucleotides in regions that are differentially methylated in 11 cancers, and approximately 724,000 sites are sequenced for each sample. The fol...

Embodiment 3

[0146] Example 3: Refinement study

[0147] This detailed study evaluated 96 markers on 203 previously diagnosed bladder cancer patients currently under surveillance, including 86 methylation marker genes from gene discovery studies, 4 methylation-independent reference genes, and 6 FGFR3 mutations. Two of the six FGFR3 mutation assays failed to generate any signal, and further evaluation of these two was excluded. In addition, 170 patients in this study had enough RNA for other evaluations, including the determination of 2 TERT mutations. The results presented here are based on these 170 patients, which evaluated 86 methylation markers, 4 reference genes, 4 FGFR3 mutations, and 2 TERT mutations. The patients included 85 patients who had not relapsed, 32 patients who were previously diagnosed with LR relapse, and 53 patients who were previously diagnosed with HR relapse.

[0148] After bisulfate conversion, RT-PCR analysis was used to determine the methylation level. Non-paramet...

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Abstract

The present disclosure relates to methods of monitoring bladder cancer patients and analyzing patient samples for presence of methylated DNA and optionally particular gene mutations. In some embodiments, analysis results are correlated with clinical outcome measures such as risk of bladder cancer recurrence.

Description

Technical field [0001] The present disclosure relates to methods for monitoring patients with bladder cancer and analyzing patient samples for the presence of methylated DNA and optionally specific gene mutations. In some embodiments, the results of the analysis are correlated with clinical outcome indicators, such as the risk of bladder cancer recurrence. Background technique [0002] Bladder cancer is one of the most common cancers in industrialized countries and one of the most expensive cancers to diagnose and monitor. (See R. Siegel et al., CA Cancer J Clin 63: 11-30 (2013); and see, for example, T. Reinert Adv. Urol., Article ID 503271, doi: 10.1155 / 2012 / 503271, pages 1-11 (2012 ); see also A. Feber et al., Clin. Epigenetics 9: 8 doi: 10.1186 / s13148-016-0303-5 (2017)). For example, most new cases are presented as non-muscle invasive bladder cancer, with a low probability of metastasis, but still have the potential to develop into more aggressive diseases (for example, hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/106C12Q2600/154A61P35/00G01N2800/52G01N2800/56G01N2800/7028
Inventor M·洛帕廷A·蒂西亚斯C·N·西尔克D·P·米勒M·克雷格P·菲波D·克内泽维奇
Owner 基美健有限公司
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