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Methods and kits for molecular subtyping of bladder cancer

A kit and technology for bladder cancer, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of lack of sensitivity, failure to lead to bladder cancer prognosis classification, etc.

Active Publication Date: 2022-02-11
百欧恩泰诊断有限责任公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, determination of individual risk by testing multiple markers by an IHC-based approach does not lead to a reliable prognostic classification of bladder cancer, which may help resolve the described diagnostic and therapeutic dilemma
In addition, IHC tests often lack sensitivity

Method used

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  • Methods and kits for molecular subtyping of bladder cancer
  • Methods and kits for molecular subtyping of bladder cancer
  • Methods and kits for molecular subtyping of bladder cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0397] Example 1: Determination of mRNA expression levels by reverse transcription (RT) quantitative PCR (RT-qPCR)

[0398] RNA was isolated from paraffin-embedded formalin-fixed tissues (=FFPE tissues). More specifically, use Total RNA was extracted from 5–10 μm curled FFPE tumor tissue with an extraction kit (BioNTech Diagnostics GmbH, Mainz, Germany) and quantified by real-time fluorescent RT-PCR using CALM2 as a reference gene. Typically, 2.5 μL (approximately 50-100 ng) of each qualified RNA extract was analyzed by qRT-PCR as described below.

[0399] For detailed analysis of gene expression by quantitative RT-PCR method, primers flanking the region of interest and a fluorescently labeled probe hybridized in between were used. Using the NCBI primer design tool ( www.ncbi.nlm.nih.go ) to select target-specific primers and probes. RNA-specific primer / probe sequences were used to enable RNA-specific measurements by targeting primer / probe sequences across exon / exon bo...

Embodiment 2

[0404] Example 2: Molecular subtypes of NMIBC tumors based on mRNA expression levels of HER2, ESR1, PGR and Ki67

[0405] For the purposes of the present invention, FFPE tissues from 82 NMIBC patients with pT1 stage who had undergone standard treatment were analyzed. Total RNA was extracted using a commercial RNA extraction kit, and mRNA expression levels were measured by one-step RT-qPCR using the RNA-specific TaqMa assay. Relative gene expression levels were determined by normalizing gene expression levels using the 40-ΔΔCT method against synthetic in vitro transcripts of target genes and two housekeeping genes (CALM2, B2M) as controls. The prognostic value of mRNA expression levels of ESR1 and PGR was analyzed by unsupervised cluster analysis, partition test, Mann Whitney test, and Kaplan Meier analysis to assess cancer-specific survival (CSS). The median follow-up time was 62 months.

[0406] The mean threshold values ​​(given as 40-AACT values) are as follows: HER2: 40....

Embodiment 3

[0417] Example 3: MIBC tumor molecular subtyping based on mRNA expression levels of HER2, ESR1, PGT and Ki67

[0418] The mRNA expression levels of ESR1, PGR, HER2, and Ki67 in FFPE tissues of patients with muscle-invasive bladder cancer (MIBC pT1-4; Nx) were analyzed by RT-qPCR.

[0419] Gene expression in FFPE tissues from 92 patients (22 females; 70 males; median age 67 [range 45-97]; 23 pT1 / 2, 48 pT3, 21 pT4) Retrospective analysis and one-step RT-qPCR were performed using the RNA-specific TaqMa assay to measure mRNA expression levels. Relative gene expression levels were determined by normalizing gene expression levels using the 40-ΔΔCT method against synthetic in vitro transcripts of target genes and two housekeeping genes (CALM2, B2M) as controls. The prognostic value of individual genes and typing algorithms was analyzed by unsupervised partition test, Mann Whitney test, and Kaplan Meier analysis to assess disease-specific survival (DSS).

[0420] Compared with HER...

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Abstract

The present invention relates to an in vitro method for identifying molecular subtypes of tumors in bladder cancer patients and methods for stratifying bladder cancer patients for tumor therapy. The present invention also relates to a corresponding kit and its use, and a prognostic biomarker for bladder cancer, especially a single prognostic biomarker.

Description

technical field [0001] The present invention relates to an in vitro method for identifying molecular subtypes of tumors in bladder cancer patients and methods for stratifying bladder cancer patients for tumor therapy. The present invention also relates to a corresponding kit and its use, and a prognostic biomarker for bladder cancer, especially a single prognostic biomarker. Background technique [0002] Based on histopathological assessment, bladder cancer is considered to be very heterogeneous and includes all the different histopathological subtypes (papillary, non-papillary, squamous cell carcinoma, adenocarcinoma, sarcoma, small cell carcinoma, etc.). Bladder cancer is also known to have a widely variable clinical outcome with marked variability in response to local or systemic therapy. Very important for the prognosis and treatment of bladder cancer is the depth of invasion. [0003] Tumors that do not invade the muscle layer are called superficial or non-muscle-inva...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/158C12Q2600/112
Inventor 拉尔夫·马库斯·维尔茨
Owner 百欧恩泰诊断有限责任公司