Methylation Analysis On Self-samples As Triage Tool For HPV-positive Women

A methylation, sample technology, used in cancer prevention and medical diagnostics, to solve problems such as inability to perform

Inactive Publication Date: 2014-09-17
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this set of tests did not perform well on self-sampled cervix-vaginal samples, primarily because the signal for CADM1 methylation in self-sampled samples from women with ≥CIN2 / 3 was higher than in physician-collected cervical samples. Below the inspection threshold more frequently in the case of

Method used

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  • Methylation Analysis On Self-samples As Triage Tool For HPV-positive Women
  • Methylation Analysis On Self-samples As Triage Tool For HPV-positive Women
  • Methylation Analysis On Self-samples As Triage Tool For HPV-positive Women

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. HPV-immortalized (immortalized) keratinocyte line (HPV-immortalized keratinocyte cell lines) and cervical cancer cell lines Hsa-miR124 methylation

[0064]Because the mature Hsa-miR124 sequence is encoded at three distinct genomic locations (hsa-miR124-1[8p23.1], hsa-miR124-2[8q12.3], and hsa-miR124-3[20q13.33]), The hsa-miR124 methylation status was therefore determined at all 3 loci by quantitative methylation-specific PCR (MSP). The following amplicons were detected: hsa-miR124-1: nt811 to 904; hsa-miR124-2: nt701 to 838; hsa-miR124-3: nt897 to 991, each involving figure 2 sequence shown in . The housekeeping gene B-actin was used as an internal reference (Harden et al., J Urology 2003; 169:1138-1142). Using these assays, we did not find hsa-miR124 methylation (EK) in primary keratinocytes isolated from three different donors, however, in three hrHPV-containing cervical cancer cell lines (SiHa, HeLa and CaSki ) methylates the corresponding CpG is...

Embodiment 2

[0065] Example 2: Overexpression of hsa-miR124 in SiHa cells has anti-proliferative and anti-migratory effects effect

[0066] To determine the possible functional relevance of reduced hsa-miR124 expression in cervical carcinogenesis, two cervical cancer cell lines, SiHa and CaSki, were transduced with a retroviral vector containing the genetic components leading to stable expression of hsa-miR124. Expression analysis by RT-PCR confirmed ectopic expression of hsa-miR124 in hsa-miR124 transductants and lack of expression in cells transduced with empty retroviral vector. After 6 days of culture, ectopic expression of hsa-miR124 in SiHa and CaSki transductants resulted in a significant decrease in cell proliferation compared to hsa-miR-ctrl transducers (p<0.05). Furthermore, using the wound healing assay, SiHa hsa-miR124 transductants were found to have reduced migratory ability compared to SiHa hsa-miR-ctrl cells.

Embodiment 3

[0067] Example 3: Hsa-miR124 promoter methylation in cervical tissue samples

[0068] To determine if and when DNA hypermethylation of all three hsa-miR124 regulatory regions occurs during the course of cervical carcinogenesis in vivo, we performed mild CIN lesions ( CIN1), severe CIN lesions (CIN3) and cervical cancers (SCCs and AdCas), quantitative MSP analysis was performed using primers and probes as defined in Table 2. In normal cervix, 6% (1 / 18) samples were positively marked for methylation on hsa-miR124-3, while in none of the normal samples, methylation was detected on the other two gene regulatory regions ( hsa-miR124-1 and hsa-miR124-2). In CIN1 lesions, 28% (10 / 36) showed methylation on hsa-miR124-1, 6% (2 / 36) on hsa-miR124-2 and 11% ( 4 / 36). Methylation positivity in CIN3 lesions ranged from 46% (19 / 41) in hsa-miR124-1, 20% (8 / 41) in hsa-miR124-2 to 10% (4 / 41) in hsa-miR124-3 different. The frequencies of hsa-miR124-1, hsa-miR124-2 and hsa-miR124-3 as detec...

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Abstract

The inventors now have developed a (molecular) diagnostic assay based on detection of alterations in markers MAL and hsa-mi R124, in particular promoter hypermethylation, to identify human papillomavirus (HPV)-induced high-grade precancerous lesions such as premalignant cervical lesions of invasive cervical cancer, within cell material obtained via self- sampling. In particular, the present invention relates to the use of the MAL gene (including its promoter) and the hsa-mi R124 gene as marker for HPV- induced high-grade premalignant lesions, allowing early detection and a better treatment option for the individual patient.

Description

technical field [0001] The present invention relates to the fields of cancer prevention and medical diagnostics; and to molecular diagnostic tests for human papillomavirus (HPV)-induced invasive (invasive) cancer and its severe precursor lesions, Such as invasive cervical cancer and premalignant (premalignant) cervical lesions. In particular, the present invention relates to the comprehensive analysis of MAL and hsa-miR124 promoter methylation in (hrHPV-positive) self-sampled samples in hrHPV-induced precancerous lesions with invasive potential (invasive potential). and hrHPV-induced invasive cancer assays. Background technique [0002] Cervical cancer is the second most common cancer in women worldwide and causes approximately 250,000 cancer deaths a year. [0003] The development of squamous cell carcinoma of the cervix is ​​characterized by a series of precancerous lesions, the so-called cervical epithelial sarcomatoid lesions (CIN), which are divided into grades 1 to 3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/178C12Q1/6886C12Q2600/154
Inventor 彼得鲁斯·约瑟夫斯·费丁安杜斯·斯尼杰德尔斯伦斯克·丹尼拉·玛丽亚·斯泰恩贝尔根丹尼勒·安妮·玛丽·海德曼克里斯托弗鲁斯·琼内斯·拉姆贝图斯·玛丽亚·迈耶
Owner SELF SCREEN
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