Journal List > J Breast Cancer > v.10(1) > 1036089

Lee, Yim, Lee, Park, Lee, and Lee: Association between Promoter Hypermethylation of the p16INK4a and hTERT Genes and Their Protein Expressions in Human Breast Cancer

Abstract

Purpose

This study was undertaken to observe the pattern of methylation of the p16INK4a and human telomerase reverse transcriptase (hTERT) genes and the p16 and hTERT protein expressions in invasive ductal carcinoma of the breast. In addition, we evaluated the relationship between the methylation status of the two genes and their protein expressions.

Methods

We performed methylation-specific PCR (MSP) and immunohistochemical staining in 63 breast cancer specimens.

Results

There was no statistical association between p16INK4a gene methylation and the histological grade (tumor grade, tumor size and lymph node status). Methylation of the hTERT promoter did show significant differences according to the histological tumor grade and tumor size, but there was no clinical significance. Methylation of the p16INK4a and hTERT genes was found in 22.2% and 31.8% of the specimens, respectively. A negative p16 protein expression (0-10% expression rate) was observed in 38.1% of the specimens (24 of 63). A positive hTERT expression (more than a 25% expression rate) was observed in 73.0% of the specimens (46 of 63). There was no statistical significance in the relationship between the methylation status and the protein expression.

Conclusion

Our data suggest that methylation of the p16 and hTERT genes is not associated with their protein expressions according to Immunohistochemisty. There seemed to be another complicated mechanism for p16 inactivation and hTERT activation in breast cancer.

Figures and Tables

Fig 1
Representative Methylation-specific PCR (MSP) results are shown for four genes (p16 and hTERT) in breast cancer sample. MSP was performed on bisulfite-treated DNA form breast cancer. MSP products were run on a 2% agarose gel. unmethylated DNA 〈U〉, methylated DNA 〈M〉, negative control (DW) 〈-〉, positive control 〈+〉, 100 bp DNA ladder 〈L〉.
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Fig 2
Examples of immunohistochemical staining: (A) p16 staining of normal breast. (B) p16 positive in breast cancer. (C) hTERT staining of normal breast. (D) hTERT positive in breast cancer. All sections is ×400.
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Fig 3
Relationship between the methylation status in promoter lesion for p16, hTERT gene and immunohistochemistry positive cell percentage for p16, hTERT protein. Notes: MSP (+)=only methylation PCR (+) or metylation PCR (+) and unmethylation PCR (+); MSP (-)=only unmethylation PCR (-).
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Table 1
Clinicopathological characteristics of breast cancer (n=63) used in this study
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*: Modified Scarff-Bloom-Richardson grading system.

Table 2
Methylation of p16 and hTERT from representative Methylation-specific PCR (MSP) assay
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MSP (+)=only methylation PCR (+) or metylation PCR (+) and unmethylation PCR(+); MSP (-)=only unmethylation PCR (-); NA=metylation PCR (-) and unmethylation PCR (-).

Table 3
Relationship between clinicopathological finding and methylation
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Table 4
Cell counting of p16 and hTERT from immunohistochemistry are shown (%)
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IHC (-)=p16 ≤10% & hTERT ≤25%; IHC (+)=p16>10% & hTERT >25%.

Table 5
Relationship between immunohistochemistry and methylation status (p16 and hTERT)
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