Elsevier

Human Pathology

Volume 39, Issue 11, November 2008, Pages 1637-1646
Human Pathology

Original contribution
Methylation of CpG islands of p16INK4a and cyclinD1 overexpression associated with progression of intraductal proliferative lesions of the breast

https://doi.org/10.1016/j.humpath.2008.04.001Get rights and content

Summary

P16INK4a is a tumor suppressor gene frequently inactivated by aberrant promoter hypermethylation. In this study, p16INK4a methylation was evaluated in intraductal proliferative lesions of the breast, using real-time quantitative polymerase chain reaction (MethyLight) and methylation-sensitive restriction endonuclease polymerase chain reaction. Immunohistochemistry was performed to compare and validate the methylation analysis. P16INK4a methylation associated with oncogene cyclinD1 expression, detected through the use of in situ hybridization and immunohistochemistry, was likewise characterized. P16INK4a methylation displayed varying significance among different types of intraductal proliferative lesions. Both the positive rate and the median quantitative methylation value increased with the evolution of intraductal proliferative lesions through the use of quantitative and qualitative assays. P16INK4a methylation was positively correlated to cyclinD1 overexpression. This study demonstrated that p16INK4a methylation served as the silencing mechanism of p16INK4a protein expression and played a crucial role in the intraductal proliferative lesions' progression. In the differential diagnosis of intraductal proliferative lesions, quantitative DNA methylation analysis of p16INK4a by MethyLight may be used as a surrogate, especially to distinguish atypical ductal hyperplasia from usual ductal hyperplasia and low-grade ductal carcinoma in situ. Furthermore, this study discovered that flat epithelial atypia do not share similar molecular profiles of p16INK4a epigenetic modification with atypical ductal hyperplasia and low-grade ductal carcinoma in situ.

Introduction

Transcriptional inactivation by cytosine methylation at promoter CpG islands of tumor suppressor genes is believed to be a mechanism vital to human carcinogenesis. The p16INK4a gene, a widely known tumor suppressor gene, is localized on chromosome 9p21; it can form complexes with CDK4, CDK6, and D-type cyclins to arrest the cell cycle's progression from the G1 to the S phase. According to earlier observations, p16INK4a is suppressed by aberrant promoter hypermethylation in diverse types of malignant tumors, including breast cancer, and in recent years, its inactivation has been gaining increased attention with the development of molecular pathology [1], [2], [3], [4]. For example, Kimberly et al [5] described a new function of p16INK4a and demonstrated that the loss of p16INK4a expression generated supernumerary centrosomes, thus driving genomic instability, which was considered an early event in tumorigenesis.

As an oncogene, cyclinD1 has been deemed important in breast carcinogenesis, whereas its protein ranks among the frequently overexpressed proteins in breast cancer [6]. Both p16INK4a and cyclinD1 are important molecules in breast carcinogenesis. However, no study to date has comprehensively examined interrelations among p16INK4a methylation, p16INK4a protein, cyclinD1 messenger RNA (mRNA), and cyclinD1 protein in the spectrum of intraductal proliferative lesions (IPL) of the breast.

IPL was divided into usual ductal hyperplasia (UDH) (Fig. 1), flat epithelial atypia (FEA) (Fig. 2), atypical ductal hyperplasia (ADH) (Fig. 3), and ductal carcinoma in situ (DCIS) (Fig. 4, Fig. 5), in accordance to the 2003 World Health Organization (WHO) histologic classification of breast tumors [7]. In most cases, the histopathological distinction between different IPL types can be made on morphological grounds alone [8]. However, establishing the distinction between some lesions remains a difficult task. Furthermore, diagnostic discrepancies persist in select cases [9], particularly in the distinction between ADH and low-grade DCIS (LDCIS).

Immunohistochemistry detection for myoepithelial markers such as α-smooth muscle actin, myosin, calponin, p63, and CD10 can distinguish in situ carcinoma from invasive lesions. If plump myoepithelial cells are evident throughout, then it may indicate that the lesion is benign [10]. However, immunostaining for myoepithelial markers cannot be used for differentiating ADH from LDCIS. Detecting the distribution pattern of different cytokeratin subtypes is sometimes diagnostic on a relatively lower magnification on the microscope [11], [12]. An observation of the distribution pattern, however, can bring forth subjectivity to a certain extent. Therefore, exploring other cost-effective molecular diagnostic methods for differential IPL diagnosis is a critical issue.

Since the completion of the Human Genome Project—which sequenced and mapped all genes—in April 2003, the clinical value of cancer epigenetics has become clearer. Aside from the DNA sequence, epigenetic information is also considered as heritable information, and it is represented by a methylation of cytosines at the CpG islands [13], [14]. Miyamoto and Ushijima [15] illustrated a series of aberrant methylation detection methods to assist cytology in spotting cancer cells in body fluids and to examine cancer-derived DNA using plasma/serum. Is it possible to apply the detection of p16INK4a methylation in the differential diagnosis of IPL as a molecular diagnosis method? This study therefore aims to explore this possibility, as well as demonstrate the molecular changes underlying IPL.

Section snippets

Tissue specimens

Tissue collection and analysis in this study was approved by the Tianjin Medical University Tumor Hospital in China. All specimens, formalin-fixed and paraffin-embedded, were retrieved from the archive of the Breast Pathology Department, Tianjin Medical University Tumor Hospital. Forty UDH cases, 20 FEA cases, 40 ADH cases, 18 LDCIS cases, and 22 high-grade DCIS (HDCIS) cases were randomly selected from the years 1998 to 2006. The availability of enough tumor tissue in each case was ensured for

Methylation-sensitive restriction endonuclease

A band of methylated DNA was located in the sample of 1(2.5%) UDH, 6 (30%) FEA, 13 (32.5%) ADH, 9 (50.0%) LDCIS, and 14 (63.6%) HDCIS. From UDH to FEA/ADH to LDCIS/HDCIS, there was a distinct increase in the presence of methylation (Table 1). A significant difference was established among 5 groups (χ2 = 29.379, P = .000); however, there were no significant differences between FEA and ADH (χ2 = 0.039, P = .844), FEA and LDCIS (χ2 = 1.586, P = .208), ADH and LDCIS (χ2 = 1.615, P = .204), and

Discussion

Aberrant promoter methylation is a common epigenetic modification in human cancer [28]. Several methods to determine DNA methylation status in tumor tissues have been developed, including qualitative and quantitative assays. Some researchers have suggested that qualitative methods may display positive results for tumors with low methylation levels, which may not be biologically important. For example, Ota et al [29] demonstrated that the prognostic significance of p16INK4a methylation is

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    This project was supported by the National Science Fund (No. 30471967) of China and by the Scientific and Technological Development Fund (No. 993607811) of Tianjin Scientific and Technological Committee of China.

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