Identification of early molecular markers for breast cancer

Abstract

Background: The ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma. Since DCIS is a curable disease, it would be highly desirable to identify molecular markers that allow early detection. Mice transgenic for the WAP-SV40 early genome region were used as a model for DCIS development. Gene expression profiling was carried out on DCIS-bearing mice and control animals. Additionally, a set of human DCIS and invasive mammary tumors were analyzed in a similar fashion. Enhanced expression of these marker genes in human and murine samples was validated by quantitative RT-PCR. Besides, marker gene expression was also validated by immunohistochemistry of human samples. Furthermore in silico analyses using an online microarray database were performed.

Results: In DCIS-mice seven genes were identified that were significantly up-regulated in DCIS: DEPDC1, NUSAP1, EXO1, RRM2, FOXM1, MUC1 and SPP1. A similar up-regulation of homologues of the murine genes was observed in human DCIS samples. Enhanced expression of these genes in DCIS and IDC (invasive ductal carcinoma) was validated by quantitative RT-PCR and immunohistochemistry.

Conclusions: By comparing murine markers for the ductal carcinoma in situ (DCIS) of the mammary gland with genes up-regulated in human DCIS-samples we were able to identify a set of genes which might allow early detection of DCIS and invasive carcinomas in the future. The similarities between gene expression in DCIS and invasive carcinomas in our data suggest that the early detection and treatment of DCIS is of utmost relevance for the survival of patients who are at high risk of developing breast carcinomas.

Figure 1

Microarray analysis of murine samples. A: Supervised hierarchical clustering using 173 probe sets (= 140 genes) overexpressed in mice taken 2-3 months after lactation and in IDC of WAP-TNP8 mice. Each row represents a probe set and each column a sample. The length and the subdivision of branches display the relation of the samples based on their expression. Each group contains samples obtained from five mice. The time point of determination of gene expression was calculated as months after lactation (1 month, 2 months,...). As a positive control IDCs (Tumor) were used. Additionally, wild type (WT = Balb/C) mice and mice without lactation (neg. contr) were used as negative controls. Red indicates upregulation, green downregulation, and black no change. B: Supervised hierarchical clustering of the murine samples using the seven marker genes clearly distinguishes between control samples and malignant samples.

Figure 2

Validation of the marker gene expression by RT-PCR. Relative expression is shown in Box - Whisker - Plots. Gray columns show a 50% range of the data surrounding the median; black lines within each column mark the median; circles mark outliers. Significance was calculated with the Mann-Whitney-U test (P < = 0.05*, P < = 0.01**, P < = 0.001 three stars). A: Panel of the murine samples. Controls are transgenic mice before lactation (H). Months are calculated from the start of lactation (2 m = 2 months; 3 m = 3 months; 4 m = 4 months; 5 m = 5 months; IDC = invasive ductal carcinoma). Each group contains 7 samples. B: Panel of human samples. Controls are healthy tissues from reduction plastics (H).

Figure 3

Microarray analysis of human samples. Supervised hierarchical clustering using of the human samples using the seven marker gene set clearly distinguishes between control samples and malignant samples. Each row represents a probe set and each column a sample. Red indicates upregulation, green downregulation, and black no change.

Figure 4

Histological analysis of markers genes. Protein expression was determined by immunohistochemistry using sections from Formalin-fixed paraffin-embedded tissue (A: MUC1, B: SPP1, C: RRM2, D: FOXM1, E: DEPDC1, F: NUSAP1). For each protein, expression is shown in human breast tissue with a rising degree of malignancy (healthy, DCIS, invasive breast tumour). Specific signals are represented by pink staining (arrowhead) (counterstained with haematoxylin, original magnification 400×, bars:100 μm). The inserts depict the negative controls as a reference.