Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

Abstract

Background: Invasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells.

Methods: We examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR.

Results: Using GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC.

Conclusion: IDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies.

Figure 1

Unsupervised hierarchical clustering of all samples using all probe sets. No tumor types are grouped together, however, two large clusters are evident: one (blue) mainly consists of tumor cells and the other (black) mainly consists of normal cells. This suggests differences in global gene expression profiles of tumor and normal cells. 1–10, case numbers; D, ductal normal cells; L, lobular normal cells; Tduc, ductal tumor; Tlob, lobular tumor.

Figure 2

Hierarchical clustering of normal ductal and lobular cells based on 82 probe sets found by both rank products and pairwise analysis. Ductal and lobular cells from the same patients tend to cluster together, and they can not be well separated from each other. This suggests expected similarities of expression signatures in normal epithelial (ductal and lobular) cells of mammary gland tree. 1–10, case numbers; D, ductal normal cells; L, lobular normal cells.

Figure 3

Hierarchical clustering of tumor and normal samples based on 25 probe sets common to ductal and lobular carcinoma versus normal cells identified by both rank products and pairwise analysis. Tumor samples are grouped together, however, gene expression profiles of normal cell types from cases 10 and 3 are different from those of other normal cells, and tumor cells from case 2 are different from those of other tumor cells. 1–10, case numbers; D, ductal normal cells; L, lobular normal cells; Tduc, ductal tumor; Tlob, lobular tumor.

Figure 4

Hierarchical clustering of invasive ductal and lobular breast carcinomas based on 325 probe sets identified by rank products and/or pairwise analysis. Tumor types are well separated. 1–10, case numbers; Tduc, ductal tumor; Tlob, lobular tumor.

Figure 5

Representative immunohistochemical staining for the selected proteins: E-cadherin, DDR1, DVL1, cytokeratin 5/6, cytokeratin 17 and EMP1. 1.1. E-cadherin is negative in lobular carcinoma; 1.2. E-cadherin is positive in ductal carcinoma; 2.1. DDR1 is negative in lobular carcinoma; 2.2. DDR1 is positive in ductal carcinoma; 3.1. DVL1 is positive in lobular carcinoma; 3.2. DVL1 is negative in ductal carcinoma; 4.1. Cytokeratin 5/6 is negative in lobular carcinoma cells, but its expression is retained in normal ductal epithelial cells; 4.2. Cytokeratin 5/6 is negative in ductal carcinoma cells; 4.3. Duct lobular unit in normal mammary gland tissue is positive for cytokeratin 5/6; 5.1. Cytokeratin 17 is negative in lobular carcinoma cells, but its expression is retained in normal ductal epithelial cells; 5.2. Cytokeratin 17 is negative in ductal carcinoma cells, but its expression is retained in normal ductal epithelial cells; 5.3. Duct lobular unit in normal mammary gland tissue is positive for cytokeratin 17; 6.1. EMP1 is positive in lobular carcinoma; 6.2. EMP1 is negative in ductal carcinoma.

Figure 6

Asporin mRNA detection by chromogenic in situ hybridization. 1–2. Various magnifications (×100, ×400, ×400) of lobular carcinoma; red arrows, lobular tumor cells are positive; blue arrows, stromal cells are negative; 3. Ductal carcinoma is negative (×200).

Figure 7

PCR validation of microarray results for CTHRC1 (1), ASPN (2), and COL3A1 (3). Fluorescence signals from Affymetrix probe sets (225681_at, 219087_at and 211161_s_at, respectively) and optical density of PCR bands (OD × cm) were transformed to a percentage of the highest value. 1–10, case numbers; Tduc, ductal tumor; Tlob, lobular tumor; D, normal ductal cells; L, normal lobular cells.