Immunohistochemical analysis of MUC5B apomucin expression in breast cancer and non-malignant breast tissues.

A deregulation of several MUC genes (MUC1, MUC2, MUC3, MUC5AC, and MUC6) was previously demonstrated in breast carcinomas. Considering that recently we found the "non-mammary" MUC5B mRNA in primary breast tumors (Berois et al. 2003), we undertook the present study to evaluate the expression profile of MUC5B protein product in breast tissues, using LUM5B-2 antisera raised against sequences within the non-glycosylated regions of this apomucin. Expression of MUC5B by breast cancer cells was confirmed by immunocytochemistry, in situ hybridization, and Western blot on MCF-7 cancer cells. Using an immunohistochemical procedure, MUC5B apomucin was detected in 34/42 (81%) primary breast tumors, in 13/14 (92.8%) samples of non-malignant breast diseases, in 8/19 (42.1%) samples of normal-appearing breast epithelia adjacent to cancer, and in 0/5 normal control breast samples. The staining pattern of MUC5B was very different when comparing breast cancer cells (cytoplasmic) and non-malignant breast cells (predominantly apical and in the secretory material). We analyzed MUC5B mRNA expression using RT-PCR in bone marrow aspirates from 22/42 patients with breast cancer to compare with MUC5B protein expression in the primary tumors. Good correlation was observed because the six MUC5B-positive bone marrow samples also displayed MUC5B expression in the tumor. Our results show, for the first time at the protein level, that MUC5B apomucin is upregulated in breast cancer. Its characterization could provide new insights about the glycobiology of breast cancer cells.

Detection of bone marrow-disseminated breast cancer cells using an RT-PCR assay of MUC5B mRNA.

The evaluation of disseminated epithelial tumor cells in breast cancer patients has generated considerable interest due to its potential association with disease recurrence. Our work was performed to analyze the usefulness of 5 mucin genes expression (MUC2, MUC3, MUC5B, MUC6 and MUC7), using RT-PCR assays, to detect disseminated cancer cells in patients with operable breast cancer. The highest frequencies of positive RT-PCR tests in breast tumor extracts were observed for MUC5B (7/15) and MUC7 (5/12). The best specificity, negative results on all peripheral blood mononuclear (PBMN) cell samples from healthy donors, were shown for MUC2, MUC5B and MUC6 RT-PCR assays. Thus, we selected MUC5B as a target gene for further evaluation. Using a nested RT-PCR, MUC5B mRNA transcripts were detected in 16/31 primary breast tumors (but not in 36 samples of normal PBMN cells) and in the human MCF-7 breast cancer cell line but not in BT20, MDA, T47D and ZR-75 breast cancer cell lines, indicating that MUC5B mRNA is expressed in a population of breast cancer cells. Using this method, 9/46 patients (19.5%) who underwent curative surgery showed positive MUC5B mRNA in bone marrow aspirates obtained prior to surgery, including 5/24 patients (20.8%) with stage I or II breast cancer, without histopathologic lymph node involvement. These results indicate that MUC5B mRNA could be a specific marker applicable to the molecular diagnosis of breast cancer cell dissemination. A comparative evaluation between MUC5B mRNA, cytokeratin 19 (CK19) mRNA and carcinoembryonic antigen (CEA) mRNA in all bone marrow aspirates suggests a putative complementation for molecular detection of disseminated carcinoma cells. Considering that breast cancer is characterized by a great phenotypic heterogeneity, the use of multimarker approach could contribute to tumor cell detection in bone marrow and blood.