Abstract
BackgroundBreast cancers are phenotypically and genotypically heterogeneous tumors containing multiple cancer cell populations with various metastatic potential. Aggressive tumor cell subpopulations might more easily be captured in lymph nodes metastases (LNM) than in primary tumors (PT). We evaluated mRNA and protein levels of master EMT regulators: TWIST1, SNAIL and SLUG, protein levels of EMT-related markers: E-cadherin, vimentin, and expression of classical breast cancer receptors: HER2, ER and PgR in PT and corresponding LNM. The results were correlated with clinicopathological data and patients outcomes.MethodsFormalin-fixed paraffin-embedded samples from PT and matched LNM from 42 stage II-III breast cancer patients were examined. Expression of TWIST1, SNAIL and SLUG was measured by reverse-transcription quantitative PCR. Protein expression was examined by immunohistochemistry on tissue microarrays. Kaplan-Meier curves for disease-free survival (DFS) and overall survival (OS) were compared using F-Cox test. Hazard ratios (HRs) with 95% confidence intervals (95% CI) were computed using Cox regression analysis.ResultsOn average, mRNA expression of TWIST1, SNAIL and SLUG was significantly higher in LNM compared to PT (P < 0.00001 for all). Gene and protein levels of TWIST1, SNAIL and SLUG were highly discordant between PT and matched LNM. Increased mRNA expression of TWIST1 and SNAIL in LNM was associated with shorter OS (P = 0.04 and P = 0.02, respectively) and DFS (P = 0.02 and P = 0.01, respectively), whereas their expression in PT had no prognostic impact. Negative-to-positive switch of SNAIL protein correlated with decreased OS and DFS (HR = 4.6; 1.1-18.7; P = 0.03 and HR = 3.8; 1.0-48.7; P = 0.05, respectively).ConclusionsLNM are enriched in cells with more aggressive phenotype, marked by elevated levels of EMT regulators. High expression of TWIST1 and SNAIL in LNM, as well as negative-to-positive conversion of SNAIL confer worse prognosis, confirming the correlation of EMT with aggressive disease behavior. Thus, molecular profiling of LNM may be used as surrogate marker for aggressiveness and metastatic potential of PT.
Highlights
Breast cancers are phenotypically and genotypically heterogeneous tumors containing multiple cancer cell populations with various metastatic potential
Relative expression of TWIST1, SNAIL and SLUG was significantly higher in lymph nodes metastases (LNM) compared to primary tumors (PT): 1.75 ± 2.41 vs. 0.25 ± 0.32 for TWIST1 (P < 0.00001), 2.8 ± 3.06 vs. 0.96 ± 2.32 for SNAIL (P < 0.00001), 1.03 ± 0.93 vs. 0.16 ± 0.19 for SLUG (P < 0.00001)
Increased mRNA expression of TWIST1 and SNAIL in LNM was associated with shorter overall survival (OS) (P = 0.04 and P = 0.02, respectively) and disease-free survival (DFS) (P = 0.02 and P = 0.01, respectively), whereas their expression in PT had no prognostic impact (Figure 2)
Summary
Breast cancers are phenotypically and genotypically heterogeneous tumors containing multiple cancer cell populations with various metastatic potential. Lymph node metastases (LNM) are considered rather a manifestation of the widespread metastatic process and a marker of aggressive phenotype of the primary tumor (PT) than the “bridge-heads” for further metastatic spread [3] It has been confirmed by the clinical observation of poorer survival after relapse in nodepositive patients compared to node-negative ones [4]. Experimental models provide further evidence that development of LNM indicates the increased potential of PT to disseminate aggressive cells and produce metastasis promoting growth factors [3], according to the recently proposed stromal progression model [5] In this model mutual regulatory interactions between stroma and tumor cells play important roles in tumor progression as genetic and epigenetic changes. Those interactions contribute to the process of epithelialmesenchymal transition (EMT), to the bone marrow mesenchymal stem cells actively recruited by tumor cells to the surrounding stroma [6]
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