Quantitative assessment of invasive mena isoforms (Menacalc) as an independent prognostic marker in breast cancer

Introduction: Mena, an Ena/VASP protein family member, is a key actin regulatory protein. Mena is up-regulated in breast cancers and promotes invasion and motility of tumor cells. Mena has multiple splice variants, including Mena invasive (Mena(INV)) and Mena11a, which are expressed in invasive or non-invasive tumor cells, respectively. We developed a multiplex quantitative immunofluorescence (MQIF) approach to assess the fraction of Mena lacking 11a sequence as a method to infer the presence of invasive tumor cells represented as total Mena minus Mena11a (called Mena(calc)) and determined its association with metastasis in breast cancer. Methods: The MQIF method was applied to two independent primary breast cancer cohorts (Cohort 1 with 501 and Cohort 2 with 296 patients) using antibodies against Mena and its isoform, Mena11a. Mena(calc) was determined for each patient and assessed for association with risk of disease-specific death. Results: Total Mena or Mena11a isoform expression failed to show any statistically significant association with outcome in either cohort. However, assessment of Mena(calc) showed that relatively high levels of this biomarker is associated with poor outcome in two independent breast cancer cohorts (log rank P = 0.0004 for Cohort 1 and 0.0321 for Cohort 2). Multivariate analysis on combined cohorts revealed that high Mena(calc) is associated with poor outcome, independent of age, node status, receptor status and tumor size. Conclusions: High Mena(calc) levels identify a subgroup of breast cancer patients with poor disease-specific survival, suggesting that Mena(calc) may serve as a biomarker for metastasis.