Quantitative assessment of invasive mena isoforms (Mena^calc) 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.