Claudin-2 is an independent negative prognostic factor in breast cancer and specifically predicts early liver recurrences

Abstract

Background: Predicting any future metastatic site of early-stage breast cancer is important as it significantly influences the prognosis of advanced disease. This study aimed at investigating the potential of claudin-2, over-expressed in breast cancer liver metastases, as a biomarker for predicting liver metastatic propensity in primary breast cancer.

Methods: Claudin-2 expression was analyzed in two independent cohorts. Cohort 1 included 304 women with metastatic breast cancer diagnosed between 2002 and 2007, while cohort 2 included 237 premenopausal women with early-stage node-negative breast cancer diagnosed between 1991 and 1994. Global transcriptional profiling of fine-needle aspirates from metastases was performed, followed by immunohistochemical analyses in archival primary tumor tissue. Associations between claudin-2 expression and relapse site were assessed by univariable and multivariable Cox regression models including conventional prognostic factors. Two-sided statistical tests were used.

Results: CLDN2 was significantly up-regulated (P < 0.001) in liver metastases compared to other metastatic sites. Claudin-2 protein was more frequently expressed in primary tumors from patients who subsequently developed liver metastases (P = 0.02) and high expression was associated with a shorter metastasis-free interval (cohort 1, HR = 1.4, 95% CI = 1.0-1.9; cohort 2, HR = 2.2, 95% CI = 1.3-3.5). Specifically, a significantly shorter interval between primary tumor diagnosis and liver-specific recurrence was observed among patients with high levels of claudin-2 expression in the primary tumor (cohort 1, HR = 2.3, 95% CI = 1.3-3.9).

Conclusion: These results suggest a novel role for claudin-2 as a prognostic biomarker with the ability to predict not only the likelihood of a breast cancer recurrence, but more interestingly, the liver metastatic potential of the primary tumor.

Keywords: BCSS; Breast cancer; CI; CLDN2; Claudin-2; ER; HR; IHC; LNM; LiMFS; Liver metastasis; OR; PR; Prognostic biomarker; RFS; TMA; breast cancer specific survival; claudin-2 mRNA; confidence interval; estrogen receptor; hazard ratio; immunohistochemistry; liver metastasis-free survival; lymph node metastasis; odds ratio; progesterone receptor; relapse-free survival; tissue microarray.

Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Figures

Figure 1

Kaplan–Meier representation of post‐recurrence survival according to site of relapse in cohort 1. A) Patients were stratified by presence (liver) or absence (other) of liver metastases. B) Patients with non‐liver metastases (breast, lymph‐node, skin, bone, lung and ascite) were further stratified into three groups according to the most distant metastatic site. P values are from two‐sided Log‐rank tests.

Figure 2

Supervised analysis comparing transcriptional profiles of liver metastases to non‐liver metastases (breast, lymph‐node, skin, bone, lung and ascite). A summary of significantly differentially altered cell adhesion and matrix‐remodeling genes is presented. Red corresponds to up‐regulated genes and green corresponds to down‐regulated genes within the heatmap. The color scale represents the mean centered Log 2 expression of the genes. Black in the top bar represents liver metastases and gray represents other metastases.

Figure 3

Claudin‐2 mRNA expression. A–B) Box plots comparing CLDN2 expression between liver and non‐liver (breast, lymph‐node, skin, bone, lung and ascite) metastatic lesions in cohort 1. The specific anatomical location of the profiled metastases was taken into consideration. C–D) Box plots comparing CLDN2 expression between patients presenting with liver metastases vs non‐liver metastases. Patients were categorized into four groups associated with prognosis and this stratification considered only the most advanced metastatic site recorded and not the specific anatomical location of the metastatic lesion profiled [local; locally advanced or regional metastases in the lymphnodes or skin, bone; skeletal metastases with or without loco‐regional metastases, lung; plural metastases with or without skeletal and loco‐regional metastases, liver; hepatic metastases with or without plural, skeletal or loco‐regional metastases]. The open circles and asterisks in the figures represent mild and extreme outliers respectively for each group in each comparison. All statistical tests are two‐sided.

Figure 4

Claudin‐2 protein expression. Representative images of immunohistochemical staining of primary breast cancers showing A) deficient (50% positive tumor cells) claudin‐2 expression, respectively.