Cytoplasmic p21WAF1/CIP1 expression is correlated with HER-2/ neu in breast cancer and is an independent predictor of prognosis

Abstract

Background: HER-2 (c-erbB2/Neu) predicts the prognosis of and may influence treatment responses in breast cancer. HER-2 activity induces the cytoplasmic location of p21WAFI/CIPI in cell culture, accompanied by resistance to apoptosis. p21WAFI/CIPI is a cyclin-dependent kinase inhibitor activated by p53 to produce cell cycle arrest in association with nuclear localisation of p21WAFI/CIPI. We previously showed that higher levels of cytoplasmic p21WAFI/CIPI in breast cancers predicted reduced survival at 5 years. The present study examined HER-2 and p21WAFI/CIPI expression in a series of breast cancers with up to 9 years of follow-up, to evaluate whether in vitro findings were related to clinical data and the effect on outcome.

Methods: The CB11 anti-HER2 monoclonal antibody and the DAKO Envision Plus system were used to evaluate HER-2 expression in 73 patients. p21WAFI/CIPI staining was performed as described previously using the mouse monoclonal antibody Ab-1 (Calbiochem, Cambridge, MA, USA).

Results: HER-2 was evaluable in 67 patients and was expressed in 19% of cases, predicting reduced overall survival (P = 0.02) and reduced relapse-free survival (P = 0.004; Cox regression model). HER-2-positive tumours showed proportionately higher cytoplasmic p21WAFI/CIPI staining using an intensity distribution score (median, 95) compared with HER-2-negative cancers (median, 47) (P = 0.005). There was a much weaker association between nuclear p21WAFI/CIPI and HER-2 expression (P = 0.05), suggesting an inverse relationship between nuclear p21WAF1/CIP1 and HER-2.

Conclusion: This study highlights a new pathway by which HER-2 may modify cancer behaviour. HER-2 as a predictor of poor prognosis may partly relate to its ability to influence the relocalisation of p21WAFI/CIPI from the nucleus to the cytoplasm, resulting in a loss of p21WAFI/CIPItumour suppressor functions. Cytoplasmic p21WAFI/CIPI may be a surrogate marker of functional HER-2 in vivo.

Figure 1

Examples of HER-2 and p21^WAF1/CIP1 immunoreactivity in infiltrating ductal carcinoma of the breast. Immunostaining was performed as described in Materials and methods, and nuclei were counterstained with haematoxylin. (a) A tumour showing HER-2 overexpression, with (b) a concomitant predominance of cytoplasmic p21^WAF1/CIP1staining and minimal or no nuclear p21^WAF1/CIP1 expression. (c) A HER-2-negative tumour, with (d) a predominance of nuclear p21^WAF1/CIP1 expression compared with cytoplasmic p21^WAF1/CIP1, which was proportionately less on intensity distribution scoring in 10 fields. Insets in panels (b) and (d) show higher-power views of the same fields. Magnification × 3–4.

Figure 2

Relationship between HER-2 and p21^WAF1/CIP1 localisation. Scatter plots according to HER-2 status and the subcellular localisation of p21^WAF1/CIP1 measured as an intensity distribution score (IDS). HER-2 staining was analysed as described in Materials and methods. (a)Distribution of nuclear p21^WAF1/CIP1 IDS and medians according to HER-2-negative and HER-2-positive breast cancers, with patient numbers shown. (b) Cytoplasmic p21^WAF1/CIP1 IDS and medians according to HER-2 status, and numbers of patients. Of the 67 patients, nuclear p21^WAF1/CIP1 was not evaluable in four patients, or in the cytoplasm in six cases. P values indicate significance.

Figure 3

Relationship between HER-2 and prognosis in 67 patients using the log-rank test. (a) Overall survival curves according to HER-2 status, classified as negative or positive according to Materials and methods. (b) Relapse-free survival curves according to HER-2 expression. Significant P values are indicated in bold.