Overexpression of Cripto and its prognostic significance in breast cancer: A study with long-term survival

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Abstract

Introduction

Cripto is a founding member of the EGF-CFC family, and plays an important role in tumourigenesis, tumour cell proliferation and migration. We aimed to determine the significance of Cripto expression on the survival of patients with breast cancer.

Methods

Immunohistochemical detection of Cripto was performed by using mAb C13 on 120 formalin-fixed paraffin-embedded breast tumour specimens in tissue microarrays. This cohort comprises a series of 120 patients with primary operable breast cancer diagnosed between 1989 and 1995, retrieved from the Concord Repatriation General Hospital breast carcinoma database.

Results

Using a cutoff value of 80%, Cripto overexpressed in 57 of the 120 (47.5%) patients. We found significant associations between overexpression of Cripto and the Nottingham Prognostic Index (NPI, p < 0.01), histological grade (p < 0.01), pathological tumour type (p = 0.04), PR (p = 0.02) as well as Ki-67 (p = 0.02). Univariate analysis reveals that there is a significant correlation between overexpression of Cripto and survival (p = 0.0003). Cox regression analysis indicates that the overexpression of Cripto is an independent prognostic factor in breast cancer (HR 2.79, 95%CI 1.20–6.50).

Conclusion

The unique epitope recognized by mAb C13 is overexpressed on breast tumour tissues. In this series of invasive breast cancers, overexpression of Cripto was more often found in high grade and poor prognosis tumours compared to low grade and good prognosis breast cancers. Moreover, overexpression of Cripto was significantly associated with decreased patient survival.

Introduction

Cripto, a growth factor with an EGF-like domain, is the first member of the EGF-CFC family of genes to be sequenced and characterized, and contributes to deregulated growth of cancer cells. A role for Cripto in tumour development has been described in human and mouse.1 Cripto expression, first found in human teratocarcinomas, was demonstrated to be overexpressed in breast, cervical, ovarian, gastric, lung, colorectal and pancreatic carcinomas in contrast to normal tissues, where Cripto expression was invariably low or absent.2, 3, 4, 5, 6, 7 Cripto may play an important role in tumourigenesis,8 since in vitro Cripto induces mammary cell proliferation, reduces apoptosis, increases cell migration, and inhibits milk protein expression. This prediction is strengthened by observations of Cripto expression in 80% of human and mouse mammary tumours.1

The EGF-like domain of Cripto is unique, as it lacks an A loop which is essential for erbB receptor binding, Cripto does not bind directly to any of the four known type IerbB receptors, but binds to its specific receptor glypican-1 with high affinity9, 10, 11 and activates ras/raf/MAPK and PI3-K/Akt/glycogen synthase pathways,10, 12 which are known to be responsible for cellular proliferation, transformation and survival.13, 14

Overexpression of Cripto enhanced the migration ability of human cervical cancer.15 The ability of Cripto to induce both scattering and branching morphogenesis in mammary epithelial cells is reminiscent of the effects on motility and epithelial mesenchymal transition (EMT) that is often characterized by decrease in cell–cell adhesion, which coincides with enhanced migration and facilitates invasion of tumour cells.16, 17 Recent data have shown that there is enhanced invasion ability in ER positive MCF-7 cells transfected with a Cripto expression vector as demonstrated by cell invasion assays by using Boyden chambers and 8 μm pore size filters coated with Matrigel.18 This suggests that overexpression of Cripto might be associated with the progression towards a more aggressive phenotype in breast cancer.

To our knowledge, there has been no previous study examining the prognostic significance of Cripto expression in breast cancer. It was, therefore, our aim to assess the potential value of Cripto in predicting clinical outcome for breast cancer patients in long-term survival.

In our present study, we examined the expression of Cripto in a cohort of 120 patients of breast cancer treated between 1989 and 1995, together with other important and commonly used prognostic factors for breast cancer: estrogen receptor (ER), progesterone receptor (PR), Her-2, which is also an EGF family member, and cell proliferation marker Ki-67.

Section snippets

Clinical specimens

The present study is based on a series of patients with primary operable breast cancer, who were treated by the multidisciplinary team according to the national breast cancer centre guidelines.19 Patient characteristics, including clinical and pathologic information, were documented and maintained prospectively in a computer database, The Nottingham Prognostic Index (NPI) scores20 were calculated and follow-up information was updated at a regular basis. Ethical approval was granted by the

Level of expression of Cripto

The staining pattern was specific, since no staining was detected in parallel control sections of benign breast tissue. The staining was cytoplasmic and generally confined to the carcinoma cells with some cell membrane staining. However, 15 (12.5%) cases exhibited some nuclear staining. The nuclear staining was not counted for scoring. Associations with clinical outcome were investigated after cutoff analysis of the receiver operating statistics curves for this data set. An optimal cutoff value

Discussion

According to a recent review, the highest significant increase in expression of Cripto compared to non-involved epithelium was in colon, stomach, pancreas, breast, lung and bladder carcinoma.21 It was noted that the level of expression increases with degree of dysplasia from pre-malignant to metastatic disease in gastric cancer.22 A previous study has shown that Cripto expression level was significantly correlated with tumour size, lymph node status and lymphovascular involvement in cervical

Conclusion

Our results suggest that the overexpression of Cripto may prove to be of clinical value in breast cancer as an independent prognostic factor for improving estimation of prognosis and guiding therapeutic decisions. There is potential for overexpression of Cripto to be combined with existing individual prognostic factors or with integrated indices such as the NPI. There is possibility for development of a new antibody targeting Cripto for treatment of breast cancer with overexpression of Cripto.

Acknowledgements

The authors thank Dr. Owen Dent for statistical advices and also thank the Department of Anatomical Pathology, Royal Prince Alfred Hospital for performing the Her-2 immunohistochemistry staining.

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    1

    Permanent address: Department of Breast Surgery, Hubei Cancer Hospital, South Zhuodaoquan Road, Wuhan, Hubei 430079, People's Republic of China.

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