HE4, A New Potential Tumor Marker for Early Diagnosis and Predicting of Breast Cancer Progression

Abstract

Background & objective: This study examined the potential of human epididymis protein 4 (HE4) as a marker in early diagnosis or as a prognostic factor for breast cancer (BC) patients.

Methods: A total of 31 patients diagnosed with BC were enrolled in the study between 2008 and 2018. The mRNA and protein expression levels of HE4 were analyzed by immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) in the BC tissue and the non-tumoral adjacent tissue. Using ELISA technique, HE4 plasma levels were also measured in 43 BC patients compared to 43 healthy individuals. The correlation between HE4 expression and clinicopathological features was then investigated.

Results: An increase in HE4 expression was observed at mRNA and protein levels in the BC group compared to the control group (P<0.01, P<0.0001, respectively). In addition, the relative expression of HE4 mRNA in BC patients showed a significant correlation with the differentiation grade of cancer cells (P<0.001). Plasma levels of HE4 was also associated with grade (P<0.0001), stage, and tumor size in BC patients (for both P<0.01). Patients with metastatic BC (P<0.01), lymphatic invasion, and lymph node involvement (for both P<0.05) showed significantly higher plasma levels of HE4 expression than patients without metastasis.

Conclusion: According to our findings, upregulation of HE4 may be related to invasive BC phenotype. Measuring plasma levels of HE4 could be useful as a screening test in early diagnosis of BC.

Keywords: Breast neoplasms; Gene expression; HE4; WAP four-disulfide core domain protein 2; WFDC2 protein.

Fig. 1

HE4 relative expression in breast cancer tissues (BCT) (n=31) compared with non-tumoral adjacent tissues (NTAT) (n=31). A: The figure shows relative quantification values in 2^-∆Ct scale. HE4 mRNA expression in BCT was significantly higher compared with NTAT (P<0.05). B: The figure shows higher plasma HE4 levels in BC patients (n=43) compared with healthy volunteers (n = 43) (P<0.0001).

Fig. 2

Correlation between HE4 expression and tumor grade. A: The relative expression of HE4 in three grades of breast cancer (Grade I (n = 6), Grade II (n=15), Grade III (n = 9)). As shown, HE4 mRNA expression in grade III was significantly higher compared with grade I (P<0.001 for both). B: Correlation between plasma HE4 expression and histological grade. As shown, plasma HE4 expression in grade III (n=13) was significantly higher compared with grade I (n=10) and grade II (n=19) (P<0.0001 for both)

Fig. 3

A: Plasma HE4 expression in four stages of breast cancer. A significant increase in plasma HE4 of stage IV (n=12) BC patients was observed compared with stage I (n=7) (P<0.01) and stage II (n=17) (P<0.05). Plasma HE4 expression was shown to have a significant increase in stage III (n=7) compared with stage I BC patients (P<0.05). Also, plasma HE4 expression has a statistically significant correlation with tumor size (P<0.01). B: Plasma HE4 expression in early stages of breast cancer (n=24) compared with healthy control group (n=43). The figure shows significant upregulation of plasma HE4 in early stages of breast cancer as compared to healthy individuals (P<0.01)

Fig. 4

Representative immunohistochemical staining for HE4 in three BCT and NTAT samples. High levels of cytoplasmic expression (++) in BCT samples were detected; A, D, and G: $\times$100; B, E, and H: $\times$400. Positive immunoreactivity for HE4 was not observed in NTAT samples; C, F, and I: $\times$400. (scale bar, 100 µm)