Human Epididymis Protein 4(HE4) As A New Tumor Marker In Small Cell Lung Cancer

Lung cancer is the leading cancer killer in the industrialized areas and in some developing countries such as China, accounting for 20 % of all cancer deaths in China and ranking highest for both incidence and mortality in the world. It is classified into small cell lung cancer(SCLC) or non-SCLC(NSCLC). SCLC accounts for 13 % to 20 % of all lung cancer and is closely linked to the intensity and duration of tobacco smoking, which is characterized by its aggressive nature with rapid growth, paraneoplastic endocrinopathies and early metastasis and is associated with poor long-term outcome. To improve the outcome of SCLC, it is critical to detect lung cancer at an early stage using sensitive diagnostic and prognostic biomarkers. To date, a number of serum biomarkers have been found to be elevated in the serum of patients with lung cancer. However, these biomarkers are not sufficiently sensitive or specific to obtain a reliable diagnosis of or prognosis for lung cancer. Thus it is urgently need to identify useful biomarkers for early non-invasive diagnosis and to monitor the progression of lung cancer.Human epididymis protein 4(HE4), a small molecule secreted glycoprotein first identified in human epididymis, has been approved by FDA as a novel serum biomarker for the early diagnosis and recurrence monitoring of ovarian cancer. Several recent studies of small-cohort samples have found the significantly increased HE4 levels in tumor tissues and sera of lung cancer with positive intratumoral HE4 protein expression or high levels of serum HE4 after chemotherapy correlating with a shorter disease-free and overall survival(OS) or worse OS after the treatment respectively. The diagnostic and prognostic value of serum HE4 is further validated in a recent study which showed that serum HE4 detection holds great potential to differentiate lung cancer from pulmonary tuberculosis(PTB) and healthy controls and higher levels of HE4 predicts poor prognosis in NSCLC patients. However, there have as yet been no in detailed studies to evaluate the expression and clinical significance of HE4 in patients with SCLC.In the present study, we aimed to evaluate the expression and clinical value of tumoral and serum HE4 levels in SCLC.Where no statistically significant difference was seen for the basic information between patients with SCLC and healthy controls, including age, gender and smoking status. Serum HE4 levels were found to be significantly higher in SCLC patients than in healthy controls(98.93 pmol/L[36.63-1782] vs. 49.06 pmol/L[34.86-194.8], p =0.0093); Furthermore, the HE level in SCLC patients was not correlated with extent of disease(93.41 pmol/L [36.63-834.7] vs.103.3 pmol/L [38.99-1782], p = 0.5896) for LD vs. ED; smoking history(95.78 pmol/L [36.63-1782] vs. 113.5 pmol/L [38.99-834.7], p = 0.9775) for smoker vs non-smoker; or gender(102.1 pmol/L [38.99-194.4] vs. 95.78 pmol/L [36.63-1782], p = 0.5305) for female vs. male.We also investigated HE4 expression of cancer tissues from SCLC patients by immunohistochemical staining. HE4 expression was detected in lung cancer tissues but not in normal lung tissues. We could not find a correlation between extent of disease and HE4 staining of lung cancer tissues(data not shown). Strong HE4 staining was detected in cytoplasmic and plasma membrane areas but not in nuclear area.To assess the clinical potential of HE4, we calculated sensitivities and specificities of HE4. The receiver operating characteristic(ROC) curve for HE4 with its cutoff points for achieving the best individual accuracy is got. The AUC for serum HE4 was 0.85 for differentiating SCLC patients from healthy adults using an optimal cut-off value of 84.19 pmol/L that achieve a sensitivity of 69.4 % and a specificity of 93.3 %. As CEA, CA125, CA153, CA199, CA724, CYFRA21-1, SCC and NSE are also widely used biomarkers for lung cancer diagnosis, we also examined the serum levels of these markers in the same SCLC patients with HE4 detection. Among 8 biomarkers examined, CEA(γ = 0.4133, p = 0.0035), CA125(γ = 0.408, p = 0.004) and CYFRA21-1(γ = 0.4783, p = 0.0006) show a correlation with serum HE4 level. Furthermore, higher serum HE4 level(>84.19 pmol/L) was observed in 23/36(63.9 %), 24/37(64.9 %), 25/37(67.6 %), 22/33(66.7 %), 32/43(74.7 %), 29/43(67.4 %), 33/47(70.2 %), 25/37(67.6 %) cases with negative CA125(<35 U/m L), CEA(<5.0 ng/m L), CA199(<37 U/m L), CA153(<30 U/m L), CA724(<10 U/m L), CYFRA21-1(<4.0 ng/m L), SCC(<1.8 ng/m L) and NSE(<24.0 ng/m L). Using the tumor marker alone, the highest sensitivity(69.4 %) and accuracy(78.5 %) was found with HE4, the highest specificity(100 %) with NSE. The combination of HE4 with other tumor markers further increased the sensitivity and accuracy with combined HE4 and NSE achieving the best sensitivity(75.5 %) and accuracy(82.3 %) among various combinations though a loss in specificity(93.3 %) compared with NSE alone.we attempted to find some relevancy between HE4 and the prognosis of SCLS by survival analysis. It was layered according to stage and the level of HE4 in patients with completing fellow-up. While the difference was not statistically significant(p > 0.5), but it seems to have a poor prognosis with high level of HE4 in ED patients.Finally, To explore the diagnostic value of plasma HE4 protein in lung cancer by meta analysis. The related articals were retrievaled in Pubmed, Embase, Google scholar, CNKI, and finally 48 articles were downloaded. Stata12.0 analysis software was used in systematically reviewing of relevant literature. A total of 8 articles and 1447 of the subjects were included in this study.Among them, 867 cases and 580 controls were included in the literature time from 2011 to 2016. Heterogeneity analysis revealed that the sensitivity, specificity, positive predictive value and negative predictive value of the I2 values were 78.3%(p <0.001), 73.2%(p <0.001), 56.9%(p =0.017), 63.2%(p <0.001),(p =0.002), respectively. The area under the ROC curve was 0.8639. The publication bias of this study was detected by funnel plot and Egger’s test. The study showed that there was no obvious asymmetry in the shape of the funnel plot. Linear test results are 0.094. This study suggests that plasma HE4 plays an important role in the diagnosis of lung cancer.As far as we know, the HE4 levels in the tumor tissues and sera of SCLC patients and found that HE4 presents a significantly elevated level in both tumor tissues and sera of SCLC patients as compared to those from healthy controls. ROC analysis revealed that serum HE4 level had a great potential to distinguish SCLC patients from healthy controls with much higher sensitivity than commonly used tumor biomarkers in current clinic at the nearly same level of specificity. As HE4 measurement can be easily performed by commercially available ELSIA kit and well-established in the clinical laboratory, our and other previous results strongly suggest that HE4 may hold great potential as a serum biomarker for the diagnosis of lung cancer, including NSCLC and SCLC.This topic due to the time and so on conditions, are preliminary proved HE4 in SCLC preliminary application value in diagnosis, but did not involve HE4 in SCLC specific molecular mechanism of biological effect and function, and its research and development of specific molecular mechanism for creating new ideas and find the corresponding effective drug targets, improve overall survival in patients with extremely essential. This topic further research will focus on further research from the following aspects:1. Use HE4 specific neutralizing antibodies inhibit HE4 levels, combined with the role of proteomics analysis clearly HE4 may approach.2. Build activity of HE4 missing mutant genes, clear HE4 enzyme activity in mediating the role of TGF –β regulation.3. Establish HE4 expression, high stability or knock out of silence expression in lung cancer cell line, further defined HE4 EMT effect to promote lung cancer cells, and EMT(mesenchymal- epithelial transition). And detection of the related gene expression in order to make clear TGF –β and related signaling pathways(Smad dependence and not rely on pathway) the role of EMT in HE4 promote lung cancer cells, clarify HE4 enzyme activity in regulating the TGF –β expression and the role of promoting the EMT.4. Lung cancer cell line established in nude mice model, testing the above indicators to verify found in vitro, and inhibit or block the effect of HE4 expression for lung cancer treatment.5. By collecting SCLC tumor tissue and serum specimens, detection of HE4, TGF –β and EMT related protein expression, evaluating the correlation between them and its relationship with metastasis of lung cancer.