Since HCC is usually asymptomatic for much of its natural history, most of HCC patients were detected at an intermediate or advanced stage, by which time surgical or oncological treatment options were limited [38]. Therefore, screening and diagnose HCC as early as possible, when it is curable, was critical important to improve the treatment of this deadly disease [9]. For this reason, surveillance in patients with high risks of developing HCC has been recommended, and US and serum AFP are commonly used [25]. Recently, studies have shown that surveillance program improved prognosis of HCC [26]. However, US examination depends on the experience of the sonographer and the technical quality of the US equipment, and is therefore subjective and nonrepetitive [24]. Furthermore, the use of AFP to screen a population with chronic liver disease who is at risk of developing HCC has been reported to have a poor sensitivity of only 20–30% at cutoff values > 100 ng/ml [27]. A better screening program is in urgent need for early HCC diagnosis.
Many novel serum diagnostic markers of HCC have been found, including DCP, GPC3, AFP-L3 and GP73 [31, 32]. Although many non-protein serum markers have been identified for HCC in the past decade such as mutated DNAs, methylated DNAs and RNAs [including microRNA, lncRNA (long non-coding RNA) and circRNA (circular RNA) or even these RNAs inside exosomes] [39–43], protein markers could be detected in serum are the most applicable for clinical routine assessments [22, 44], for their advantages including non-invasive, requiring less than 100µL serum, low dependence on operator expertise, low cost, high reproducibility, and no samples need pretreatment (such as extraction, purification or reverse transcription) [22]. Egfl7, a recently identified protein involved in the progression of HCC, maybe a satisfactory marker for HCC: it is a secretory protein, is specifically overexpressed in HCC cells instead of normal cells such as vascular endothelial cells or cholangiocytes within HCC tissues, and is hardly detectable in human adult normal liver tissues [32, 33]. More importantly, Egfl7 has already been evidenced to be upregulated in the serum of patients with HCC or other type of cancers [33, 45–47], suggesting its possible application in the diagnosis of HCC. However, the levels of serum Egfl7 in patients with early HCC are still unknown.
In the present study, we detected the serum levels of Egfl7 in the patients with early HCC. The results showed a significantly higher level of serum Egfl7 in these patients than healthy individuals (increased by 3.71 folds), which was consist with the elevated levels of serum Egfl7 in HCC patients [33, 45]. In differentiating early HCC from the healthy individuals, Egfl7 had a significantly higher sensitivity than AFP (75.9%-77.4% vs. 58.9%-65.3%%) and their accuracy and AUROC were similar indicating a generally superior of Egfl7 in the detection of early HCC to AFP as well as some other serum marker for early HCC, such as GPC3 (sensitivity ranged from 47.9–66.2%) [48] and DKK1 (sensitivity ranged from 70.9–73.8%) [25, 26].
Our results showed serum Egfl7 levels was modestly elevated in patients with CLD compared with healthy individuals (1600.3 ng/mL vs. 1082.80 ng/mL) but much lower than those with early HCC (4017.28 ng/mL). Furthermore, in surveillance of early HCC from CLD, Egfl7 had a much better AUROC (0.787-0.800 vs. 0.563–0.675), sensitivity (75.2%-75.9% vs. 36.1%-61.8%) and accuracy (73.5%- 74.1% vs. 59.7%-61.9%) than AFP, indicating Egfl7 as a better serum marker than AFP in the surveillance of early HCC from CLD. Moreover, Egfl7 also exhibited an advantage in the aspect of sensitivity compared with some recently reported serum markers for distinguishing early HCC from CLD such as GPC3 (sensitivity of 55%) [22], GP73 (sensitivity of 62%) [49] and DKK1 (sensitivity ranged from 54.8–73.8%) [25, 26].
Although AFP is often recommended as a serum marker for the surveillance of HCC, the use of AFP to screen a population with chronic liver disease who is at risk of developing HCC has been reported to have a poor sensitivity of only 20–30% at cutoff values > 100 ng/mL [40]. In this study, to distinguish early HCC from chronic liver disease, AFP had a specificity which ranged from 62–90.8% when the cutoff value increased from 20 ng/mL to 400 ng/mL but the sensitivity correspondingly decreased from 61.8–38.4%, which meaning 61.6% of early HCC was missed at a cutoff value of 400 ng/mL. Therefore, the recognition of AFP-negative HCC is important to improve the efficacy of early detection of HCC. In the present study, 67.7%-70.8% of patients with early HCC who had a negative AFP could be diagnosed by Egfl7, which was slightly better than AFP-L3 (50%) [50], GP73 (66%-67%) [23, 50] and comparable with DKK1 (67.3%-73.1%) [25, 26]. In consideration of a high specificity of AFP in the surveillance and early detection of HCC, Egfl7 might be helpful to make up the deficiency of AFP in sensitivity and further improve the diagnostic efficacy of early HCC. The combination of Egfl7 and AFP showed a significantly increased sensitivity ranged from 88.9–91.0%, which was remarkably higher than the sensitivity of Egfl7 or AFP alone and also the sensitivity of AFP combined with other serum markers such as DKK1 (63.8%-90.8%) [25, 26] and GPC3 (81%-89%) [22].
In addition, we have found that the level of serum Egfl7 was also elevated in patients with metastatic liver cancer (increased by 2.50 folds) or benign liver tumors (increased by 1.62 folds) also had a moderately elevated compared with the healthy individuals, although the magnitude is smaller than that in early HCC (3.71 folds), suggesting serum Egfl7 might be helpful to determine the nature (benign tumor or HCC) and origin (primary or secondary) of liver tumors.
To our knowledge, this is the first large-scale study to report the performance of Egfl7 as a serum diagnostic marker for early HCC in a test cohort and an independent validation cohort. The results indicate Egfl7 as a novel and effective serological marker for the early detection of HCC, with a significantly higher sensitivity and accuracy than AFP, especially in the surveillance of a high-risk population with CLD. In addition, serum Egfl7 was positive in most early HCC patients with negative AFP and could be used combined with AFP to further improve the diagnostic efficacy.