Detection of Circulating Antibodies to p16 Protein-Derived Peptides in Hepatocellular Carcinoma

Yangchun Xu, MM; Litong Gu, MM; Jiaxin Wang, MD; Zhenqi Wang, MD; Ping Zhang, MD, PhD; Xuan Zhang, MD, PhD


Lab Med. 2020;51(6):574-578. 

In This Article

Abstract and Introduction


Objective: This study aimed at confirming the alteration of circulating anti-p16 immunoglobulin G (IgG) levels in hepatocellular carcinoma (HCC).

Methods: An in-house-developed enzyme-linked immunosorbent assay was used for determining plasma IgG antibodies against p16-derived antigens in 122 HCC patients and 134 healthy controls.

Results: Plasma anti-p16 IgG levels were significantly higher in HCC patients than in the controls (Z = 3.51, P = 0.0004), with no difference between males and females. A trend of increasing plasma anti-p16 IgG levels was associated with increasing HCC stage, with group 3 patients having the highest anti-p16 IgG levels (Z = 3.38, P = 0.0008). Group 3 exhibited the best sensitivity (19.6%) and specificity (95%) for plasma anti-p16 IgG detection, with an area under the receiver operating characteristic curve of 0.659 (95% confidence interval, 0.564–0.754).

Conclusion: Circulating IgG antibody to p16 protein might be a useful biomarker for HCC prognosis assessment rather than for early malignancy diagnosis.


Hepatocellular carcinoma (HCC) is a common malignant disorder associated with a very unfavorable prognosis and a high incidence and fatality rate.[1] Despite the significance of early HCC diagnosis in its treatment, robust biomarkers for early clinical HCC diagnosis remain unavailable. To date, alpha-fetoprotein (AFP) levels in circulation and abdominal ultrasound examination are recommended for routine HCC surveillance and diagnosis. However, due to their low specificity and sensitivity and limited prognostic value, their clinical usefulness is debatable.[2,3] Therefore, the need for more sensitive and specific early HCC diagnosis methods remains urgent. Numerous studies have suggested using the overexpression of tumor-associated antigens (TAAs) in many cancers, which triggers the secretion of corresponding autoantibodies, potentially in cancer diagnosis.[4–6] Although several circulating anti-TAA antibodies have served as serological biomarkers for early HCC diagnosis and HCC prognosis prediction in the last 2 decades,[7–11] the identification of robust antibody markers with optimal specificity and sensitivity for early HCC diagnosis remains vital.

The malfunction of the cyclin-dependent kinase (CDK) inhibitor called cyclin-dependent kinase inhibitor 2A (CDKN2A), also called p16, considered to be an important tumor suppressor, may result in uncontrolled cell proliferation.[12,13] Promoter hypermethylation of p16 is reportedly a characteristic of HCC patients.[14] Several studies have demonstrated that hypermethylation-induced p16 expression inhibition plays crucial roles in HCC progression and is closely related to a poor prognosis in recurrent early-stage HCC.[14–16] Similarly, a meta-analysis revealed that hypermethylation-induced p16 gene loss is related to the risk of increased liver cirrhosis and HCC development.[17] Notably, p16 is reportedly overexpressed in many cancers, including cervical, breast, and non–small cell lung (NSCL) cancers,[18–20] and compared with controls, patients with such cancers highly express circulating antibodies against p16 protein, suggesting the clinical diagnostic value of anti-p16 immunoglobulin G (IgG) levels. Previous studies have proved the reliability and sensitivity of an in-house enzyme-linked immunoassay (ELISA) (using linear peptides as antigens) in detecting circulating autoantibodies against some TAAs, including circulating anti-p16 IgG.[21,22] Therefore, the in-house ELISA was applied for confirming the associations between plasma anti-p16 IgG levels and HCC in this study in order to further elucidate the clinical applicability of plasma anti-p16 IgG levels in HCC diagnosis.