Diagnosis of hepatocellular carcinoma (HCC) has recently increased in many low incidence countries including Portugal [10, 16, 37]. The incidence and mortality rates are heterogeneous, but mortality during the last decades increased and according to World Health Organization (WHO) has a mortality rate of about 47,000 deaths/year in Europe, close to the rate of incidence. Early detection of HCC opens doors for various effective treatments such as surgical resection and transplantation, which can subsequently lead to long-term survivals in a greater number of patients. Although surveillance and better diagnostic techniques have improved outcomes, the overall 5 years survival rate is only 8.6 % in Europe [7, 39].
In 80–90 % of cases, HCC develops on underlying liver cirrhosis or inflammation . Worldwide, approximately 54 % of cases can be attributed to HBV infection while 31 % can be attributed to HCV infection, leaving approximately 15 % associated with other causes. The percentage of alcohol related HCCs is not well defined, but alcoholic cirrhosis is clearly a risk factor for HCC , and in one study, alcoholic liver disease accounted for 32 % of all HCC’s . In Portugal, for instance, alcoholic cirrhosis (AC) is the most common cause of chronic liver disease being responsible for 84 % of a total of 81 543 hospital admissions for cirrhosis between 1993 and 2008 .
Implementation of surveillance programs to identify at-risk candidate populations and identification of biomarkers for early HCC detection are a major public health goal to decrease HCC-related deaths. According to the EASL–EORTC Clinical Practice Guidelines on Management of HCC, patients with cirrhosis are at high risk and should be enrolled in surveillance programs. The current recommendation for HCC surveillance consists of ultrasound that should be performed every 6 months in patients at high risk. This method has a sensitivity of 60 % [9, 28], although the presence of cirrhosis, with fibrous septa and regenerative nodules, produce a coarse pattern which may impair identification of small tumours. .
Alpha-fetoprotein (AFP) is the most widely used serological marker but has a suboptimal performance and is considered an inadequate screening test for HCC. The association of AFP and ultrasound significantly increases costs and the number of false positives, seeming to have no advantage in practice and therefore is not currently recommended [11, 13].
Other serological biomarkers have been or are under investigation for early diagnosis of HCC, including des-gamma-carboxy prothrombin (DCP) (also known as prothrombin induced by Vitamin K Absence II–PIVKA II), the ratio of glycosylated AFP (L3 fraction) to total AFP, alpha-fucosidase, and glypican 3. None of these have better performance characteristics than AFP.
Recently osteopontin (OPN) attracted attention as a promising biomarker for HCC diagnosis in patients with virus related cirrhosis with better sensitivity than AFP in differentiating HCC cases from cirrhosis controls as suggested by the results of two major studies [27, 36], which included patients with liver disease, particularly chronic HBV or HCV infections.
Kim J and coworkers determined plasma levels of OPN (ELISA), as well as AFP and PIVKA II, in 62 patients with HCC (69 % HBV, 10 % HCV, 3 % alcohol related), in 60 patients with chronic liver disease without tumor (83 % HBV, HCV 3 %, 10 % alcohol related) and in 60 healthy controls. Significantly higher plasma OPN levels (p <0.001) where detected in patients with HCC (median 954 ng/ml, range 168–5742) than in chronic liver disease (381 ng/ml, 29–1688) and healthy controls (155 ng/ml, 10–766). OPN levels correlated with progressive deterioration of underlying liver function in terms of Child-Pugh class and advancing degree of tumor stage. The sensitivity and specificity of OPN for the diagnosis of HCC were 87 % and 82 %, respectively, for a cut-off of 617.6 ng/mL. OPN had an AUC (0.898) greater than AFP (0.745) or PIVKA II (0.578), suggesting a better diagnostic accuracy. Immunohistochemistry showed OPN expression of in 92 of 285 tumors (32.3 %) and was found in malignant hepatocytes and macrophages, which invade the tumor, but not in normal hepatocytes or in Kupffer cells .
Shang S et al. performed proteomic profiles of plasma from patients with cirrhosis or HCC and validated selected candidate HCC biomarkers in two geographically distinct cohorts to include HCC of different etiologies. Mass spectrometry profiling identified OPN as significantly up-regulated in HCC cases. OPN levels were subsequently measured in 312 plasma samples from 131 patients with HCC, 76 cirrhotics, 52 with chronic hepatitis B or C and 53 healthy controls, belonging to two independent cohorts. OPN has a higher sensitivity than AFP in the diagnosis of HCC in all groups and was also useful in HCC patients with normal AFP. A prospective pilot study involving 22 patients who developed HCC during follow-up found that OPN was already increased one year before diagnosis, thus suggesting a potential predictive role of this biomarker for the occurrence of the tumor .
However, it is known that OPN correlates to other tumors and pathological conditions, which can impose a strong limitation to its use as a HCC marker. Indeed, some studies have demonstrated its role in tumorigenesis and metastasis formation, and expression of OPN has been detected in several types of carcinomas in humans. Despite that, OPN is an attractive potential tumor marker, found in the extra-cellular matrix secreted and also in body fluids, including plasma .
OPN expression is found physiological in bone and kidney, but can also be detected in many organs in pathological conditions. Hepatic expression of OPN was first found in Kupffer cells, stellate cells and macrophages in inflammatory and necrotic areas, in rats with carbon tetrachloride intoxication . Subsequently, it was shown to have increased expression in patients with AC, and also in cirrhosis of other etiologies, like NASH, primary biliary cirrhosis, autoimmune hepatitis, primary sclerosing cholangitis, suggesting that chronic liver injury is the main factor for the induction of OPN response . Serum OPN levels are correlated with hepatic inflammation and fibrosis in heavy alcohol drinkers, and hepatic OPN expression levels are strongly correlated with hepatic neutrophils accumulation, the pro-fibrogenic factor TGF-beta and fibrosis [5, 6, 19, 33].
Circulating levels of OPN are elevated in patients with liver lesions associated with HCV and HBV infections. For example, higher levels were an excellent indicator of cirrhosis in patients with chronic hepatitis B  and correlated with liver fibrosis in chronic hepatitis C, as found by Huang W et al. .
Although data suggests a better performance of plasma OPN in the diagnosis of HCC, the role of this biomarker needs validation. Moreover, data are lacking in alcoholic liver disease (only 2 in 62 patients with HCC have alcoholic cirrhosis in the study of Kim J et al.), the most common risk factor of HCC among us. So we conducted a study with the following objectives: (1) evaluate the usefulness of plasma OPN in the diagnosis of HCC in patients with alcoholic cirrhosis, and compare its accuracy with AFP; (2) investigate whether increased OPN is due to the tumor or underlying disease; and determine if there is any relationship between plasma OPN levels and the activity or severity of liver disease.