Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide contributing to approximately 600,000 deaths each year with this number on the rise in the developing world. The aetiology of HCC has been well characterised, including chronic hepatitis B and C infection as well as alcoholic cirrhosis. Current screening programs for patients at high risk of developing HCC, including ultrasound and alpha-fetoprotein analysis, are neither sufficiently sensitive nor specific to detect early HCC.  This reduces the likelihood of detecting HCC when curative treatment is effective. One genetic marker which has been shown to be associated with HCC carcinogenesis is the p16 INK4a/ARF locus. If further research confirms this mutation as a common step in early hepatocarcinogenesis then this maker could be utilised to screen for early HCC lesions in at risk populations. Further research in this area could facilitate the early diagnosis of HCC, improving the efficacy of treatment.

Introduction

Liver cancer is the sixth most common cancer worldwide and the second largest cause of cancer mortality [1]. It has several subtypes including hepatocellular carcinoma (HCC), bile duct cancer, hepatoblastoma, and various other liver sarcomas and carcinomas [2]. Of those subtypes, HCC is the most common, comprising about 78% of all liver cancers [1,2]. HCC is unequally distributed globally with over 80% of cases occurring in either Sub-Saharan Africa or Eastern Asia; predominantly in China [3,4]. When considering Western countries, there is strong evidence from the United States that the incidence of HCC is rising, with nine cancer registries reporting via the National Cancer Institute that there has been a 41% rise in mortality from primary liver cell cancer and a 70% rise overall in incidence between 1980 and 1995 [5].

A similar rise in HCC incidence and death rates in Australia have also been identified; possibly linked to the increased prevalence of Hepatitis B and Hepatitis C infection in Australia [6,7]. Moreover, there is evidence that HCC incidence rates in Australia may be up to two-fold higher than the rates reported by cancer registries such as the Victorian Cancer Registry [8].  A higher rate of HCC has also been reported in Aboriginal and Torres Strait Islander populations; estimated at 2% and 8% in urban and rural areas, respectively, compared with less than 1% for the total Australian population [9].

The aetiology of HCC is well documented in the literature with high rates linked to both hepatitis B and hepatitis C exposure [9]. Other significant causes that may cause patients to present include alcoholic liver disease, non-alcoholic steatohepatitis as well as hereditary conditions such as haemochromatosis, alpha-1 antitrypsin deficiency, and autoimmune disorders (Figure 1) [10,11]. These conditions result in significant parenchymal loss, increased fibrogenesis and inflammatory signalling resulting in cirrhosis; a condition associated with 70-90% of all detectable HCC cases [10,11].

The natural history of HCC growth begins as small asymptomatic nodules which can often take years to develop depending on the aetiological exposure [5]. Small HCCs at detection have relatively long tumour doubling times with tumours of less than five centimetres associated with a survival of 81-100% at one year, and 17-21% at three years without therapy, which suggests that early diagnosis may allow for a greater intervention window [5].

These features make HCC an insidious and difficult disease to clinically detect and investigate. Patients who develop HCC are usually asymptomatic, mostly displaying symptoms related to their chronic liver disease which can become modified with disease progression [6,12]. Examples of this include signs of decompensation such as ascites, encephalopathy, jaundice, and variceal bleeding [12]. Advanced lesions also can present more conspicuously causing obstructive symptoms such as jaundice, diarrhoea, weight loss, and fatigue [13]. These signs are a result of local tumour invasion and growth inside the liver. However, systemic signs can also occur as a result of metastases which can develop in the lung, portal vein, periportal nodes, bone or brain [13]. As a result of these features, HCC is typically diagnosed late in its course with a median survival following diagnosis of 6 to 20 months, and a five-year survival ranging from 12% in those outside major cities and 17% within major cities in Australia. [13,14].

 

Screening and detection of HCC

Accordingly, screening programs for HCC in at risk groups, those with chronic liver disease or chronic hepatitis infection, is recommended with specialist review forming part of a 6-12 monthly management plan [15]. These programs involve using alpha-fetoprotein (AFP) and ultrasound to screen for the presence of cancer lesions.

AFP is a widely studied screening test marker for HCC with a level above 400 ng/mL regarded as diagnostic [5]. However, two thirds of HCCs less than 4 cm have AFP levels less than 200 ng/mL and up to 20% of HCCs do not produce AFP even when they are very large [5]. Moreover, there is evidence that fluctuating levels of AFP in patients with cirrhosis might reflect flares of HBV or HIV infection or liver disease exacerbation rather than HCC development [16]. Some studies investigating the clinical utility of AFP have suggested it lacks the sensitivity to be useful, with one study suggesting it rarely assisted in a diagnosis [5,17,18]. As a result of this, it has been suggested that AFP testing alone should only be used if ultrasound is unavailable, with the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver recommending it not be used at all [16,18,20]. This therefore limits the use of AFP as a reliable test to screen for HCC in at risk groups. Due to these limitations, other serum markers which include plasma microRNAs such as miR-122 and miR-192, des-gamma-carboxy prothrombin, AFP isoforms and glypican-3 are currently being investigated and evaluated for future use [19,20].

Another modality that is used in HCC screening is ultrasound. Ultrasound can detect large HCCs with high sensitivity and specificity; however, it is less able to reliably identify smaller lesions, which are required if more effective therapy is to be offered [6,19,20]. Ultrasound has been shown to detect 85-95% of lesions 3-5 cm in diameter, but can only achieve a 60-80% sensitivity of 1 cm lesions [5]. A large meta-analysis investigating this in 2009 found similar results demonstrating that surveillance with ultrasound showed a limited sensitivity (64%) for early HCC detection [19]. Combined use of AFP and ultrasound has been shown to increase detection rates, but had a raised combined false positive rate of 7.5% compared to AFP and ultrasound alone at 5.0% and 2.9% respectively [18].  Despite the limitations of these tests, the Royal Australian College of General Practitioners guidelines suggest that patients with chronic liver disease or chronic hepatitis infection should be considered for 6-12 monthly AFP and ultrasound screening [15]. The Asian Pacific Association for the Study of the Liver also recommends surveillance for HCC with both AFP and ultrasound every 6 months [21].

The importance of early HCC detection cannot be understated. The natural history of early tumours is poorly known as most are treated upon diagnosis; however, surgical resection, tranplantation and ablation offer high rates of complete responses and a potential cure in all patients with early HCC [22]. Advanced course HCC has a survival of less than six months without treatment with prognostic factors for survival including anatomical extension of the tumour, performance status and functional hepatic reserve based on the Child-Pugh Score [22-24]. It is from this that researchers are currently investigating superior screening techniques which can identify tumours earlier and with greater sensitivity and specificty to enable earlier intervention and better treatment outcomes.

 

Cancer biology as the pathway for a HCC screening test

One approach that is currently being investigated in the medical literature focusses on the biology of HCC hepatocarcinogenesis to develop a sensitive early screening test that can guide and detect treatment before the cancer can extend and spread. Mature hepatocytes have an average lifespan of between 200-400 days and rarely proliferate unless stimulated by acute injury [25]. The observation of normally quiescent hepatocytes and cholangiocytes proliferating after partial hepatectomy has highlighted the significant regenerative ability of the liver after acute insult [26]. If this regenerative capacity is compromised, the liver has liver progenitor cells (LPCs) which can expand and regenerate the chronically damaged liver [27,28]. LPCs can propagate and differentiate into two types of liver epithelial cells; hepatocytes and cholangiocytes [27]. These cells have been defined as stem cells because they are clonogenic, with a high growth potential and are able to be induced to differentiate into both types of liver cells and have shown capability in repopulating the liver on transplantation [27-30].

The proliferation and differentiation of LPCs into hepatocytes render them a target population for hepatocarcinogenesis [30]. LPCs have been traced to hepatocytes and are markedly elevated in chronic liver disease [28]. Recent laboratory experimentation has shown a link between induced liver damage in mice and the development of HCC, suggesting a tenable link between LPCs and HCC development [31,32]. LPCs have also been documented in chronic human liver pathologies, such as chronic hepatitis C, which is highly associated with hepatocarcinogenesis [29,33-35]. Analysis of premalignant lesions in HCC have also identified the presence of LPCs, with up to 50% of developed HCCs being shown to express markers of progenitor cells including CK7, CK19, OV6 [35-38] These findings have also been found in further studies on both human and mouse liver cells [38,39]. These results suggest a significant link exists between LPCs and HCC carcinogenesis.

From a molecular analysis of HCC progression, it has been shown that hepatocarcinogenesis is a multistep process that is heterogeneous and not well understood [40]. Progressive genetic alterations have been shown to cause a spectrum of cellular changes starting from cell hyperplasia, proliferation to dysplasia and eventually cancer [28,40]. This model is widely accepted and has been applied to many types of cancer, including HCC [28]. Multiple studies have demonstrated that two tumour suppressor pathways are important in controlling cell proliferation including the retinoblastoma protein pathway and the p53 pathway [28,41]. Most human tumours have genetic mutations, deletions, deregulated methylation or alterations in microRNA signalling in their Retinoblastoma and p53 pathways; making these genes likely candidates in the transformation of non-tumourigenic LPCs to tumourigenic LPCs [42-44].

Future HCC screening with the marker p16 INK4a/ARF

One gene involved in the p53 tumour suppressor pathway which may play a crucial role in hepatocarcinogenesis is the INK4a/ARF locus. Studies have identified variable rates of inactivation of the p16 INK4a/ARF gene in HCC with inactivation ranging in the literature from 35-82% of HCCs depending on the aetiology [45-50]. Studies on HCC mouse models have highlighted the important role of INK4a/ARF in tumourigenesis with concomitant loss of p53 and INK4a/ARF accelerating tumourigenesis and the progression to metastatic lung lesions [51-53]. Furthermore, tumours lacking both p53 and INK4a/ARF demonstrated strong migration and invasion capabilities. This was not demonstrated when p53 itself was inactive; suggesting that INK4a/ARF inactivation may be a critical step in HCC development (Figure 2) [52,53]. Significant evidence suggests that INK4a/ARF are important tumour suppressors encoded at 9p21 [9,46,52-54]. Kaneto et al. suggested that the methylation of the INK4a/ARF locus promoter is an early event in hepatocarcinogenesis, making this gene an ideal candidate for further study in the pursuit for an accurate diagnostic tool for HCC [46].

The nature of how genes are inactivated in tumourigenesis is biologically complex. The INK4a/ARF gene can be affected by many different forms of inactivation notably mutations, homozygous deletions or gene methylation [54]. Tannapefel et al. showed that of 71 carcinomas examined, 59% showed aberrant methylation of the INK4a gene [54]. Another recent review suggested that as many as 40-70% of HCCs demonstrate an INK4a methylation resulting in the downregulation of protein expression [55]. The pathogenesis of the ARF gene in HCC was not as clear, with studies finding ARF gene methylations in non-cancerous liver tissue as well as low rates of ARF methylation in human HCC samples [54,55]. Despite this, evidence of promotor methylation has arisen from investigations into the cause of INK4a/ARF inactivation in other tumours such as human cutaneous squamous cell carcinoma and colorectal carcinomas [56,57]. These studies provide evidence that the methylation of the INK4a/ARF gene may be an important step in the hepatocarcinogenesis of HCC and therefore could be an effective clinical marker for the identification of HCC development in at risk patients.

 

Conclusion

While it is clear from this review that further research needs to be conducted to better understand the role of the INK4a/ARF gene locus in HCC development, the potential for a clincial application remains a potent driver for further research in this area of molecular medicine. While the current screening methods using AFP and ultrasound are considered to be clincially useful in detecting HCC, there exists a space for more accurate modalities to detect early HCC lesions.  If future research shows that the INK4a/ARF gene is a common early mutation in HCC hepatocarcinogenesis, then future tests may be developed which can sample high probability sites in the liver or the blood to investigate for DNA which contains this mutation. This could potentially improve the prognosis of patients with HCC development and allow early directed treatment with the possibility of cure.

 

Acknowledgements

Thank you to Professor George Yeoh for his assistance in proofreading this article and providing support and advice.

Conflicts of interest

None declared.

 

References

[1]       Laursen L. A preventable cancer. Nature. 2014;516(7529):S2-3.

[2]       Australian Government. Liver cancer. Australia: Cancer Australia; 2014 [cited 2016 April]. Available from: http://canceraustralia.gov.au/affected-cancer/cancer-types/liver-cancer

[3]       El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132(7):2557-76.

[4]       Han ZG. Functional genomic studies: insights into the pathogenesis of liver cancer. Annu Rev Genomics Hum Genet. 2012; 9c;13:171-205.

[5]       Ryder SD. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut. 2003;S3:iii1-iii8.

[6]       Law MG, Roberts SK, Dore GJ, Kaldor JM. Primary hepatocellular carcinoma in Australia, 1978–1997: increasing incidence and mortality. Med J Aust. 2000;173:403–5.

[7]       El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N. Engl. J. Med. 1999;340:745–50.

[8]       Hong TP, Fink M, Dev A, Roberts S Nicoll A, Lubel J, Kronborg I, et al. Novel population-based study finding higher than reported hepatocellular carcinoma incidence suggests an updated approach is needed. Hepatology. 2016;63(4):1205-1212.

[9]       Cunningham J, Rumbold AR, Zhang X, Condon JR. Incidence, aetiology, and outcomes of cancer in Indigenous peoples in Australia. Lancet Oncology. 2008;9(6):585–95.

[10]     Lee YA, Wallace MC, Friedman SL. Pathobiology of liver fibrosis: a translational success story. Gut. 2015;64(5):830-41.

[11]     Intrahepatic arteriovenous shunting due to hepatocellular carcinoma and cirrhosis, and its change by transcatheter arterial embolization. AU Sugano S, Miyoshi K, Suzuki T, Kawafune T, Kubota M SO Am J Gastroenterol. 1994;89(2):184.

[12]     Schwartz J, Carithers R, Chopra S. Clinical features and diagnosis of primary hepatocellular carcinoma. In: UpToDate, Post TW (Ed), UpToDate, Waltham, MA. (Accessed 2016 June.)

[13]     Saunders, WB. A new prognostic system for hepatocellular carcinoma: a retrospective study of 435 patients: the cancer of the liver Italian program (CLIP) investigators. 1998;28(3):751-5.

[14]     Refshuage A, Kalisch D. Cancer survival and prevalence in Australia period estimates from 1982 to 2010. Australian Government: Australian Institute of Health and Welfare. 2012 [cited 2016 Sep]. Available from: http://www.aihw.gov.au/WorkArea/DownloadAsset.aspx?id=10737422721

[15]     Lusis N. Clinical guidelines: prevention and early detection of liver (hepatocellular) cancer. Royal Australian College of General Practitioners. 2012 [cited 2016 May] Available from: http://www.racgp.org.au/your-practice/guidelines/national-guide/prevention-and-early-detection-of-cancer/prevention-and-early-detection-of-liver-(hepatocellular)-cancer/

[16]     Llovet JM, Ducreux M, Lencioni R, Bisceglie AM, Galle PR, Dufour JF, et al. EASL-EORTC Clinical practice guidelines: management of hepatocellular carcinoma. European Association for the study of liver, European Organisation for Research and Treatment of Cancer. Journal of Hepatology. 2012; 56;908-943.

[17]     Ebara M, Ohto M, Shinagawa T, Sugiura N, Kimura K, Matsutani S, et al. Natural history of minute hepatocellular carcinoma smaller than three centimeters complicating cirrhosis. A study in 22 patients. 1986;90(2):289-98.

[18]     Bruix J, Sherman M. American association for the study of liver diseases: management of hepatocellular carcinoma: an update. Hepatology. 2011;53(3):1020-2.

[19]     Singal A, Volk ML, Waljee A, Salgia R, Higgins P, Rogers M, et al. Meta-analysis: surveillance with ultrasound for early-stage hepatocellular carcinoma in patients with cirrhosis. Alimentary Pharmacology & Therapeutics. 2009; (30):37–47.

[20]     Colombo M, Chopra S. Prevention of hepatocellular carcinoma and recommendations for surveillance in adults with chronic liver disease. In: UpToDate, Post TW (Ed), UpToDate, Waltham, MA. (Accessed 2016 June.)

[21]     Omata M, Lesmana L Tateishi R, Chen PJ, Lin S, Yoshida H, et al. Asian pacific association for the study of liver consensus recommendations on hepatocellular carcinoma. Hepatology International. 2010;4:439-474.

[22]     Okuda K, Ohtsuki T, Obata H, Tomimatsu M, Okazaki N, Hasegawa H, et al. Natural history of hepatocellular carcinoma and prognosis in relation to treatment. Study of 850 patients. Cancer. 1985;56(4):918-28.

[23]     Llovet JM, Bruix J. Systematic review of randomized trials for unresectable hepatocellular carcinoma: chemoembolization improves survival. Hepatology. 2003; 37(2):429-42.

[24]     Llovet JM, Brú C, Bruix J. Prognosis of hepatocellular carcinoma: the BCLC staging classification. Semin Liver Dis. 1999;19(3):329-38.

[25]     Malato Y, Naqvi S, Schurmann N, Ng R, Wang B, Zape J, et al. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. J Clin Invest. 2011;121(12):4850-60.

[26]     Mishra L, Banker T, Murray J, Byers S, Thenappan A, He AR, et al. Liver stem cells and hepatocellular carcinoma. Hepatology. 2009;49(1):318-29.

[27]     Miyajima A, Tanaka M, Itoh T. Stem/Progenitor cells in liver development, homeostasis, regeneration and reprogramming. Cell Stem Cell. 2014;14(5):561-574.

[28]     Chen C, Wang G. Mechanisms of hepatocellular carcinoma and challenges and opportunities for molecular targeted therapy. World J Hepatol. 2015; 7(15):1964-1970.

[29]     Kumar M, Zhao X, Wang XW. Molecular carcinogenesis of hepatocellular carcinoma and intrahepatic cholangiocarcinoma: one step closer to personalized medicine? Cell Biosci. 2011;1(1):5.

[30]     Yamashita T, Wang XW. Cancer stem cells in the development of liver cancer. J Clin Invest. 2013;123(5):1911-8.

[31]     Viebahn CS, Benseler V, Holz LE, Elsegood CL, Vo M, Beroloino P, et al. Invadin macrophages play a major role in liver progenitor cell response to chronic liver injury. J Hepatol. 2010;53(3):500-507.

[32]     Passman A, Strauss R, McSpadden S, Finch-Edmondson M, Woo K, Diepeveen L, et al. A modified choline-deficient, ethionine-supplemented diet reduces morbidity and retains a liver progenitor cell response in mice. Dis Model Mech. 2015;8(12):1635-41.

[33]     Zhang A, London R, Schulz F, Giguère-Simmonds P, Delriviere L, Chandraratana H, et al. Human liver progenitor cell lines are readily established from non-tumorous tissue adjacent to hepatocellular carcinoma. Stem Cells Dev. 2010; 19(8):1277-84.

[34]     Davies R, Knight B, Tian Y, Yeoh G, Olynyk J. Hepatic oval cell response to the choline-deficient, ethionine supplemented model of murine liver injury is attenuated by the administration of a cyclo-oxyenase 2 inhibitor. Carcinogenesis. 2006;27(8):1607-1616.

[35]     Roskams T. Liver stem cells and their implication in hepatocellular and cholangiocarcinoma. Oncogene. 2006;25(27):3818-22.

[36]     Mishra L, Banker T, Murray J, Byers S, Thenappan A, He AR, et al. Liver stem cells and hepatocellular carcinoma. Hepatology. 2009;49(1):318-29.

[37]     Roskams T. Liver stem cells and their implication in hepatocellular and cholangiocarcinoma. Oncogene. 2006; 25(27):3818-22.

[38]     Li C, Wang Y, Dong W, Xiang S, Liang H, Wang H, et al. Hepatic oval cell lines generate hepatocellular carcinoma following transfection with HBx gene and treatment with aflatoxin B1 in vivo. Cancer Letters. 2011;311(1):1-10.

[39]     Mishra L, Banker T, Murray J, Byers S, Thenappan A, He AR, et al. Liver stem cells and hepatocellular carcinoma. Hepatology. 2009;49(1):318-329.

[40]     Cha C, Dematteo RP. Molecular mechanisms in hepatocellular carcinoma. Best Pract Red CL GA. 2004;19(1):25-37.

[41]     Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell. 2002;2(2):103-112.

[42]     Ito T, Nishiada N, Fukuda Y, Nishimura T, Komeda T, Nakao K. Alterations of the p14(ARF) gene and p53 and p53 status in human hepatocellular carcinoma. J Gastroenterol. 2004;39(4):355-361.

[43]     Gramantieria L, Fornaria F, Challengaria E, Sabbioni S, Lanza G, Croce CN, et al. MicroRNA involvement in hepatocellular carcinoma. J Cell Mol Med. 2008; 12(6):2189-2204.

[44]     Tannapfel A, Busse C, Weinans L, Benicke M, Katalinic A, Geissler F, et al. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas. Oncogene. 2001;20(48):7104-7109.

[45]     Tannapfel A, Wittekind C. Genes involved in hepatocellular carcinoma: deregulation in cell cycling and apoptosis. Virchows Arch. 2002;440(4):345-52.

[46]     Kaneto H, Sasaki S, Yamamoto H, Itoh F, Toyota M, Suzuki H, et al. Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut. 2001;48(3):372-7.

[47]     Jin M, Piao Z, Kim NG, Park C, Shin EC, Park JH, et al. p16 is a major inactivation target in hepatocellular carcinoma. Cancer. 2000;89(1):60-8.

[48]     Lee S, Lee HJ, Kim JH, Lee HS, Jang JJ, Kang GH. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol. 2003;163(4):1371-8.

[49]     Li B, Liu W, Wang L, Li M, Wang J, Huang L, et al. CpG island methylator phenotype associated with tumor recurrence in tumor-node-metastasis stage I hepatocellular carcinoma. Ann Surg Oncol. 2010;17(7):1917-26.

[50]     Roncalli M, Bianchi P, Bruni B, Laghi L, Destro A, Di Gioia S, et al. Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma. Hepatology. 2002;36(2):427-32.

[51]     Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer. 2006 Sep;6(9):674-87.

[52]     Chen YW, Klimstra DS, Mongeau ME, Tatem JL, Boyartchuk V, Lewis BC. Loss of p53 and Ink4a/ARF cooperate in a cell autonomous fashion to induce metastasis of hepatocellular carcinoma cells. Cancer Res. 2008; 67(16):7589-7596.

[53]     Chen YW, Paliwal S, Draheim K, Grossman SR. p19ARF inhibits the invasion of hepatocellular carcinoma cells by binding to CtBP. Cancer Res. 2009;68(2):476-482.

[54]     Tannapfel A, Busse C, Weinans L, Benicke M, Katalinic A, Geissler F, et al. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas. Oncogene. 2001;20(48):7104-7109.

[55]     Nishida N, Goel A. Genetic and epigenetic signatures in human hepatocellular carcinoma: a systemic review. Curr Genomics. 2011;12(2):130-137.

[56]     Brown VL, Harwood CA, Crook T, Cronin JG, Kelsell DP, Proby CM. p16INK4a and p14ARF tumor suppressor genes are commonly inactivated in cutaneous squamous cell carcinoma. J Invest Dermatol. 2004;122:1284-1292.

[57]     Esterller M, Tortola S, Toyota M, Capella G, Peinado MA, Baylin SB, et al. Hypermethylation-associated inactivation of p14ARF is independent of p16INK4a methylation and p53 mutational status. Cancer Res. 2000;60:129.