Global Journal of Pathology and Microbiology, 2018, 6, 15-20 15

E-ISSN: 2310-8703/18 © 2018 Scientific Array

Minireview

Hepatocellular Carcinoma: A Brief Overview of Epidemiology, Risk Factors and Histopathological Features

Noha Said Helal*

Pathology Department, Theodor Bilharz Research Institute, Giza, Egypt Abstract: Liver cancer is one of the most prevalent cancers worldwide with hepatocellular carcinoma [HCC] represents the major histological subtype. Many risk factors were identified, including: hepatitis B, hepatitis C infection, chronic alcohol consumption, and non-alcoholic fatty liver disease. Histopathologically, HCC has variable architectural and cytological features.

Keywords: Hepatocellular carcinoma, Epidemiology, Risk factors, Histopathology.

EPIDEMIOLOGY

Worldwide, liver cancer is the fifth most common cancer in men and the ninth most common cancer in women. Liver cancer is much more common in countries in sub-Saharan Africa and Southeast Asia. In 2018, Mongolia had the highest rate of liver cancer, followed by Egypt. There were over 840,000 new cases in 2018. Liver cancer is also a leading cause of cancer deaths worldwide, accounting for more than 700,000 deaths each year [1]. Among primary liver cancers, hepatocellular carcinoma (HCC) represents the major histological subtype accounts for 85%-90% of primary liver cancers [2]. Men have a higher prevalence of HCC than women; the ratio of affected men to affected women varies between 2:1 and 4:1, depending on the geographic region [3]. The reasons for the disparity between men and women are obscure, but they may

include environmental factors such as a higher prevalence of persistent HBV or HCV infection, alcohol abuse, smoking in men than in women, genetic and hormonal factors [4]. The incidence of HCC increases with age, reaching its highest prevalence among those aged over 65 years [5]. However, a shift in incidence towards younger persons has been noted with acquisition of both hepatitis B and C virus infection at younger age [6].

ETIOLOGY AND RISK FACTORS Hepatitis B Virus [HBV]

The WHO has reported HBV to be second only to tobacco as a known human carcinogen [7]. HBV is the *Address correspondence to this author at the Theodor Bilharz Research Institute, El-Nile Street, Warrak El-Hadar, Imbaba, PO Box 30, Giza 12411, Egypt; Tel: +20225401019; Fax: +20235408125; E-mail: nohasaidhelal@yahoo.com

most common cause for HCC and accounts for an estimated 54% of all liver cancers. HBV increases the relative risk for developing HCC 15–20-fold with a mortality rate of 30%–50% among all cases of chronic HBV infection [8], furthermore patients with HBV-cirrhosis have 1000 times higher risk of developing HCC, compared to HBsAg negative individuals. Thus, it is likely that the probability of acquiring HCC increases with severity of underlying liver disease [9]. Based on genetic sequencing, human HBV is currently grouped into 10 genotypes (A-J). Patients with genotypes C and D are more likely to progress to cirrhosis and HCC [10].

However, because of the increasing utilisation of HBV immunization, since the early 1980s, a significant decrease in HCC incidence in adults was observed, 3-4 decades later, so HCC has regarded the first human cancer amenable to prevention using mass vaccination programmes [9].

Hepatitis C Virus [HCV]

HCV was known to be the second most common risk factor for HCC, with an estimated 10%–25% of all cases attributed to it around the world [8] and epidemiological studies have shown up to 70% of patients with HCC have anti-HCV antibody in the serum [11]. Chronic HCV infection is associated with a 20–30-fold increased risk of developing HCC as compared to uninfected individuals. In addition, HCV and HBV co-infection puts one at higher risk of developing HCC [3]. Nowadays, by the introduction of direct-acting antiviral therapy for HCV, these agents have revolutionized patient care, with cure rates of more than 90% [12].

16 Global Journal of Pathology and Microbiology, 2018 Vol. 6 Noha Said Helal

Aflatoxin B1 [AFB1]

Exposure to aflatoxin leads to development of HCC, through damage of DNA in liver cells and mutation in p53 tumor suppressor gene [13]. AFB1 is produced by a fungus of the genus, Aspergillus spp, in Asia and sub-Saharan Africa in which climatic factors and storage techniques favour the fungus to be a common contaminant of foods, such as grain, corn, peanuts and legumes. Areas with high exposure of AFB1 coincide with areas with a high prevalence of HCC. It has also been suggested that a high intake of AFB1 in HBV-infected patients is an added risk factor for HCC development [14].

Schistosomiasis

Schistosomiasis is a common parasitic infestation in some parts of the world. There is some epidemiological evidence that the presence of schistosomal infection may modify the course of hepatitis C genotype 4 co-infection and may lead to significantly more complications, such as portal hypertension at an earlier stage with accelerated progression to hepatitis C-associated fibrosis and thus quicker progression to HCC [15].

Diabetes Mellitus

Diabetes can act as an independent risk factor for HCC, regardless of chronic HCV or HBV infection, alcoholic liver disease, or non-specific cirrhosis. The occurrence of HCC is 2-3 times higher in patients with diabetes mellitus [16]. Diabetes, as part of the insulin resistance syndrome, has been implicated as a risk factor for non-alcoholic fatty liver disease (NAFLD), including in its most severe form, non-alcoholic steatohepatitis (NASH). NASH has been identified as a cause of both “cryptogenic cirrhosis” and HCC [17]. Type 2 diabetes mellitus is associated with central obesity, which promotes carcinogenesis through the secretion of proinflammatory cytokines by visceral adipose tissue [18].

Obesity

Obesity has been implicated as risk factors for HCC, most likely through the development of nonalcoholic steatohepatitis [NASH] [19].

Hemochromatosis

Patients with hemochromatosis, especially in the presence of cirrhosis, are at an increased risk of developing HCC. HCC accounts for about 30% of all iron-related deaths in hemochromatosis [20].

Diet

Many epidemiological studies have examined the relationship between diet and HCC risk. Some studies reported an inverse relationship between HCC and diets which are high in milk, wheat, fish, vegetable, and fruit content [21]. Regarding egg, meat and animal protein consumption, there are studies reported an inverse relation with HCC risk [12,22], while others reported an increased risk [23,24].

Coffee Drinking

There is an inverse relation between coffee consumption and risk of HCC. Coffee has been shown to affect liver enzymes and development of cirrhosis, and therefore could protect against liver carcinogenesis [25]. Coffee drinking might also protect against HCC by reducing levels of insulin and thereby the risk for type 2 diabetes [26].

Pesticides

Pesticides are considered to be possible epigenetic carcinogens through mechanisms, such as spontaneous initiation of genetic changes, cytotoxicity with persistent cell proliferation, oxidative stress, inhibition of apoptosis, suppression of intracellular communication and construction of activated receptors

[27].

Cirrhosis

HCC tends to occur in the background of cirrhosis which is found in 80%–90% of patients with HCC. Macronodular cirrhosis associated with viral hepatitis carries a greater risk of malignant transformation than alcohol related micronodular cirrhosis. Among patients with viral hepatitis- related cirrhosis, HCV-related cirrhosis seems to carry a higher risk for developing HCC than HBV- related cirrhosis [28].

Alcohol Intake

Alcohol abuse is a leading cause of cirrhosis in western countries, which in turn is linked with an increased risk of liver cancer. Chronic consumption of alcohol from 40 to 60 grams of alcohol on a daily bases are highly associated with HCC [29].

Tobacco Use

Smoking increases the risk of liver cancer. Former smokers have a lower risk than current smokers, but both groups have a higher risk than those who never smoked [30].

Overview of Epidemiology, Risk Factors and Histopathological Features Global Journal of Pathology and Microbiology, 2018 Vol. 6 17

Vinyl Chloride and Thorium Dioxide (Thorotrast)

Vinyl chloride is a chemical used in making some kinds of plastics. Thorotrast is a chemical that in the past was injected into some patients as part of certain x-ray tests. Exposure to these chemicals raises the risk of angiosarcoma of the liver and also increases the risk of developing cholangiocarcinoma and hepatocellular cancer, but to a far lesser degree [31].

Drugs

Several drugs have come under suspicion as possible hepatocarcinogens.

Anabolic steroids are male hormones used by some athletes to increase their strength and muscle mass. Long-term anabolic steroid use can slightly increase the risk of hepatocellular cancer. Cortisone-like steroids, such as hydrocortisone, prednisone, and dexamethasone, do not carry this same risk. Contraceptive steroids have also been incriminated in the development of HCC. Tamoxifen and danazol have been implicated in hepatic carcinogenesis [31].

HISTOPATHOLOGY

HCCs consist of tumor cells that resemble hepatocytes. The tumor shows loss of hepatic architecture i.e. loss of portal tracts and reduction of normal reticulin framework [32]. The stroma is composed of sinusoid-like blood spaces lined by a single layer of endothelial cells resembling capillary vessels. This phenotypic change of sinusoids is called 'capillarization'. Unlike the sinusoidal endothelial cells

in normal liver tissue, those of HCC are immunohistochemically positive for CD34 and factor-VIII-related antigen [33].

Hepatocellular carcinoma has four principal histological growth patterns: trabecular, pseudoglandular (pseudoacinar), solid (compact) and macrotrabecular. The different architectural patterns and cytological variants frequently occur in combination [32].

ARCHITECTURAL PATTERNS Trabecular [plate-like] Pattern

This pattern is the most common in well and moderately differentiated HCCs. Tumor composed of cells grow in cords of variable thickness separated by sinusoid-like blood spaces (Figure 1A). Well-differentiated tumors have a thin trabecular pattern and trabeculae become thicker with de-differentiation [34].

Pseudoglandular and Acinar [Adenoid] Pattern

This pattern is formed mostly by a single layer of tumour cells, and some glandular or acinar structures are formed by dilatation of the bile canaliculus-like structure between cancer cells (Figure 1B). Pseudoglands may be dilated and large, infrequent to numerous, empty or filled with eosinophilic material [35].

Compact [Solid] Pattern

Sinusoid-like blood spaces are inconspicuous and slit-like, giving the tumor a solid appearance [34].

Figure 1: Architectural patterns of HCC. A, Trabecular pattern; B, Pseudoglandular pattern (Hematoxylin & Eosin×100).

18 Global Journal of Pathology and Microbiology, 2018 Vol. 6 Noha Said Helal

Macrotrabecular Pattern

Tumor composed of trabeculae more than 10 cells thick. It has been associated with worse prognosis [32].

CYTOLOGICAL VARIANTS Fibrolamellar Carcinoma

It is a distinct clinical pathological entity with a better prognosis than typical HCC. The vast majority occur in patients between 18 and 45 years (median 27 years) of age. It is not related to HCV infection [36]. Microscopically, the tumor cells grow in sheets or small trabeculae that are separated by hyalinized collagen fibers with a characteristic lamellar pattern (Figure 2A). They are large and polygonal and have a deeply eosinophilic and coarsely granular cytoplasm and distinct nucleoli [37].

Pleomorphic Cell Variant

Tumor cells show marked variation in cellular and nuclear size and shape. Bizarre multinucleated or mononuclear giant cells are often present. Pleomorphic tumor cells lack both cohesiveness and distinct trabecular pattern [34].

Clear Cell Variant

The tumor consists predominantly of cells with clear cytoplasm due to the presence of abundant glycogen and variable amount of fat, arranged in acinar, trabecular or solid patterns [32].

Sarcomatous [Sarcomatoid] Change

HCC occasionally appears sarcomatous, characterized by the proliferation of spindle cells or

bizarre giant cells (Figure 2B) [34]. In many cases, the sarcomatous change is present in a part of the tumor, and transitional features between trabecular HCC and sarcomatous components are frequent. Sarcomatous change is more frequent in cases with repeated chemotherapy or transchemo-arterial embolization [39].

Scirrhous Carcinoma

This uncommon type is characterized by marked fibrosis along the sinusoid-like blood spaces with varying degrees of atrophy of tumour trabeculae [32].

Undifferentiated Carcinoma

Undifferentiated carcinoma is a rare variant with male preponderance. Compared to HCC, undifferentiated carcinomas are postulated to have a worse prognosis [34].

HCC with Variants of Changes which Include

Fatty change is most frequent in small, early-stage tumors less than 2 cm in diameter. Bile production is occasionally seen as plugs in dilated canaliculi or pseudoglands. Globular hyaline bodies are small, round, homogeneous, and strongly acidophilic intracytoplasmic bodies. They are PAS-positive and stain orange to red with Masson trichrome stain. Mallory hyaline bodies are intracytoplasmic, eosinophilic aggregated intermediate PAS-negative filaments. Pale bodies are intracytoplasmic, round to ovoid, amorphous and lightly eosinophilic materials accumulate in cystically dilated endoplasmic reticulum, and are commonly seen in the fibrolamellar variant of HCC. Ground glass inclusions are rarely observed in tumors of HBsAg-positive patients. They stain with

Figure 2: Cytological variants of HCC. A, Fibrolamellar; B, Sarcomatous variant (Hematoxylin & Eosin×100).

Overview of Epidemiology, Risk Factors and Histopathological Features Global Journal of Pathology and Microbiology, 2018 Vol. 6 19

modified orcein, Victoria blue, or aldehyde fuchsin, and show immunohistochemical positivity with anti-HBsAg antibody. They are thought to be HBsAg-positive hepatocytes entrapped in a tumor [34].

Combined Hepatocellular-Cholangiolar Carcinoma [HCC/CC]

It accounts for up to 5% of all primary carcinomas of the liver [40]. Combined HCC/CC contains a mixture of hepatocellular and ductular elements scattered throughout the tumor [41].

REFERENCES [1] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA,

Jemal A. Global Cancer Statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68(6): 394-424. https://doi.org/10.3322/caac.21492

[2] Kim H, Ho-Lim HY. Novel EGFR-TK Inhibitor EKB-569 Inhibits Hepatocellular Carcinoma Cell Proliferation by AKT and MAPK Pathways. J Korean Med Sci 2011; 26: 1563-1568 https://doi.org/10.3346/jkms.2011.26.12.1563

[3] El-Serag HB. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterol 2012; 142(6): 1264-1273.e1 https://doi.org/10.1053/j.gastro.2011.12.061

[4] Korah ET, Badr AE, Emara MM, Kohla AS, Michael GS. Relation between sex hormones and hepatocellular carcinoma. Andrologia 2016; 48: 948-55 https://doi.org/10.1111/and.12536

[5] Motola-Kuba D, Zamora-Valdes D, Uribe M, Mendez-Sanchez N. Hepatocellular carcinoma. An overview. Ann Hepatol 2006; 5(1): 16-24 https://doi.org/10.1016/S1665-2681(19)32034-4

[6] Velazquez RE, Rodriguez M, Navascues CA, Linares A, Perez R, Sotorrios NG, et al. Prospective analysis of risk factors for hepatocellular carcinoma in patients with liver cirrhosis. Hepatology, 2003; 37: 520-527 https://doi.org/10.1053/jhep.2003.50093

[7] Chitapanarux T, Phornphutkul K. Risk Factors for the Development of Hepatocellular Carcinoma in Thailand. J Clin Transl Hepatol 2015; 3(3): 182-188 https://doi.org/10.14218/JCTH.2015.000025

[8] Huang YT, Jen CL, Yang HI, Lee MH, Su J, Lu SN, et al. Lifetime risk and sex difference of hepatocellular carcinoma among patients with chronic hepatitis B and C. J Clin Oncol. 2011; 29(27): 3643-50. https://doi.org/10.1200/JCO.2011.36.2335

[9] Liu CJ, Kao JH. Hepatitis B virus-related hepatocellular carcinoma: epidemiology and pathogenic role of viral factors. J Chin Med Assoc 2007; 70: 141-145 https://doi.org/10.1016/S1726-4901(09)70346-6

[10] Sonneveld MJ, Rijckborst V, Cakaloglu Y, Simon K, Heathcote EJ, Tabak F, et al. Durable hepatitis B surface antigen decline in hepatitis B e antigen-positive chronic hepatitis B patients treated with pegylated interferon-α2b: relation to response and HBV genotype. Antivir Ther 2012; 17(1): 9-17. https://doi.org/10.3851/IMP1887

[11] Whittaker S, Marais R, Zhu AX. The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene 2010; 29: 4989-5005. https://doi.org/10.1038/onc.2010.236

[12] Baumert TF, Berg T, Lim JK, Nelson DR. Status of Direct-Acting Antiviral Therapy for Hepatitis C Virus Infection and Remaining Challenges. Gastroenterology 2019; 156(2): 431-445. https://doi.org/10.1053/j.gastro.2018.10.024

[13] Szab E, Paska C, Kaposi Novak P, Schaff Z, Kiss A. Similarities and Differences in Hepatitis B and C Virus Induced Hepatocarcinogenesis. Pathol Oncol Res 2004; 1: 5-11 https://doi.org/10.1007/BF02893401

[14] Parkin DM. The global health burden of infection-associated cancers in the year 2002. Int.J Canc 2006; 118: 3030-3044. https://doi.org/10.1002/ijc.21731

[15] El-Zayadi A, Badran HM, Barakat EM, Attia M, Shawky S, Mohamed MK, et al. Hepatocellular carcinoma in Egypt: A single center study over a decade. World J. Gastroenterology 2005; 11(33): 5193-5198.

[16] Li X, Wang X, Gao P. Diabetes Mellitus and Risk of Hepatocellular Carcinoma. BioMed Res Inter 2017, Article ID 5202684, 10 pages. https://doi.org/10.1155/2017/5202684

[17] Davila JA, Morgan RO, Shaib Y, McGlynn KA, El-Serag HB. Diabetes increases the risk of hepatocellular carcinoma in the United States: a population based case control study. Gut 2005; 54: 533-539 https://doi.org/10.1136/gut.2004.052167

[18] Fujita K, Iwama H, Miyoshi H, Tani J, Oura K, Tadokoro T, et al. Diabetes mellitus and metformin in hepatocellular carcinoma. World J Gastroenterol. 2016; 22(27): 6100-13. https://doi.org/10.3748/wjg.v22.i27.6100

[19] Calle EE, Teras LR, Thun MJ. Obesity and mortality. N Engl J Med 2005; 353(20): 2197-9 https://doi.org/10.1056/NEJM200511173532020

[20] Manos M, Murphy R. Trends in the incidence and etiology of hepatocellular carcinoma in a managed care population: the roles of viral hepatitis and fatty liver disease. Hepatol J 2007; 46: 400.

[21] Talamini R, Polesel J, Montella M, Dal Maso L, Crispo A, Tommasi L, et al. Food groups and risk of hepatocellular carcinoma: A multicenter case-control study in Italy. Int J Cancer 2006; 119: 2916-2921 https://doi.org/10.1002/ijc.22267

[22] Yu SZ, Huang XE, Koide T, Cheng G, Chen GC, Harada K, et al. Hepatitis B and C viruses infection, lifestyle and genetic polymorphisms as risk factors for hepatocellular carcinoma in Haimen, China. Jpn J Cancer Res 2002; 93: 1287-1292 https://doi.org/10.1111/j.1349-7006.2002.tb01236.x

[23] Braga C, La Vecchia C, Negri E, Franceschi S. Attributable risks for hepatocellular carcinoma in northern Italy. Eur J Cancer 1997; 33: 629-634 https://doi.org/10.1016/S0959-8049(96)00500-X

[24] Kurozawa Y, Ogimoto I, Shibata A, Nose T, Yoshimura T, Suzuki H, et al. Dietary habits and risk of death due to hepatocellular carcinoma in a large scale cohort study in Japan. Univariate analysis of JACC study data. Kurume Med J 2004; 51(2): 141-149. https://doi.org/10.2739/kurumemedj.51.141

[25] Bravi F, Bosetti C, Tavani A, Gallus S, La Vecchia C. Coffee reduces risk for hepatocellular carcinoma: an updated meta-analysis. Clin Gastroenterol Hepatol 2013; 11(11): 1413-1421 https://doi.org/10.1016/j.cgh.2013.04.039

[26] Huxley R, Lee CM, Barzi F, Timmermeister L, Czernichow S, Perkovic V, et al. Coffee, decaffeinated coffee, and tea consumption in relation to incident type 2 diabetes mellitus: a systematic review with meta-analysis. Arch Intern Med 2009; 169(22): 2053-63. https://doi.org/10.1001/archinternmed.2009.439

[27] Ezzat S, Abdel-Hamid M, Eissa SA, Mokhtar N, Labib NA, El-Ghorory L, et al. Associations of pesticides, HCV, HBV, and

20 Global Journal of Pathology and Microbiology, 2018 Vol. 6 Noha Said Helal

hepatocellular carcinoma in Egypt. Int J Hy. Environ Health 2005; 208: 329-339 https://doi.org/10.1016/j.ijheh.2005.04.003

[28] Ishak KG, Goodman ZD, Stocker JT. Tumors of the liver and intrahepatic bile ducts. In Atlas of Tumor Pathology, third edition, Armed Forced Institute of Pathology, Washington DC 2001: 199-230

[29] Ascha MS, Hanouneh IA, Lopez R, Abu-Rajab Tamini T, Feldstein AF, Zein NN. The Incidence and Risk Factors of Hepatocellular Carcinoma in Patients with Nonalcoholic Steatohepatitis. Hepatology. 2010; 51(6): 1972-8 https://doi.org/10.1002/hep.23527

[30] American Cancer Society American Cancer Society, available at: (https: //www.cancer.org/cancer/liver-cancer/causes-risks-prevention/risk-factors.html), Last Revised: April 1, 2019,

[31] David E. Kleiner, in Macsween's Pathology of the Liver (Seventh Edition), 2018, Pages 673-779 https://doi.org/10.1016/B978-0-7020-6697-9.00012-1

[32] Torbenson MS, Ng IO, Park YN, Roncalli M, Sakamato M. Hepatocellular carcinoma. In: World Health Organization Classification of Tumors: Pathology and Genetics of Tumors of the Digestive System. 5th edition. IARC Press, Lyon, France 2019: pp229-240

[33] Kimura H, Nakajima T, Kagawa K, Deguchi T, Kakusui M, Katagishi T, et al. Angiogenesis in hepatocellular carcinoma as evaluated by CD34 immunohistochemistry. Liver 1998, 18: 14-19 https://doi.org/10.1111/j.1600-0676.1998.tb00121.x

[34] Theise ND, Curado MP, Franceschi S, Hytiroglou P, Kudo M, Park YN, et al. Tumors of the Liver and Intrahepatic Bile Ducts. In: World Health Organization Classification of

Tumors: Pathology and Genetics of Tumors of the Digestive System. 4th edition. IARC Press, Lyon, France 2010: pp205-216

[35] Brunt EM. Histopathologic Features of Hepatocellular Carcinoma. Clini Liver Dis 2012; 1(6): 194-199 https://doi.org/10.1002/cld.98

[36] Klein WM, Molmenti EP, Colombani PM. Primary liver carcinoma arising in people younger than 30 years. Am J Clin Pathol 2005, 124[4]: 512-8 https://doi.org/10.1309/TT0R7KAL32228E99

[37] Lin CC, Yang HM. Fibrolamellar Carcinoma: A Concise Review. ARCH Pathol Lab MED 2018; 142(9): 1141-1145. https://doi.org/10.5858/arpa.2017-0083-RS

[38] Schlageter M, Terracciano LM, D'Angelo S, Sorrentino P. Histopathology of hepatocellular carcinoma. World J Gastroenterol 2014;20: (43): 15955-15964 https://doi.org/10.3748/wjg.v20.i43.15955

[39] Kojiro M. Histopathology of liver cancers. Best practice and research. Clin Gastroenterol 2005; 19[(1): 39-62 https://doi.org/10.1016/j.bpg.2004.10.007

[40] Tang D, Nagano H, Nakamura M, Wada H, Marubashi S, Miyamoto A, et al. Clinical and pathological features of Allen's type C classification of resected combined hepatocellular and cholangiocarcinoma: a comparative study with hepatocellular carcinoma and cholangiocellular carcinoma. J Gastrointest Surg 2006, 10: 987-998. https://doi.org/10.1016/j.gassur.2006.01.018

[41] Yin X, Zhang BH, Qiu SJ, Ren ZG, Zhou J, Chen XH, et al. Combined hepatocellular carcinoma and cholangiocarcinoma: clinical features, treatment modalities, and prognosis. Ann Surg Oncol 2012, 19(9): 2869-76. https://doi.org/10.1245/s10434-012-2328-0

Received on 24-11-2018 Accepted on 11-12-2018 Published on 31-12-2018 DOI: http://dx.doi.org/10.20941/2310-8703.2018.06.4

© 2018 Noha Said Helal; Licensee Scientific Array. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.