Laboratory evidence of Helicobacter species infection in hepatocellular
carcinoma
Gian Paolo CAVIGLIA1, Antonella OLIVERO1, Chiara ROSSO1, Caterina BOSCO1, Davide G.
RIBALDONE1, Sharmila FAGOONEE2
1Department of Medical Sciences, University of Turin, Turin, Italy
2Institute for Biostructures and Bioimages (CNR) c/o Molecular Biotechnology Center, Turin, Italy
*Corresponding author: Gian Paolo Caviglia, Department of Medical Sciences, University of Turin, Turin 10100, Italy. E-mail: caviglia.giampi@libero.it
Abstract
Chronic Helicobacter pylori infection is considered the main cause of gastric cancer and it has been associated to several extra-gastric manifestations. In addition, Helicobacter species (Helicobacter spp) have been associated to chronic liver disease progression and hepatocellular carcinoma (HCC)
development, both in the animal model and in human beings. Helicobacter spp may be responsible of cancer development through both a direct mechanism involving the production of toxins causing hepatocellular damage and an indirect mechanism of induction of a chronic inflammatory state, a well-known risk factor of progression towards cancer.
To date, most studies investigated Helicobacter spp presence in the liver tissue by means of PCR techniques targeting specific sequences of the 16Sribosomal DNA of the bacteria, while culturing the microorganism from the liver has always been difficult. For this reason, it is still to be proven whether the presence of DNA sequences of Helicobacter spp in the liver represents a true colonization or only a genomic trace related to an entero-hepatic circulation of the bacterium.
Here, we review the current available data regarding the potential association between Helicobacter spp infection and HCC development addressing the issue concerning the detection methods for
Helicobacter spp in liver tissue.
Helicobacter pylori (H. pylori) is a microaerophilic, spiral-shaped, gram-negative bacterium acquired
mainly during childhood.1 From an epidemiological point of view, H. pylori infection global
prevalence is approximately of 50%, with substantial country-to-country variation.2 A review of 37
studies with national coverage in 22 countries reported prevalence peak of approximately 70% or higher around age 60 years in Central and South America and Asia in the late 1990s and early 2000s, with a decreasing trend in most countries where data are available for different time periods.3 More
recently, a meta-analysis of 184 full-text articles comprising data from 62 countries, showed that Africa had the highest pooled prevalence of H. pylori infection (70.1%), whereas Oceania had the lowest prevalence (24.4%).4 Among individual countries, the prevalence of H. pylori infection varied greatly,
from a low prevalence in Switzerland (18.9%) and the highest in Nigeria (87.7%), with nearly 4.4 billion individuals with H. pylori infection worldwide in 2015.4
Chronic infection with H. pylori is associated with around 90% of non-cardia gastric cancer cases in the world;5 in 1994, the International Agency for Research on Cancer classified H. pylori as
group I carcinogen, i.e. a definite cause of gastric cancer.6 As a matter of fact, improved sanitation
together with implementation of national endoscopic screening programs led to a significant reduction of gastric cancer incidence and tumor related death, respectively.7, 8 However, the bacterium has been
associated with the development of gastric mucosa-associated lymphoid tissue lymphomas and a variety of other extra-gastric manifestations, based on potential mechanism involving a low-grade inflammatory state, molecular mimicry patterns, and interference with absorption of nutrients.9-13
In the mouse model, the discovery of Helicobacter (H.) hepaticus, a novel helical
microorganism that selectively and persistently colonizes the biliary tree, able to cause an histological distinctive pattern of chronic active hepatitis and associated with a high incidence of hepatocellular neoplasms in infected animals,14, 15 led to further studies on the involvement of Helicobacter spp in the
Hepatocellular carcinoma: mechanisms and risk factors
Liver cancer is largely a problem of the East Asia and sub-Saharan Africa where 83% (50% in China alone) of the estimated 782,000 new cancer cases worldwide occurred in 2012 (Table I, Figure 1). However, the incidence of liver cancer is increasing also in Europe, particularly in the Southern regions. It is the fifth most common cancer in men (554,000 cases, 7.5% of the total) and the ninth in women (228,000 cases, 3.4%).16 Liver cancer is the second most common cause of death from cancer
worldwide, estimated to be responsible for nearly 746,000 deaths in 2012 (9.1% of the total). The prognosis for liver cancer is very poor (mortality/incidence: 0.95), and as such the geographical patterns in incidence and mortality are similar.16 Hepatocelluar carcinoma (HCC) represents more than
90% of primary liver cancers.17
HCC occurrence results form a complex interplay among genetic predisposition, environmental factors and mechanisms of malignant transformation inducing HCC in predisposing conditions.18-21
Understanding in molecular pathogenesis of HCC has improved in the last decade following the collaborative efforts of researchers sharing data and combining data sets of large number of samples from patients with HCC. The genetic landscape and molecular biomarkers of HCC have been described and data from genomic profile enabled the classification of HCC in 2 major molecular classes
(proliferative and non-proliferative) with different prognostic profiles, activation pathways and tumor phenotype.22
More than 90% of HCCs arise in the setting of an established chronic liver disease. To note, cirrhosis is the principal risk factor for HCC development;23,24 indeed, one-third of cirrhotic patients
will develop HCC during their lifetime.25 Major underlying etiologic factors include infections
(hepatitis B virus [HBV], hepatitis C virus [HCV], liver flukes in endemic areas), behavioral factors (alcohol consumption), metabolic factors (obesity) and aflatoxins; currently, HBV is the leading cause of incident cases of HCC, deaths, and disability-adjusted life-years, followed by alcohol and HCV.26
Beside environmental factors, several host-related factors are associated to a greater risk of HCC development; advanced age and male gender are the main demographic factor significantly associated with increased risk of tumor development.27,28 Also comorbidities play a crucial role in liver disease
progression and thus in higher risk of HCC development; increased hepatic iron content, metabolic syndrome and cigarette smoking represent additional carcinogenic co-factors.29-31Not last, several
studies investigated the potential role of bacterial infections of the liver as a synergic trigger of chronic liver disease progression towards a more severe outcome.32
Helicobacter spp and hepatocellular carcinoma: in vivo studies
Apart from H. pylori that colonizes human gastric environment, several Helicobacter spp have been identified in the intestinal tract and in liver of different vertebrate species, including human beings.33 In many cases, these entero-hepatic Helicobacter spp have been linked with inflammation or
malignant transformation in competent hosts and with more severe clinical disease in
immune-compromised humans and animals.33
Animal studies (A/JCr mouse model) have shown that H. hepaticus infection cause chronic active hepatitis in susceptible mice; histopathological lesions are characterized by focal non-suppurative necrotizing hepatitis progressing to active chronic hepatitis with oval cell hyperplasia, cholangitis and minimal necrosis.14,15 Between 12 and 18 months of age, most mice developed
pre-neoplastic nodular hyperplasia and hepatocellular tumors.14,15 Interestingly, authors succeeded in
isolation of H. hepaticus from the liver and in culturing the microorganism in anaerobic or
microaerobic conditions at 37°C.34 Subsequent studies performed in different mouse models showed
similar hepatic lesions in mice experimentally infected, although no carcinoma was detected.35,36
However, studying the H. hepaticus-resistant C57BL/6 mouse model following the inoculation with the bacteria, it has been showed a genetic basis of resistance to Helicobacter spp-induced liver disease.37
Likely, genetic susceptibility, environmental conditions and specific bacterial strains are key determinants of disease outcome.
From a pathogenic point of view, it is supposed that Helicobacter spp may cause liver damage and consequently HCC trough both a direct and indirect mechanism. H. pylori has a direct oncogenic effect on gastric cells exerted by the cytotoxin-associated gene A (CagA) protein; phosphorylated CagA is able to interact with the pro-oncogenic tyrosine phosphatase 2 which in turn leads to the activation of theRas-mitogen-activated protein kinase pathway which provokes pro-mitogenic cellular response.38 Similarly, it has been reported that H. hepaticus produces a cytolethal distending toxin
(Ctd), an holotoxin composed by three subunits (CdtA, CdtB, and CdtC) of which CdtB exhibits a DNase activity, thus able to trigger tumor development.39,40 Apart from a direct mechanism, chronic
inflammation is known to be a factor of progression towards cancer.41 Indeed, Helicobacter spp
infection results in a strong induction of pro-inflammatory cytokines, such as interleukin 8, via the nuclear factor kappa B pathway.42 Moreover, H. hepaticus infection also results in oxidative liver stress
damage where reactive oxygen and nitrogen species may contribute to carcinogenesis.43
Several clinical studies investigated the potential association between Helicobacter spp infection and HCC (Table II).44-57 Despite a high degree of heterogeneity concerning the background
disease of the patients, the type of control patients and the methods used for Helicobacter spp
assessment in the liver, almost all studies reported a significant association between the presence of the bacteria in the liver and the development of tumor. In addition, a meta-analysis including 9 case-control studies and 1 retrospective cross sectional study, reported an overall prevalence of H. pylori infection of 53.3% (129 out of 242) in the liver of patients with HCC and 10.4% (29 out of 280) in the liver of control patients.58 The summary odds ratio (OR) for the association of H. pylori infection with the risk
Methods for Helicobacter spp detection
Most studies investigated the presence of Helicobacter spp by means of molecular approaches. Precisely, PCR methods were adopted to amplify bacterial DNA sequences located in the 16S
ribosomal DNA (rDNA), using different sets of primers.32 Next, direct sequencing of the 16S rDNA
was used for speciation of the bacteria.32 However, from a microbiological point of view, this
molecular approach could not be considered the “gold standard” for detecting Helicobacter spp infection in the liver. Indeed, bacterial culturing is more reliable and precise in comparison to PCR methods targeting 16S rDNA; more importantly, culture is the ultimate test to prove the presence of viable organismrather than the mere presence of genomic traces. Unfortunately, isolation procedures from liver and culturing of the organism have always been difficult. Avenaud et al. failed to detect any bacterial growth from culture of frozen hepatic tissue under anaerobic or microaerobic conditions; likely, the freezing of the liver tissue at -80°C, which considerably decreases bacterial viability, or the unusual niche of the bacteria, increased the difficulty of culturing.44 Nevertheless, in the case of H.
hepaticus-induced carcinoma, it was not always possible to grow the bacteria despite their presence in
the liver.14 Fox et al.,using frozen liver tissues from 44 mice without characteristic hepatic lesions,
detected H. hepaticus DNA by PCR in the livers of 21 mice (47%), compared to 14 mice (32%) having
H. hepaticus cultured from their frozen liver tumors.59 In addition, the results of H. hepaticus culture
and PCR were consistent in only 84% of the cases.54 Xuan et al., using frozen liver specimens from
clinical samples, found a significant lower positive rate of Helicobacter spp cultured in HCC (10.7%; 3 out of 28) compared to the positive rate of PCR detection (60.7%; 17 out of 28).58
Since the bacterial load in the liver seemed very low,60 another relevant issue was the extraction
method used for DNA isolation. Rabelo-Goncalves et al. compared different protocols for DNA extraction from formalin-fixed paraffin-embedded tissue from patients with HCC in order to detect H.
method. Interestingly, the authors observed that all commercial protocols had similar results in terms of DNA yield, purity and H. pylori detection rate, while phenol-chloroform method resulted in higher amount and purity of extracted DNA, with a trend towards an higher detection rate of H. pylori specific sequences (4 out of 10 samples positive with 3 commercial methods, 5 out of 10 with 1 commercial method and 7 out of 10 with phenol-chloroform-based method, p=0.466).61 Despite phenol-chloroform
protocol is the most laborious, time consuming and potentially toxic method, due to its higher
efficiency in producing a high yield of DNA,62 may be recommended when target DNA it is supposed
to exist in small quantity in the investigated tissue.
Conclusions
To date, there is a growing evidence of H. pylori involvement in several extra-gastric
manifestations.6 In the epidemiological and clinical setting the higher seroprevalence of Helicobacter
spp associated with more advanced stages of liver disease supports the hypothesis of their role in the progression of chronic hepatitis towards cirrhosis and HCC.63-66Also in the biological setting,
Helicobacter spp resulted in almost all studies associated to a more severe disease and to the onset of
cancerous lesions. Despite technical improvements in the fields of microbiology and molecular biology, the difficulty in culturing the microorganism rises doubts on the role of the infection. In fact, the presence of DNA sequences of Helicobacter spp in the liver is not sufficient to demonstrate a causal relationship between the microorganism infection and the progression of chronic liver disease towards cirrhosis and HCC. Moreover, it remains unsolved the issue regarding the route of potential colonization of the liver; the bacterium may arrive in the liver from the circulation through the hepatic portal vein or, more likely, may pass from the stomach to the liver through the duodenum and biliary tract (Figure 2). Well designed and controlled interventional studies are needed to clarify the strength
of association and the potential causal role of Helicobacter spp infection in patients with advanced liver disease and HCC.
Conflicts of interest. The authors certify that there is no conflict of interest with any financial
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Table I. Prevalence and number of deaths for liver cancer in 2012.
Estimated numbers (thousands) Cases Deaths
World 782 746
More developed regions 134 123
Less developed regions 648 622
WHO Africa region 39 37
WHO Americas region 63 58
WHO East Mediterranean region 29 28
WHO Europe region 71 69
WHO South-East Asia region 80 77
WHO Western Pacific region 501 477
IARC membership (24 Countries) 176 159
United States of America 30 24
China 395 383
India 27 27
European Union (EU-28) 52 48
Abbreviations: IARC,International Agency for Research on Cancer; WHO, World Health
Table II. Case-control studies investigating the association between Helicobacter spp infection in liver of patients with hepatocellular carcinoma and controls
Study Country Year Helicobacter positivity Control group
Cases Controls
Avenaud et al.44 France 2000 8/8 1/8 Benign liver tumours (n=6) and
sclerosing cholangitis (n=2)
Nilsson et al.45 Sweden 2001 22/30 0/20 Metastatic liver carcinoma (n=20)
Fan et al.46 China 2002 9/15 0/13 Bening liver tumours (n=8) and
cholelithiasis (n=5)
Dore et al.47 Italy 2002 6/11 5/30 Chronic viral hepatitis without (n=18)
or with cirrhosis (n=12)
Coppola et al.48 Italy 2003 0/21 0/7 Metastatic liver carcinoma (n=7)
Verhoef et al.49 Netherlands 2003 9/20 3/31 Metastatic liver carcinoma (n=31)
Pellicano et al.50 Italy 2004 17/20 2/6 Metastatic liver carcinoma (n=6)
Zhang et al.51 China 2004 16/48 2/37 Chronic viral hepatitis without (n=6)
and with cirrhosis (n=12), benign liver tumours (n=9) and peritumoral liver tissue (n=10)
Ito et al.52 Japan 2004 13/15 0/17 Cirrhotic liver tissue specimen (n=10)
and normal liver tissue specimen (n=7)
Huang et al.53 China 2004 8/20 0/16 Cholelitiasis (n=8), cholecystitis (n=2)
and benign liver tumours (n=6)
Huang et al.54 China 2004 16/38 0/30 Liver cirrhosis (n=15) and benign liver
tumours (n=15)
Rocha et al.55 France 2005 19/31 19/79 Benign liver tumours (n=12), metastatic
liver carcinoma (n=12), chronic hepatitis C without (n=29) and with cirrhosis (25)
Xuan et al.56 China 2006 17/28 0/22 Liver trauma (n=5), cavernous liver
hemangioma (n=7), liver cyst (n=6) and hepatolithiasis (n=4)
Esmat et al.57 Egypt 2012 12/16 18/69 HCV infection without histological
activity (n=16), chronic active hepatitis C (n=25), HCV-related cirrhosis (n=17)
and gastro duodenal and gall bladder disease (n=11)
Figure 1. Worldwide estimated incidence of liver cancer in 2012.
Modified from: GLOBOCAN 2012 (IARC)
In the figure are reported percentages of the estimated number of new cases of liver cancer in selected World regions (USA, Europe, India and China).
