- Email: [email protected]
Helicobacter pylori seroprevalence in patients with cirrhosis of the liver and hepatocellular carcinoma
Cancer Detection and Prevention 27 (2003) 494–497
Helicobacter pylori seroprevalence in patients with cirrhosis of the liver and hepatocellular carcinoma Nicola Leone, MD a , Rinaldo Pellicano, MD a , Franco Brunello, MD a , Miguel Angel Cutufia b , Mara Berrutti, MD a , Sharmila Fagoonee b , Mario Rizzetto, MD, PhD a , Antonio Ponzetto, MD, PhD a,∗ a
Department of Gastro-Hepatology, Molinette Hospital, Via Chiabrera 34, 10126 Turin, Italy b Department of Biology, Biochemistry and Genetics, University of Turin, Turin, Italy Accepted 28 July 2003
Abstract Background: Infection by Helicobacter hepaticus causes chronic hepatitis and hepatocellular carcinoma (HCC) in mice, and Helicobacter pylori (H. pylori) genomic sequences have been demonstrated in the liver of patients with HCC. H. pylori infection reportedly occurs with high frequency in patients with cirrhosis but none of the studies has investigated it in subjects with cirrhosis and superimposed HCC. In this case-control study, we searched for the seroprevalence of H. pylori infection in patients with HCC. Patients and method: Forty-six patients (30 males, 16 females, mean age 69 years) with HCC and hepatitis C virus (HCV)-related cirrhosis were compared to 46 sex and age (±1 year) matched patients presenting consecutively to the Emergency Department of Molinette Hospital of Torino. All subjects were tested for presence in serum of IgG antibodies against H. pylori and the result was analyzed using the chi-square test. Results: H. pylori seropositivity was more prevalent among patients with HCC (36/46, 78.2%) than in controls (25/46, 54%) (P < 0.05) (OR 3.02, 95% confidence interval [1.12–8.34]). Twenty-five out of 30 (83.3%) male patients showed seropositivity at a variance with 16/30 (53%) in the controls (P < 0.05); 11 out of 16 (68.7%) female patients were seropositive versus 9 out of 16 (56.2%) control subjects (P = n.s.). Conclusion: Seroprevalence of antibodies to H. pylori was found to be higher in patients with HCC than in controls. © 2003 Published by Elsevier Ltd. on behalf of International Society for Preventive Oncology. Keywords: Cirrhosis; Liver disease; HCC; Helicobacter pylori; Hepatocellular carcinoma
1. Introduction Hepatocellular carcinoma (HCC) is the fourth cause of death due to cancer worldwide [1] and recent reports indicate that its incidence is rapidly and steadily rising both in the United States [2] and in United Kingdom [3]. HCC arises from chronic liver inflammation (hepatitis) and cirrhosis, a diffuse process with fibrosis and nodule formation, resulting from hepatocellular necrosis [4]. Viral, toxic, metabolic and autoimmune causes have been associated with such necrosis. In western countries, hepatitis C virus (HCV) infection represents the most frequent cause of liver cirrhosis. The majority of HCV-infected individuals become chronic carriers of the virus and long-term follow-up studies have
∗ Corresponding author. Tel.: +39-11-633-6255/6250; fax: +39-11-645-936. E-mail address: [email protected] (A. Ponzetto).
demonstrated that a significant percentage (4–25%) develops cirrhosis leading ultimately to hepatocellular carcinoma [5,6]. It is presently unknown, however, why only a proportion of cirrhotic patients develops HCC. Viral infection, by itself, can only partially explain the pathogenesis of HCC; as for HCV, a direct transforming mechanism remains yet to be established. Furthermore, quite a large percentage of the general population in Italy is infected by HCV and the majority of these infected subjects do not show any sign of liver disease and HCC. Guadagnino et al. reported that 34% of females and 32% of males in a city in southern Italy have circulating HCV RNA in their serum, while a minority have signs of liver disease [7]. There is evidence that viral and host characteristics influence the outcome of HCV infection. The viral genotype 1b has been repeatedly connected with severity of liver outcome, although in few reports this association has been rejected [8].
0361-090X/$30.00 © 2003 Published by Elsevier Ltd. on behalf of International Society for Preventive Oncology. doi:10.1016/j.cdp.2003.07.004
N. Leone et al. / Cancer Detection and Prevention 27 (2003) 494–497
As far as host characteristics are concerned, HLA class II haplotype DR5 in particular has been shown to protect the patient from developing severe disease [9]. A striking finding indicated by Ward et al. was that a bacterial infection of the liver in healthy A/JCr male mice was capable of inducing a strong inflammatory change in the parenchyma (i.e. hepatitis) leading to hepatocellular carcinoma [10]. This bacterial pathogen was demonstrated to belong to Helicobacter genus, and named Helicobacter hepaticus (H. hepaticus) from its ecological niche. The presence of genomic sequences of Helicobacter pylori (H. pylori) has been demonstrated in France in the liver of patients with primary HCC [11] confirming our previous data on HCC arising in patients suffering of liver cirrhosis [12]. H. pylori infection is also very common in subjects suffering from liver cirrhosis [12], but its prevalence has never been reported in HCC patients. Therefore, we undertook a case-control study to verify the occurrence of antibodies against H. pylori in the serum of a cohort of patients affected by HCC, in comparison to a group of patients admitted to the Emergency Department of the same Hospital, the San Giovanni Battista Hospital (Molinette) in Torino.
495
A commercial enzyme linked immunosorbent assay (ELISA, Helori-test® Eurospital, Trieste, Italy) was used to detect anti-H. pylori (IgG) antibodies in serum. This is a test of large diffusion validated in a survey on 3281 patients including also our population, endoscoped for upper gastrointestinal symptoms [13]. The assay sensitivity and specificity versus histology were 70.6 and 90.5%, respectively; positive predictive value was 87.2% [13]. Briefly, calibrators, positive controls, negative controls and diluted (1:200) serum samples were added to wells coated with purified H. pylori group-specific antigens. Plates were incubated for 60 min at 37 ◦ C. After incubation the liquid was removed completely and three washes with 200 l per well of washing solution were made; the liquid was removed and 100 l of anti-IgG conjugate was pipetted into each well; wells were incubated for 60 s at 37 ◦ C; the washing step was repeated, 100 l of chromogenic substrate was added to each well; wells were incubated for 30 s at 37 ◦ C, the reaction was stopped by adding 25 l of stopping solution. Reading was performed at 405 nm and the mean optical density expressed as a percentage of the optical density of a positive control serum assayed on the same plate. The prevalence of H. pylori infection in cases and controls was compared using the chi-square test (χ2 ) by means of 2 × 2 contingency table. Fisher’s exact test was used for small sample size. Results were considered significant when P < 0.05.
2. Materials and methods The presence of anti-H. pylori antibodies was evaluated in 46 consecutive patients (30 males, 16 females, mean age 69 years, range 51–85) with cirrhosis and superimposed HCC, attending the Department of Gastro-Hepatology, Hospital San Giovanni Battista of Torino. HCC was diagnosed either by biochemical (serum levels of alpha fetoprotein and desgamma-carboxyprothrombin) or by ultrasonographic or spiral computed tomography (CT) signs. Angiography, magnetic resonance imaging (MRI) and histology were performed when considered necessary in view to plan for surgical strategy. All these patients were infected by HCV virus. HCV infection was diagnosed when serum samples resulted positive in the test for antibodies against HCV (Axsym System, Abbott Diagnostics). This was confirmed by one of the following assays: RIBA II, LIA, HCV3 (Sorin, Saluggia, Italy) and circulating HCV-RNA was detected by polymerase chain reaction (PCR). Other causes of liver disease, such as viral (HBV), autoimmune (anti-nuclear, anti-mitochondrial, anti-smooth muscle and anti-microsome antibodies) or metabolic (serum iron, percentage of transferrin saturation, ferritin, ceruloplasmin, alpha-1 antitripsin) hepatitis, were ruled out. The controls were 46 sex and age (±1 year) matched patients admitted to the Department of Emergency Care of our hospital (mean age 69, range 51–85 years) and residing in the same area.
3. Results The prevalence of antibodies to H. pylori was significantly higher (78.2%) in the patients with HCC than (54%) in the controls (P < 0.05) (OR 3.02, 95% confidence interval [1.12–8.34]) (Table 1). Thirty-six out of 46 HCC patients were seropositive compared to 25 out of 46 control subjects. In the youngest age group (less than 50 years old), the prevalence of H. pylori infection among HCC patients (100%, 3/3) was higher than in controls (33% 1/3), but due to the small sample this difference was not significant: for the age group 50–59 years, 3/6 (50%) subjects, both among patients than controls, were anti-H. pylori positive. In the group over 60 year, the prevalence of H. pylori infection was higher in HCC patients, 30/37 (81%), than in controls, 21/37 (57%) (P < 0.05) (OR 3.27, 95% confidence interval 1.03–10.70) (Table 1). Table 1 Seroprevalence of anti-H. pylori antibodies among patients with HCC and controls Age (years)
HCC Hp(+)/total
Controls Hp(+)/total
≤49 50–59 >60
3/3 3/6 30/37
1/3 3/6 21/37∗
36/46 (78.2%)
25/46 (54%)∗
∗P
< 0.05.
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N. Leone et al. / Cancer Detection and Prevention 27 (2003) 494–497
Of the male patients, 25 out of 30 (83.3%) seropositivity versus 16 out of 30 (53%) of the (P < 0.05); of female patients, 11 out of 16 were seropositive versus 9 out of 16 (56.2%) of (P = n.s.).
showed controls (68.7%) controls
4. Discussion We found a higher seroprevalence of anti-H. pylori antibodies in patients with hepatocellular carcinoma and liver cirrhosis as compared to subjects admitted to the Emergency Department of the same hospital from 14 to 30 August 1994. This difference cannot be explained by a dissimilar socio-economic status either at time of diagnosis or during childhood, since Emergency Care admission is open and free for everyone. In northern Italy, patients admitted to this unit are more frequently unskilled industry workers, low-level workers who take advantage of free medical care. Furthermore, we matched cases and controls on the basis of occupation and level of education (year of school). We felt therefore that such control population could compare with the cirrhotic patients and this choice is not associated with the same bias that could have arisen if the controls were blood donors. H. pylori infection is a chronic infection. In most instances, it is acquired during childhood, and is often associated with low socio-economic class [14]. The presence of this bacterium has been strongly established as the main cause of several gastroduodenal diseases, including peptic ulcer disease [15], gastric carcinoma [16], and gastric MALT lymphoma [17]. A very high prevalence of H. pylori infection in patients with cirrhosis of the liver has been mentioned in several reports: a higher prevalence of H. pylori infection was noted by Siringo et al. in cirrhotics, compared to blood donors (P < 0.0005) [18] and by Spinzi et al. [19] in two north Italian towns. Our study group has demonstrated a significantly higher seroprevalence of anti-H. pylori antibodies among HCV-infected cirrhotic patients [20]. By a multivariate analysis, Calvet et al. found that male sex and H. pylori seropositivity (OR 1.7, 95% CI 1.02–2.81) were variables independently related to peptic ulcer in cirrhotics with different etiologies [21]. In Taiwan, no association was found by Chen et al. between peptic ulcer and H. pylori infection in cirrhotic patients [22], but on the contrary, Fan et al. demonstrated a higher seroprevalence of H. pylori in Chinese patients with HBV-related chronic hepatitis than in controls matched for age and socio-economic status [23]. Selection bias and methodological diagnosis could possibly be the origin of the heterogeneity of the studies. The prevalence of H. pylori infection in patients with cirrhosis of the liver and superimposed HCC had not been reported thus far. Clinical and experimental considerations hence suggested the rational for such a study [24]. From a clinical point of view, the medical history of cirrhotic patients is punctuated by frequent and recurrent hos-
pitalizations due to high rate of complications. Among the most relevant of them, peptic ulcer and upper GI hemorrhage are of peculiar relevance, being life-threatening for the patient and of high cost for Health Care Services, requiring both emergency care and subsequent long hospital stay [25]. Kirk et al. demonstrated in 1980 a frequency of 33% of peptic ulcer in patients with chronic liver disease [26]. In an Italian multicentre study, between 12 and 20% of cirrhotic patients were demonstrated to bear gastric or duodenal ulcer with a high prevalence in the gastric site [27]. Since peptic ulcer is related to the presence of H. pylori infection in non-cirrhotic patients, it is logical to suppose a role for the same bacterium also in subjects with cirrhosis. To search for the presence of the bacterium and to cure from it, stems from the rationale to prevent the development of peptic ulcer and its complications in cirrhotics, too, as we usually do in non-cirrhotic patients. From an epidemiological point of view, several data indicated that only a proportion of patients infected by HCV develops liver cirrhosis [7], and among these only a minority ultimately succumbed to liver cancer, and also that “classical” prognostic features do not explain all the variations of the disease [8]. These observations suggested that other factors besides the viral pathogen could concur in generating HCC. After the experimental demonstration by Ward et al. [10] that H. hepaticus causes hepatitis and HCC in male A/JCr mice, a number of Helicobacter sp. have been isolated from the liver of cats and dogs with hepatitis [28] and from the human biliary tract and gallbladder [29]. In western countries, HCC arises almost invariably on the background of cirrhosis representing a long-term complication of the disease, after decades of continuing inflammation. One “new” possible mechanism capable of inducing pro-inflammatory cytokines and lymphoid proliferation might indeed be liver or biliary tract infection by bacteria belonging to Helicobacter genus. A few papers in the last years reported the finding of genomic sequences belonging to Helicobacter spp. in the liver of patients with HCC. We have shown sequences of Helicobacter spp. in 23 of 25 human livers with cirrhosis and HCC [12]. Avenaud et al. confirmed these data by demonstrating genomic sequences of Helicobacter sp. in eight of eight liver specimens from patients with HCC and the sequenced PCR products confirmed H. pylori and Helicobacter felis [11]. Moreover, Agha-Amiri et al. found, in seven out of 20 patients with HCC, genomic sequences of a bacterium belonging to the RNA superfamily VI (Campylobacter, Helicobacter, Arcobacter) with highest homology to Arcobacter spp. [30]. By PCR analysis, hybridization studies and partial DNA sequencing, Nilsson et al. have recently demonstrated genomic sequences of H. pylori and Helicobacter spp. in human liver from patients with primary sclerosing cholangitis or primary biliary cirrhosis [31].
N. Leone et al. / Cancer Detection and Prevention 27 (2003) 494–497
The role of Helicobacter spp. in the evolution of cirrhosis and HCC in humans is unknown, but a potential mechanism has been reported by Taylor et al., who described a new liver-specific toxin produced by several Helicobacter spp. [32]. In conclusion, we found that the seroprevalence of H. pylori in subjects with cirrhosis of the liver and superimposed HCC is much more frequent than in controls. This fact raises the question on its correlation with the higher prevalence of peptic ulcer and bleeding in such patients. Moreover, the recent data on the presence of genomic sequences of the Helicobacter spp. in the liver of patients with HCC should alert us on a possible, new prognostic factor for HCV carriers, which in mice is able to induce HCC, and in humans is related to gastric carcinogenesis. Acknowledgements The financial support of Associazione Italiana per la Ricerca sul Cancro (AIRC) is gratefully acknowledged. We are deeply indebted to Miss Debora Pellicano for the strong commitment and continuing support. References [1] Pisani P, Parkin DM, Bray F, et al. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer 1999;83:18–29. [2] El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. NEJM 1999;340:745–50. [3] Taylor-Robinson SD, Foster GR, Arora S, et al. Increase in primary liver cancer in the UK, 1974–1994. Lancet 1997;350:1142–3. [4] Hepatic cirrhosis. In: Sherlock S, Dooley J. Diseases of the liver and biliary system, 10th ed. Oxford: Blackwell Scientific Publications; 1997. [5] Yano M, Kumada H, Kage M, et al. The long-term pathological evolution of chronic hepatitis C. Hepatology 1996;23:1334– 40. [6] Chiaramonte M, Stroffolini T, Vian A, et al. Rate of incidence of hepatocellular carcinoma in patients with compensated viral cirrhosis. Cancer 1999;85:2132–7. [7] Guadagnino V, Stroffolini T, Rapicetta M, et al. Prevalence, risk factors and genotype distribution of hepatitis C virus infection in the general population: a community-based survey in southern Italy. Hepatology 1997;26:1006–11. [8] Wiese M, Berr F, Lafrenz M, et al. Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20-year multicenter study. Hepatology 2000;32:91–6. [9] Peano G, Menardi G, Ponzetto A, et al. HLA-DR5: a genetic factor influencing the outcome of hepatitis C virus infection? Arch Int Med 1994;154:2733–6. [10] Ward JM, Fox JG, Anver MR, et al. Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter sp.. J Natl Cancer Inst 1994;86:1222–7. [11] Avenaud P, Marais A, Monteiro L, et al. Detection of Helicobacter spp. in the liver of patients with and without primary liver carcinoma. Cancer 2000;89:1431–9.
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[12] Ponzetto A, Pellicano R, Leone N, et al. Helicobacter infection and cirrhosis in hepatitis C virus carriage: is it an innocent bystander or a troublemaker? Med Hypoth 2000;54:275–7. [13] Palli D, Vaira D, Menegatti M, et al. A serologic survey of Helicobacter pylori infection in 3281 Italian patients endoscoped for upper gastrointestinal symptoms. Aliment Pharmacol Ther 1997;11:719–28. [14] Mendall MA, Goggin PM, Molineaux N, et al. Association between childhood living conditions and Helicobacter pylori seropositivity in adult life. Lancet 1992;i:896–7. [15] Hopkins RJ, Girardi LS, Turney EA. Relationship between Helicobacter pylori eradication and reduced duodenal and gastric ulcer recurrence: a review. Gastroenterology 1996;110:1244–52. [16] International Agency for Research on Cancer. World Health Organization. Infection with Helicobacter pylori. In: Schistosomes, liver flukes and Helicobacter pylori. Lyon: IARC; 1994. p. 177–202. [17] Wotherspoon AC, Doglioni C, Diss TC, et al. Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori. Lancet 1993;342:575–7. [18] Siringo S, Vaira D, Menegatti M, et al. High prevalence of Helicobacter pylori in liver cirrhosis: relationship with clinical and endoscopic features and the risk of peptic ulcer. Dig Dis Sci 1997;42:2024–30. [19] Spinzi G, Pellicano R, Minoli G, et al. Helicobacter pylori seroprevalence in hepatitis C virus positive patients with cirrhosis: the Como cross-sectional study. Panminerva Med 2001;43:85–7. [20] Pellicano R, Leone N, Berrutti M, et al. Helicobacter pylori seroprevalence in hepatitis C virus positive patients with cirrhosis. J Hepatol 2000;33:648–50. [21] Calvet X, Navarro M, Gil M, et al. Epidemiology of peptic ulcer disease in cirrhotic patients: role of Helicobacter pylori infection. Am J Gastroenterol 1998;93:2501–7. [22] Chen J-J, ChangChien C-S, Tai D-I, et al. Role of Helicobacter pylori in cirrhotic patients with peptic ulcer. Dig Dis Sci 1994;39:1565–8. [23] Fan XG, Zou YY, Wu AH, et al. Seroprevalence of Helicobacter pylori infection in patients with hepatitis B. Br J Biomed Sci 1998;55:176–8. [24] Fagoonee S, Pellicano R, Rizzetto M, et al. The journey from hepatitis to hepatocellular carcinoma. Bridging role of Helicobacter sp.. Panminerva Med 2001;43:279–82. [25] Rosina F, Alaria P, Castelli S, et al. Effect of patient characteristics on hospital costs for cirrhosis: implications for the disease-related group (DRG) reimbursement system. Ital J Gastroenterol 1996;28:401–5. [26] Kirk AP, Dooley JS, Hunt RH. Peptic ulceration in patients with chronic liver disease. Dig Dis Sci 1980;25:756–60. [27] Gottardello L, Dalri L, Di Mario F, et al. Peptic ulcer and liver cirrhosis: clinical and therapeutical features. Minerva Med 1991;82:81–5. [28] Fox JG, Drolet R, Higgins R, et al. Helicobacter canis isolated from a dog liver with multifocal necrotizing hepatitis. J Clin Microbiol 1996;34:2479–82. [29] Fox JG, Dewhirst FE, Shen Z, et al. Hepatic Helicobacter sp. identified in bile and gallbladder tissue from chileans with chronic cholecystitis. Gastroenterology 1998;114:755–63. [30] Agha-Amiri K, Meier H, Rocken C, et al. New bacterial species in human hepatocellular carcinoma. Gut 1998;43(2):A89. [31] Nilsson H-O, Taneera J, Castedal M, et al. Identification of Helicobacter pylori and other Helicobacter spp. by PCR, hybridization and partial DNA sequencing in human liver samples from patients with primary sclerosing cholangitis or primary biliary cirrhosis. J Clin Microbiol 2000;38:1072–6. [32] Taylor NS, Fox JG, Yan L. In vitro hepatotoxic factor in Helicobacter hepaticus, H. pylori and other Helicobacter sp.. J Med Microbiol 1995;42:48–52.
Helicobacter pylori seroprevalence in patients with cirrhosis of the liver and hepatocellular carcinoma Nicola Leone, MD a , Rinaldo Pellicano, MD a , Franco Brunello, MD a , Miguel Angel Cutufia b , Mara Berrutti, MD a , Sharmila Fagoonee b , Mario Rizzetto, MD, PhD a , Antonio Ponzetto, MD, PhD a,∗ a
Department of Gastro-Hepatology, Molinette Hospital, Via Chiabrera 34, 10126 Turin, Italy b Department of Biology, Biochemistry and Genetics, University of Turin, Turin, Italy Accepted 28 July 2003
Abstract Background: Infection by Helicobacter hepaticus causes chronic hepatitis and hepatocellular carcinoma (HCC) in mice, and Helicobacter pylori (H. pylori) genomic sequences have been demonstrated in the liver of patients with HCC. H. pylori infection reportedly occurs with high frequency in patients with cirrhosis but none of the studies has investigated it in subjects with cirrhosis and superimposed HCC. In this case-control study, we searched for the seroprevalence of H. pylori infection in patients with HCC. Patients and method: Forty-six patients (30 males, 16 females, mean age 69 years) with HCC and hepatitis C virus (HCV)-related cirrhosis were compared to 46 sex and age (±1 year) matched patients presenting consecutively to the Emergency Department of Molinette Hospital of Torino. All subjects were tested for presence in serum of IgG antibodies against H. pylori and the result was analyzed using the chi-square test. Results: H. pylori seropositivity was more prevalent among patients with HCC (36/46, 78.2%) than in controls (25/46, 54%) (P < 0.05) (OR 3.02, 95% confidence interval [1.12–8.34]). Twenty-five out of 30 (83.3%) male patients showed seropositivity at a variance with 16/30 (53%) in the controls (P < 0.05); 11 out of 16 (68.7%) female patients were seropositive versus 9 out of 16 (56.2%) control subjects (P = n.s.). Conclusion: Seroprevalence of antibodies to H. pylori was found to be higher in patients with HCC than in controls. © 2003 Published by Elsevier Ltd. on behalf of International Society for Preventive Oncology. Keywords: Cirrhosis; Liver disease; HCC; Helicobacter pylori; Hepatocellular carcinoma
1. Introduction Hepatocellular carcinoma (HCC) is the fourth cause of death due to cancer worldwide [1] and recent reports indicate that its incidence is rapidly and steadily rising both in the United States [2] and in United Kingdom [3]. HCC arises from chronic liver inflammation (hepatitis) and cirrhosis, a diffuse process with fibrosis and nodule formation, resulting from hepatocellular necrosis [4]. Viral, toxic, metabolic and autoimmune causes have been associated with such necrosis. In western countries, hepatitis C virus (HCV) infection represents the most frequent cause of liver cirrhosis. The majority of HCV-infected individuals become chronic carriers of the virus and long-term follow-up studies have
∗ Corresponding author. Tel.: +39-11-633-6255/6250; fax: +39-11-645-936. E-mail address: [email protected] (A. Ponzetto).
demonstrated that a significant percentage (4–25%) develops cirrhosis leading ultimately to hepatocellular carcinoma [5,6]. It is presently unknown, however, why only a proportion of cirrhotic patients develops HCC. Viral infection, by itself, can only partially explain the pathogenesis of HCC; as for HCV, a direct transforming mechanism remains yet to be established. Furthermore, quite a large percentage of the general population in Italy is infected by HCV and the majority of these infected subjects do not show any sign of liver disease and HCC. Guadagnino et al. reported that 34% of females and 32% of males in a city in southern Italy have circulating HCV RNA in their serum, while a minority have signs of liver disease [7]. There is evidence that viral and host characteristics influence the outcome of HCV infection. The viral genotype 1b has been repeatedly connected with severity of liver outcome, although in few reports this association has been rejected [8].
0361-090X/$30.00 © 2003 Published by Elsevier Ltd. on behalf of International Society for Preventive Oncology. doi:10.1016/j.cdp.2003.07.004
N. Leone et al. / Cancer Detection and Prevention 27 (2003) 494–497
As far as host characteristics are concerned, HLA class II haplotype DR5 in particular has been shown to protect the patient from developing severe disease [9]. A striking finding indicated by Ward et al. was that a bacterial infection of the liver in healthy A/JCr male mice was capable of inducing a strong inflammatory change in the parenchyma (i.e. hepatitis) leading to hepatocellular carcinoma [10]. This bacterial pathogen was demonstrated to belong to Helicobacter genus, and named Helicobacter hepaticus (H. hepaticus) from its ecological niche. The presence of genomic sequences of Helicobacter pylori (H. pylori) has been demonstrated in France in the liver of patients with primary HCC [11] confirming our previous data on HCC arising in patients suffering of liver cirrhosis [12]. H. pylori infection is also very common in subjects suffering from liver cirrhosis [12], but its prevalence has never been reported in HCC patients. Therefore, we undertook a case-control study to verify the occurrence of antibodies against H. pylori in the serum of a cohort of patients affected by HCC, in comparison to a group of patients admitted to the Emergency Department of the same Hospital, the San Giovanni Battista Hospital (Molinette) in Torino.
495
A commercial enzyme linked immunosorbent assay (ELISA, Helori-test® Eurospital, Trieste, Italy) was used to detect anti-H. pylori (IgG) antibodies in serum. This is a test of large diffusion validated in a survey on 3281 patients including also our population, endoscoped for upper gastrointestinal symptoms [13]. The assay sensitivity and specificity versus histology were 70.6 and 90.5%, respectively; positive predictive value was 87.2% [13]. Briefly, calibrators, positive controls, negative controls and diluted (1:200) serum samples were added to wells coated with purified H. pylori group-specific antigens. Plates were incubated for 60 min at 37 ◦ C. After incubation the liquid was removed completely and three washes with 200 l per well of washing solution were made; the liquid was removed and 100 l of anti-IgG conjugate was pipetted into each well; wells were incubated for 60 s at 37 ◦ C; the washing step was repeated, 100 l of chromogenic substrate was added to each well; wells were incubated for 30 s at 37 ◦ C, the reaction was stopped by adding 25 l of stopping solution. Reading was performed at 405 nm and the mean optical density expressed as a percentage of the optical density of a positive control serum assayed on the same plate. The prevalence of H. pylori infection in cases and controls was compared using the chi-square test (χ2 ) by means of 2 × 2 contingency table. Fisher’s exact test was used for small sample size. Results were considered significant when P < 0.05.
2. Materials and methods The presence of anti-H. pylori antibodies was evaluated in 46 consecutive patients (30 males, 16 females, mean age 69 years, range 51–85) with cirrhosis and superimposed HCC, attending the Department of Gastro-Hepatology, Hospital San Giovanni Battista of Torino. HCC was diagnosed either by biochemical (serum levels of alpha fetoprotein and desgamma-carboxyprothrombin) or by ultrasonographic or spiral computed tomography (CT) signs. Angiography, magnetic resonance imaging (MRI) and histology were performed when considered necessary in view to plan for surgical strategy. All these patients were infected by HCV virus. HCV infection was diagnosed when serum samples resulted positive in the test for antibodies against HCV (Axsym System, Abbott Diagnostics). This was confirmed by one of the following assays: RIBA II, LIA, HCV3 (Sorin, Saluggia, Italy) and circulating HCV-RNA was detected by polymerase chain reaction (PCR). Other causes of liver disease, such as viral (HBV), autoimmune (anti-nuclear, anti-mitochondrial, anti-smooth muscle and anti-microsome antibodies) or metabolic (serum iron, percentage of transferrin saturation, ferritin, ceruloplasmin, alpha-1 antitripsin) hepatitis, were ruled out. The controls were 46 sex and age (±1 year) matched patients admitted to the Department of Emergency Care of our hospital (mean age 69, range 51–85 years) and residing in the same area.
3. Results The prevalence of antibodies to H. pylori was significantly higher (78.2%) in the patients with HCC than (54%) in the controls (P < 0.05) (OR 3.02, 95% confidence interval [1.12–8.34]) (Table 1). Thirty-six out of 46 HCC patients were seropositive compared to 25 out of 46 control subjects. In the youngest age group (less than 50 years old), the prevalence of H. pylori infection among HCC patients (100%, 3/3) was higher than in controls (33% 1/3), but due to the small sample this difference was not significant: for the age group 50–59 years, 3/6 (50%) subjects, both among patients than controls, were anti-H. pylori positive. In the group over 60 year, the prevalence of H. pylori infection was higher in HCC patients, 30/37 (81%), than in controls, 21/37 (57%) (P < 0.05) (OR 3.27, 95% confidence interval 1.03–10.70) (Table 1). Table 1 Seroprevalence of anti-H. pylori antibodies among patients with HCC and controls Age (years)
HCC Hp(+)/total
Controls Hp(+)/total
≤49 50–59 >60
3/3 3/6 30/37
1/3 3/6 21/37∗
36/46 (78.2%)
25/46 (54%)∗
∗P
< 0.05.
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N. Leone et al. / Cancer Detection and Prevention 27 (2003) 494–497
Of the male patients, 25 out of 30 (83.3%) seropositivity versus 16 out of 30 (53%) of the (P < 0.05); of female patients, 11 out of 16 were seropositive versus 9 out of 16 (56.2%) of (P = n.s.).
showed controls (68.7%) controls
4. Discussion We found a higher seroprevalence of anti-H. pylori antibodies in patients with hepatocellular carcinoma and liver cirrhosis as compared to subjects admitted to the Emergency Department of the same hospital from 14 to 30 August 1994. This difference cannot be explained by a dissimilar socio-economic status either at time of diagnosis or during childhood, since Emergency Care admission is open and free for everyone. In northern Italy, patients admitted to this unit are more frequently unskilled industry workers, low-level workers who take advantage of free medical care. Furthermore, we matched cases and controls on the basis of occupation and level of education (year of school). We felt therefore that such control population could compare with the cirrhotic patients and this choice is not associated with the same bias that could have arisen if the controls were blood donors. H. pylori infection is a chronic infection. In most instances, it is acquired during childhood, and is often associated with low socio-economic class [14]. The presence of this bacterium has been strongly established as the main cause of several gastroduodenal diseases, including peptic ulcer disease [15], gastric carcinoma [16], and gastric MALT lymphoma [17]. A very high prevalence of H. pylori infection in patients with cirrhosis of the liver has been mentioned in several reports: a higher prevalence of H. pylori infection was noted by Siringo et al. in cirrhotics, compared to blood donors (P < 0.0005) [18] and by Spinzi et al. [19] in two north Italian towns. Our study group has demonstrated a significantly higher seroprevalence of anti-H. pylori antibodies among HCV-infected cirrhotic patients [20]. By a multivariate analysis, Calvet et al. found that male sex and H. pylori seropositivity (OR 1.7, 95% CI 1.02–2.81) were variables independently related to peptic ulcer in cirrhotics with different etiologies [21]. In Taiwan, no association was found by Chen et al. between peptic ulcer and H. pylori infection in cirrhotic patients [22], but on the contrary, Fan et al. demonstrated a higher seroprevalence of H. pylori in Chinese patients with HBV-related chronic hepatitis than in controls matched for age and socio-economic status [23]. Selection bias and methodological diagnosis could possibly be the origin of the heterogeneity of the studies. The prevalence of H. pylori infection in patients with cirrhosis of the liver and superimposed HCC had not been reported thus far. Clinical and experimental considerations hence suggested the rational for such a study [24]. From a clinical point of view, the medical history of cirrhotic patients is punctuated by frequent and recurrent hos-
pitalizations due to high rate of complications. Among the most relevant of them, peptic ulcer and upper GI hemorrhage are of peculiar relevance, being life-threatening for the patient and of high cost for Health Care Services, requiring both emergency care and subsequent long hospital stay [25]. Kirk et al. demonstrated in 1980 a frequency of 33% of peptic ulcer in patients with chronic liver disease [26]. In an Italian multicentre study, between 12 and 20% of cirrhotic patients were demonstrated to bear gastric or duodenal ulcer with a high prevalence in the gastric site [27]. Since peptic ulcer is related to the presence of H. pylori infection in non-cirrhotic patients, it is logical to suppose a role for the same bacterium also in subjects with cirrhosis. To search for the presence of the bacterium and to cure from it, stems from the rationale to prevent the development of peptic ulcer and its complications in cirrhotics, too, as we usually do in non-cirrhotic patients. From an epidemiological point of view, several data indicated that only a proportion of patients infected by HCV develops liver cirrhosis [7], and among these only a minority ultimately succumbed to liver cancer, and also that “classical” prognostic features do not explain all the variations of the disease [8]. These observations suggested that other factors besides the viral pathogen could concur in generating HCC. After the experimental demonstration by Ward et al. [10] that H. hepaticus causes hepatitis and HCC in male A/JCr mice, a number of Helicobacter sp. have been isolated from the liver of cats and dogs with hepatitis [28] and from the human biliary tract and gallbladder [29]. In western countries, HCC arises almost invariably on the background of cirrhosis representing a long-term complication of the disease, after decades of continuing inflammation. One “new” possible mechanism capable of inducing pro-inflammatory cytokines and lymphoid proliferation might indeed be liver or biliary tract infection by bacteria belonging to Helicobacter genus. A few papers in the last years reported the finding of genomic sequences belonging to Helicobacter spp. in the liver of patients with HCC. We have shown sequences of Helicobacter spp. in 23 of 25 human livers with cirrhosis and HCC [12]. Avenaud et al. confirmed these data by demonstrating genomic sequences of Helicobacter sp. in eight of eight liver specimens from patients with HCC and the sequenced PCR products confirmed H. pylori and Helicobacter felis [11]. Moreover, Agha-Amiri et al. found, in seven out of 20 patients with HCC, genomic sequences of a bacterium belonging to the RNA superfamily VI (Campylobacter, Helicobacter, Arcobacter) with highest homology to Arcobacter spp. [30]. By PCR analysis, hybridization studies and partial DNA sequencing, Nilsson et al. have recently demonstrated genomic sequences of H. pylori and Helicobacter spp. in human liver from patients with primary sclerosing cholangitis or primary biliary cirrhosis [31].
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The role of Helicobacter spp. in the evolution of cirrhosis and HCC in humans is unknown, but a potential mechanism has been reported by Taylor et al., who described a new liver-specific toxin produced by several Helicobacter spp. [32]. In conclusion, we found that the seroprevalence of H. pylori in subjects with cirrhosis of the liver and superimposed HCC is much more frequent than in controls. This fact raises the question on its correlation with the higher prevalence of peptic ulcer and bleeding in such patients. Moreover, the recent data on the presence of genomic sequences of the Helicobacter spp. in the liver of patients with HCC should alert us on a possible, new prognostic factor for HCV carriers, which in mice is able to induce HCC, and in humans is related to gastric carcinogenesis. Acknowledgements The financial support of Associazione Italiana per la Ricerca sul Cancro (AIRC) is gratefully acknowledged. We are deeply indebted to Miss Debora Pellicano for the strong commitment and continuing support. References [1] Pisani P, Parkin DM, Bray F, et al. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer 1999;83:18–29. [2] El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. NEJM 1999;340:745–50. [3] Taylor-Robinson SD, Foster GR, Arora S, et al. Increase in primary liver cancer in the UK, 1974–1994. Lancet 1997;350:1142–3. [4] Hepatic cirrhosis. In: Sherlock S, Dooley J. Diseases of the liver and biliary system, 10th ed. Oxford: Blackwell Scientific Publications; 1997. [5] Yano M, Kumada H, Kage M, et al. The long-term pathological evolution of chronic hepatitis C. Hepatology 1996;23:1334– 40. [6] Chiaramonte M, Stroffolini T, Vian A, et al. Rate of incidence of hepatocellular carcinoma in patients with compensated viral cirrhosis. Cancer 1999;85:2132–7. [7] Guadagnino V, Stroffolini T, Rapicetta M, et al. Prevalence, risk factors and genotype distribution of hepatitis C virus infection in the general population: a community-based survey in southern Italy. Hepatology 1997;26:1006–11. [8] Wiese M, Berr F, Lafrenz M, et al. Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20-year multicenter study. Hepatology 2000;32:91–6. [9] Peano G, Menardi G, Ponzetto A, et al. HLA-DR5: a genetic factor influencing the outcome of hepatitis C virus infection? Arch Int Med 1994;154:2733–6. [10] Ward JM, Fox JG, Anver MR, et al. Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter sp.. J Natl Cancer Inst 1994;86:1222–7. [11] Avenaud P, Marais A, Monteiro L, et al. Detection of Helicobacter spp. in the liver of patients with and without primary liver carcinoma. Cancer 2000;89:1431–9.
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