Original Article
Polymerase Chain Reaction to Detect Hepatitis B Virus DNA and RNA Sequences in Primary Liver Cancers from Patients Negative for Hepatitis B Surface Antigen
N Engl J Med 1990; 323:80-85July 12, 1990
- Abstract
- Abstract
Background and Methods.
The role of hepatitis B virus (HBV) in the course of patients with primary liver cancer who are negative for hepatitis B surface antigen has been debated. We used the polymerase chain reaction to evaluate 28 such patients for the presence of DNA and RNA sequences of the virus; 22 of these patients had associated cirrhosis. The patients were from areas with different prevalences of HBV infection (South Africa, Italy, France, and Japan).
Results.
Antibodies to the surface and core antigens of HBV were detected in 10 of the 23 patients tested. HBV DNA sequences were detected in 17 of the 28 patients, including 8 of the 10 with HBV antibodies and 6 of 13 without HBV serologic markers. HBV RNA molecules were found in four of five tumors tested.
Conclusions.
Our investigation indicates that transcriptionally active HBV genomes are present in various geographic areas among patients with liver cancer who are negative for hepatitis B surface antigen. This observation is consistent with an etiologic role for the virus in the development of these tumors. (N Engl J Med 1990; 323:80–5.)
Article Activity
- Article
EXTENSIVE epidemiologic studies1 2 3 4 have shown a clear association between chronic infection with hepatitis B virus (HBV) and primary liver cancer. The evidence for this association includes the geographic congruence between areas with a high incidence of primary liver cancer and those with a high prevalence of HBV infection, the increase in the number of serologic markers of HBV in patients with liver cancer as compared with the general population residing in the same area, and the increased risk of primary liver cancer in persons positive for hepatitis B surface antigen (HBsAg), according to prospective studies conducted in Taiwan2 and Japan.3 HBV is thought to induce liver cancer in chronic carriers of HBsAg by a combination of at least two mechanisms. HBV infection is a factor contributing to cirrhosis of the liver, which is associated with primary liver cancer in 60 to 90 percent of patients with this disease; thus, cirrhosis appears to be a major cofactor in liver cancer. In addition, molecular studies have revealed other potential mechanisms that may account for a direct role of HBV in carcinogenesis; integrated HBV DNA sequences are present in hepatoma cells and can modify cellular expression of the gene by both extensive chromosomal rearrangement and insertional mutagenesis.5 6 7 8 9 10 11 12 13 14 15 16 Moreover, the viral protein X may act as a transactivating factor on cellular genes associated with transformation.17 , 18
The role of HBV in HBsAg-negative patients with liver cancer has been debated. We and others19 20 21 22 23 24 25 26 27 28 29 30 have previously detected HBV DNA sequences in the liver and serum of blood donors and patients with chronic hepatitis and hepatocellular carcinoma, although HBsAg was not identified in serum by conventional assays. However, the frequency of these findings remains controversial, since other investigators have been unable to demonstrate the presence of viral DNA and RNA in HBsAg-negative patients.31 32 33 34 35 36 37 These discrepancies may be linked to epidemiologic differences in the prevalence of exposure to HBV or possibly technical problems with Southern blotting (including both specificity and sensitivity19). Comparing HBsAg-positive with HBsAg-negative tumors clearly shows that in the latter, the number of viral genomes per cell is much lower.19
This issue is of major importance in the assessment of etiologic factors in liver cancer. Therefore, we have taken advantage of the sensitivity and specificity of the polymerase chain reaction (PCR)23 , 38 39 40 to search for HBV DNA and RNA sequences in the tumors of HBsAg-negative patients with liver cancer who were from different geographic areas.
Methods
Patients
We studied 28 HBsAg-negative patients with primary liver cancer from different geographic areas: 9 were from Italy, 10 from France, 8 from South Africa, and 1 from Japan. Cirrhosis was found to be associated with the tumor in six of the Italian patients, seven of the French patients, all eight South African patients, and the Japanese patient. We were particularly interested in the six patients without any serologic markers for HBV (see below). Liver samples were obtained at surgery in France, Italy, and Japan and at autopsy in South Africa. The presence of hemochromatosis, Wilson's disease and autoimmune diseases, and α1–antitrypsin deficiency was ruled out. Six patients had a history of chronic alcohol abuse.
Peripheral-blood leukocytes were obtained from 15 blood donors without HBV serologic markers and used as control samples.
Serologic Assays
Testing for HBsAg, the antibody to hepatitis B core antigen (anti-HBc), and the antibody to hepatitis B surface antigen (anti-HBs) was performed according to standard procedures (radioimmunoassay; Abbott Laboratories, North Chicago).
PCR Testing
The extraction and amplification of DNA were performed as previously described5 , 23 , 38; special care was taken to avoid any contamination. By comparing nucleotide sequences in hepadna viruses, we defined different sets of conserved DNA sequences to be used as HBV-specific primers and probes (Table 1Table 1
Oligonucleotide Sequences Used as HBV Primers and Probes (from the ayw Subtype of HBV4).*).RNA was extracted from 25 to 50 mg of frozen tumor samples according to the guanidium isothiocyanate—cesium chloride method.41 Samples weighing 1 to 3 μg were incubated with 4 units of ribonuclease-free deoxyribonuclease (Promega, Biotec, Madison, Wis.) at 37°C for 30 minutes. The enzyme was subsequently heat inactivated (at 95°C for 5 minutes), and reverse transcription was carried out in a volume of 20 μl, which contained 20 pmol of the downstream primer, 200 μmol of each of four deoxynucleotides per liter, 50 mmol of TRIS hydrochloric acid per liter (pH 8.3), 75 mmol of potassium chloride per liter, 10 mmol of dithiothreitol per liter, 3 mmol of magnesium chloride per liter, 2 μg of nuclease-free bovine serum albumin, and 200 U of cloned Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.), incubated at 42°C for 30 minutes. The complementary DNA product was amplified in 100 μl containing 20 pmol of upstream primer, 200 μmol of each of four deoxynucleotides per liter, and the PCR buffer. Forty cycles were carried out as in the PCR for DNA. In each experiment and for each sample, a "control test" omitting the reverse transcriptase was performed.
After amplification of the RNA sequences, 14 μl of the PCR mixture was subjected to electrophoresis on 1.5 percent agarose gel in TRIS borate buffer and transferred to a nylon membrane (Gene-Screen Plus, NEN, Boston). The filters were prehybridized and hybridized under stringent conditions as previously described.23
Control of Sensitivity and Specificity
PCR testing has been shown to be highly sensitive in detecting HBV DNA. We are able to detect as little as 1 fg of reference cloned HBV DNA.42 However, this high level of sensitivity may easily lead to false positive results due to minute contaminations. Therefore, the following precautions were taken to ensure the validity of the results. (1) Precautions to avoid carryover of the PCR product included physical separation of pre-PCR and post-PCR mixtures and aliquots of reagents, the use of positive displacement pipettes, and the avoidance of aerosols. (2) DNA and RNA extraction were repeated twice for each tumor, with special care to avoid cross-contamination. (3) Results were considered valid only if they were consistent in repeated (at least twice) independent experiments. (4) Contamination by plasmids containing HBV insert was excluded. (5) Negative controls including leukocyte and liver DNA samples as well as a reaction mixture without DNA were run in each experiment. In addition, preparations of peripheral-blood leukocyte DNA from 15 blood donors were tested. (6) Hybridization was performed under stringent conditions. (7) Different sets of HBV primers were used in the same experiment for each DNA sample. (8) To test whether the quality of the purified cellular DNA was sufficient for PCR, primers specific for single cellular coding sequences were used — i.e., β-globin and HLA. (9) Positive results obtained with the pre-S—specific primers (located on both sides of the EcoRI site of HBV) precluded amplification from a contaminating linear HBV insert.
Results
Ten of 23 patients tested had anti-HBc, anti-HBs, or both, and 13 had no serologic marker of HBV (Table 2Table 2
Results of Serologic and PCR Testing in HBsAg-Negative Patients with Primary Liver Cancer.). Most of the patients with anti-HBc and anti-HBs were from South Africa (all eight South African patients were positive for those antibodies).PCR testing allowed us to identify HBV DNA sequences reproducibly in the liver of 17 of the 28 patients: 5 of the 9 patients from Italy, 6 of the 8 from South Africa, 5 of the 10 from France, and the single patient from Japan. The 17 patients included 8 of the 10 positive for anti-HBc or anti-HBs and 6 of the 13 without HBV markers. Of the 17 tumors positive for HBV DNA, 14 were associated with cirrhosis and 3 were adjacent to a normal liver. On the other hand, HBV DNA sequences were identified in 14 of the 22 patients with cirrhosis associated with the primary liver cancer and 3 of the 6 without cirrhosis.
Fourteen of the 17 HBV DNA—positive tumors were tested with five different sets of HBV primers covering the regions coding for the pre-S, S, C, and X genes. To confirm the results obtained with each primer, the tumor DNA was repeatedly tested with all five combinations of primers in the same experiment and analyzed in the same gel (Fig. 1Figure 1
Detection of HBV DNA in Liver Tumors by PCR Testing.). Two different patterns were evident (Table 3Table 3
Results of PCR Testing for HBV DNA with Different Sets of HBV Primers.). In 6 of the 14 tumors (including 4 from patients from South Africa), results were positive with the use of all primers specific for pre-S, S, X, and C coding regions. In the other 8 tumors, results were positive with only two, three, or four of the five sets of primers (Fig. 1).Five tumors were available for assay for HBV RNA. PCR testing was performed with primers specific for the S gene; it clearly showed HBV messenger RNA molecules in four of five tumors tested (Fig. 2Figure 2
Detection of HBV RNA by PCR Testing.). After treatment with DNase I, a positive result was observed only when the reverse transcriptase reaction was performed before PCR amplification. This procedure ruled out the possibility that positive results were due merely to DNA contamination of the RNA preparation.The potential risks of false positive and false negative results in PCR tests were eliminated, since extensive control experiments (described in the Methods section) were repeatedly conclusive. Consistent results were also obtained in independently repeated evaluations of extracts from the same tumors. No positive results were obtained in normal liver samples tested in parallel or in 15 samples of peripheral-blood leukocytes from HBV-negative blood donors. There was no evidence of contamination with plasmid DNA. Finally, the detection of HBV RNA in four of five tumors tested further validated our findings.
Discussion
This study demonstrates, by means of a highly sensitive procedure (PCR), the frequent presence of HBV transcriptionally active genomes in a limited number of tumor cells in HBsAg-negative patients with liver cancer.
Two different patterns of HBV DNA molecules were identified. In tumors (6 of 14) for which positive results were obtained with the use of all primers covering the different HBV coding regions, the findings were consistent with the absence of large deletions in the viral DNA. The state of HBV DNA (i.e., free or integrated) could not be determined with PCR testing. In contrast, in tumors (8 of the 14) for which positive results were repeatedly obtained with only two, three, or four of the five sets of primers tested in the same experiment, the findings were consistent with deletions in the HBV DNA. This may have reflected either the presence of free, deleted HBV genomes or the integration of defective viral DNA. In view of these possibilities, it is important to consider the results obtained with the primers specific for the X gene. Integration has previously been shown to occur frequently in the cohesive ends of the HBV DNA, which partly include the X coding region.7 8 9 10 11 12 , 15 Integration in this gene may yield negative results with PCR when this set of primers is used. The relatively low frequency of tumors that are shown to be negative with these X-specific primers may reflect the presence of free HBV DNA (possibly associated with the integrated sequence). Alternatively, localization of these X primers in the 5′ part of the coding region may still allow positive results on PCR testing when the integration site is more distal.
The identification of HBV DNA in the tumors is strongly reinforced by the detection of HBV RNA molecules. It was not possible to distinguish on the basis of PCR testing which HBV RNA molecules were expressed in the tumor cells. However, these results indicate a persistent infection with a low rate of HBV transcription.
We were able to study patients from different geographic areas, including some with a high prevalence of HBV infection (South Africa) and others with an intermediate or a low prevalence (Italy, Japan, and France). Italian and French patients with no HBV markers were selected so that a group of liver cancers currently thought not to be related to HBV could be studied. HBV DNA was identified in 8 of 10 patients with anti-HBs or anti-HBc and in 6 of 13 without these antibodies. This indicates that as might be expected, patients with primary liver cancer and HBV antibodies have a high rate of persistence of HBV DNA in the liver; however, this is also true of a large number of patients with the tumor and no HBV markers. As was consistent with our results, DNA sequences of woodchuck hepatitis virus were detected in woodchucks in which primary liver cancer developed despite an apparent resolution of infection with this virus (as shown by serologic negativity for the surface antigen of woodchuck hepatitis virus and positivity for the antibody to this antigen).43 The number of viral genomes per cell was much lower than in tumors positive for the surface antigen of woodchuck hepatitis virus (e.g., 0.1 to 0.2 vs. 1000 genomes per cell). Likewise, a prospective study of patients with hepatocellular carcinoma44 showed that liver cancer developed in some who were HBsAg-negative.
Therefore, in a patient positive for anti-HBc or anti-HBs in whom liver cancer develops, a decline in the expression of the viral genome, possibly partly related to the age of the patient during the development of the tumor,45 as well as masking of HBsAg in immune complexes46 , 47 may account for serologic negativity for HBsAg. In patients from areas with a low prevalence of HBV who do not have any detectable HBV serum marker, a decline in the titers of anti-HBs and anti-HBc may also be involved. Other possible explanations include the existence of immune complexes, the absence of HBV antigens or a low level of host response to them, or genetic variations in HBV. It should be noted that HBsAg epitopes were previously found in serum from such patients, including some with primary liver cancer,48 49 50 with the use of radioimmunoassays based on the use of monoclonal anti-HBs. Nucleotide sequences have been determined in such serum isolates, after the disease was transmitted to chimpanzees.23 , 27 We found a low rate of point mutations in the viral DNA as compared with that of known subtypes of HBV.23
Our findings are consistent with an etiologic link between HBV and primary liver cancer in two different situations. In HBsAg-positive persons, clonal proliferation of tumor cells containing HBV DNA may reflect the cellular effects of viral DNA integration, the expression of viral proteins such as the X transactivator, or both events. In HBsAg-negative persons, exposure to HBV and the persistence of HBV DNA sequences (integrated or free molecule) would also represent a risk factor for liver cancer. The role of the viral DNA, however, is not known. Indeed, HBV may act through the development of cirrhosis or through a "hit-and-run" phenomenon, or both.51 , 52 It is noteworthy that in the present study, HBV DNA sequences were also identified in a few patients with tumors that developed in a histologically normal liver. Coinfection with other viruses should also be considered as a possible cofactor. It is important that antibodies to hepatitis C virus (HCV)53 have recently been detected in up to 60 to 70 percent of patients with primary liver cancer.54 , 55 HCV has been shown to be an RNA virus that is related to the flavivirus family and that has a high prevalence among cases of non-A, non-B hepatitis.53 Infection with HCV may act as a cofactor by inducing active chronic hepatitis and cirrhosis. In the present study, eight tumor samples (four from South African patients and four from French patients) were available for testing for the antibody to HCV (by enzyme-linked immunosorbent assay; Ortho Diagnostic, Raritan, N.J.).53 Only two of eight samples were found to contain the antibody (Table 2). In further investigations, the potential interactions between HBV and HCV can be tested with new molecular approaches that employ both the PCR technique and tests for the HCV antibody.
Supported by grants from the Association pour la Recherche sur le Cancer, League National contre le Cancer, Caisse National d'Assurance Maladie, Institut National de la Santé et de la Recherche Médicale, Ministère de la Recherche et de la Technologie, and the Ministère des Relations Internationales, France.
We are indebted to Valérie Thiers (Laboratoire Hybridotest) for initiating the work on the PCR in John Sninsky's laboratory (Cetus Company); to Jacques Belghiti, Henri Bismuth, Didier Houssin, and P. Gozzetti for providing tissue samples; to Pierre Berthelot for helpful discussion; to Françoise Driss for the test for antibodies to hepatitis C virus; and to Marie-Hélène Rémy for assistance in the preparation of the manuscript.
Source Information
From INSERM Unité 75, Centre hospitalier universitaire Necker (P.P., G.G., S.T., C.B.), INSERM Unité 163, Laboratoire Hybridotest. Institut Pasteur (E.N., P.T., C.B.), and Unité d'Hépatologie, Hôpital Laennec (INSERM Unité 99) (B.N., C.B.), Paris; the Department of Medicine (P.P., E.P.) and the Department of Pathology (A.D., W.G.), Istituto di Clinica Medica, Bologna, Italy; the Department of Pathology and Surgery (D.F.), Evry, France; the Department of Medicine (G.G.), University of Mainz, Mainz, Federal Republic of Germany; the Department of Medicine (M.K.), University of Johannesburg, Johannesburg, South Africa; and the Department of Gastroenterology (J.W.), Massachusetts General Hospital, Boston. Address reprint requests to Dr. Paterlini at INSERM U.75, CHU Necker, 156 rue de Vaugirard, 75015 Paris, France.
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