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Hepatitis c virus genotypes and viremia and hepatocellular carcinoma in the united states
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 1999 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 94, No. 6, 1999 ISSN 0002-9270/99/$20.00 PII S0002-9270(99)00209-9
Hepatitis C Virus Genotypes and Viremia and Hepatocellular Carcinoma in the United States Andrea E. Reid, M.D., Margaret James Koziel, M.D., Ignasio Aiza, M.D., Lennox Jeffers, M.D., Rajender Reddy, M.D., Eugene Schiff, M.D., Johnson YN Lau, M.D., Jules L. Dienstag, M.D., and T. Jake Liang, M.D. Gastrointestinal Unit and Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; Liver Diseases Section, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; Center for Liver Diseases, University of Miami School of Medicine, Miami, Florida; and Division of Gastroenterology, Hepatology and Nutrition, University of Florida, Gainesville, Florida
OBJECTIVE: Hepatitis C virus (HCV) is a well recognized cause of hepatocellular carcinoma (HCC). The pathogenic significance of HCV genotypes in hepatocarcinogenesis is undefined. The aim of this study was to investigate the genotypic distribution and viremic level of HCV in patients with HCV-associated cirrhosis with or without HCC. METHODS: A total of 28 HCV-infected patients with HCC (HCC1) and 38 patients with HCV-associated cirrhosis without HCC (HCC2) were studied. HCV genotype was assessed by the genotype-specific polymerase chain reaction (PCR) method of Okamoto and restriction fragment length polymorphism (RFLP) of the 59 untranslated region (59 UTR). Hepatitis C viremia was quantitated with the branched-chain DNA (bDNA) assay. RESULTS: Using the Okamoto method, we found genotype 1b in 64% of the HCC1 group and 74% of the HCC2 group, 36% of the HCC1 group and 16% of the HCC2 group were coinfected with a combination of genotype 1b and another genotype. Using the RFLP method, we found genotype 1b in 41% of the HCC1 group and in 24% of the HCC2 group. Other genotypes accounted for 18% of the HCC1 group and 55% of the HCC2 group; no combination genotypes were identified. Poor concordance occurred between the two genotyping methods. Mean bDNA levels were not significantly different between the two groups. CONCLUSIONS: Our study demonstrates that no particular HCV genotypes were associated with HCC and genotype did not appear to influence the development of HCV-associated HCC. (Am J Gastroenterol 1999;94:1619 –1626. © 1999 by Am. Coll. of Gastroenterology)
INTRODUCTION More than 70% of patients infected with HCV progress to chronic hepatitis and, during the first two decades of chronic hepatitis C, cirrhosis develops in at least 20%. Individuals
with HCV-associated cirrhosis have a 1.5% annual risk of developing hepatocellular carcinoma (1). Attempts to identify any clinical and virological factors that may influence the development of cirrhosis and hepatocellular carcinoma (HCC) in individuals infected with HCV have been largely inconclusive, but several studies have suggested a role for HCV genotypes (2–5). Substantial divergence in nucleotide sequences occur among isolates of HCV. Early investigations revealed a $70% overall homology among various isolates of HCV, with genetic heterogeneity in both structural and nonstructural regions (6). Phylogenetic analyses classify HCV into at least six major genotypes on the basis of overall sequence similarity in both coding and noncoding regions of the viral genome (7). One genotyping method was developed by Okamoto et al. (8), which divides HCV isolates into genotypes I, II, III, and IV on the basis of variations in the nucleotide sequences within the core (C) gene. A consensus nomenclature system with six major genotypes and 11 subtypes based on complete or partial genome analyses has been proposed by Simmonds et al. (7). In addition, variation exists in geographic distribution of HCV genotypes. The genotype distribution in the United States appears to be region-dependent; types 1a and 1b occur in almost equal prevalence in some populations (9 –11) but not in others (1a . 1b) (12). The clinical importance of viral genotype remains a subject of considerable debate. Several investigators have reported that genotype 1b is associated with older patient age (2), longer duration of infection (2, 11), acquisition of infection by routes other than injection drug use (13), poor responsiveness to interferon-a therapy (9, 14), higher levels of viremia (10), and more severe liver disease, including cirrhosis and HCC (2– 4, 15–17). In contrast, other investigators have failed to demonstrate any relation between genotype and severity of liver disease, including HCC (18 –20). In this study, we determined the prevalences of various HCV genotypes in patients with HCV-associated HCC in
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the US and investigated the relationship between the levels of hepatitis C viremia and development of HCC.
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Table 1. Clinical Characteristics of 66 Anti-HCV–Positive Patients With or Without HCC
MATERIALS AND METHODS Serum samples from 28 HCV-infected patients with cirrhosis and hepatocellular carcinoma (HCC1 group) were collected from the Massachusetts General Hospital (n 5 13) and the University of Miami (n 5 15). Serum samples collected at the same institutions between 1990 and 1994 from 38 unselected, random patients with HCV infection and histologically proven cirrhosis without HCC were analyzed as controls (HCC2 group). Serum samples were stored at 280° C with minimal freezing and thawing prior to analysis. Analysis of HCV Genotype and Level of Viremia Total RNA was extracted from 100 ml of serum using RNAzol B (Biotecx Laboratories, Houston, TX) or Trizol (Life Technologies, Gaithersburg, MD). The extracted RNA was dissolved in 20 ml of DEPC water and stored at 280° C. Genotyping according to the methods of Okamoto et al. was performed as described (8, 21). PCR products were subjected to electrophoresis and visualized by Southern blot analysis using an HCV cDNA probe containing the core sequence. Negative controls were included at the RNA extraction, reverse transcription, and first and second PCR steps to monitor for contamination. Results were considered valid if all negative controls were negative through the second PCR step. An alternative genotyping method was performed for comparison. In this method, restriction fragment length polymorphism (RFLP) of the 59 untranslated region (59 UTR) PCR product was performed (22) with minor modification (10). As specificity controls, one serum sample from an HCV-seronegative control and one serum sample from a patient previously shown to be positive for anti-HCV and HCV RNA were included for every eight samples tested. The 59UTR PCR amplicons were digested by restriction enzymes HaeIII/Rsal and MvaI/HinfI in order to differentiate HCV into major genotypes 1– 6. Subtypes 1a/c and 1b were further differentiated by restriction with BstUI. Subtypes 2a/c, 2b, 3a, and 3b were further differentiated by digestion with ScrfI. Previous studies have shown that most of the 1a/c samples were 1a, and 2a/c samples were 2c (Lau et al., unpublished data). Hence, these samples were referred to as 1a and 2c, respectively. For selected samples, HCV genotype was confirmed serologically using a commercially available serologic genotyping assay (Provided by Murex, Kent, UK). Previous studies have shown a high concordance of genotypes between the RFLP method and this serotype assay (10). HCV RNA levels were quantitated with a second generation, branched chain DNA (bDNA) assay (Quantiplex HCV RNA 2.0; Chiron, Emeryville, CA) (23) and quantitated as HCV RNA equivalents/ml (Eq/ml).
Mean age (yr) Female: n (%) Race: n (%) Caucasian Black Hispanic Asian Route of HCV transmission IVDA Transfusion Needle sticks, tattoos Unknown Clinical information missing
HCC1 (n 5 28); n (%)
HCC2 (n 5 38); n (%)
61.4 10 (35.7)
48.1 4 (10.5)
14 (50) 1 (3.6) 7 (25) 6 (21.4)
32 (84.2) 1 (2.6) 5 (13.2) 0 (0)
1 (3.7) 9 (33.3) 1 (3.7) 16 (59.3) 1 (3.7)
7 (18.4) 12 (31.6) 6 (15.8) 13 (34.2) 0 (0)
p Value ,0.001* 0.017† ,0.001†
0.076†
* Two sample t test. † Fisher’s exact test. HCC 5 hepatocellular carcinoma; HCV 5 hapatitis C virus; IVDA 5 intravenous drug abuse.
HGV RNA Assay In our evaluation of potential cofactors predisposing to HCC in patients with HCV infection, we considered the role of Hepatitis G virus (HGV) coinfection. HGV is a recently discovered flavivirus-like RNA virus with ,25% homology with HCV. To determine the prevalence of HGV coinfection in our patients, RNA was extracted from serum and resuspended in 15 ml water, and 4 ml was used for the generation of cDNA by random priming. Nested PCR was performed using primers from the 59 untranslated region of the HGV genome (24). The final PCR product was subjected to electrophoretic analysis. Statistical Analysis A two tailed x2 test or Fisher’s exact test was used for statistical analysis of categorical variables. Comparisons were made with a two sample t test when data were normally distributed or with Mann-Whitney test when the distribution of values was not normal. Logistic regression analysis was performed to control for potential confounders (e.g., age, sex, and race) in predicting the relationship between genotype and HCC. All analyses were done using the SAS System (Cary, NC) on a Sparc 20 UNIX Workstation.
RESULTS Patient Characteristics The clinical characteristics of the HCC1 and HCC2 patients are listed in Table 1. Patients with HCC were significantly older and included more women than did the HCC2 patients. The HCC2 group consisted predominately of Caucasians and included no Asians, whereas the HCC1 group, only half of which was Caucasian, included six (21%) Asians. The mode of transmission was identified in only 39% of the HCC1 group and 66% of the HCC2 group. The
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Hepatitis C Genotypes, Viremia, and Hepatocellular Carcinoma
Table 2. Genotype Analysis of 66 HCV-Infected Patients With or Without HCC by the Method of Okamoto et al. Genotype 1b Mixed infection 1b and 1a 1b and 2a 1b and 2b Untypable
HCC1 (n 5 28); n (%)
HCC2 (n 5 38); n (%)
18 (64.3)
28 (73.7)
4 (14.3) 4 (14.3) 2 (7.1) 0 (0)
2 (5.3) 4 (10.5) 0 (0) 4 (10.5)
The difference in genotype distribution between the HCC1 and HCC2 groups was not significant by Fisher’s exact test (p 5 0.08). Abbreviations are as in Table 1.
duration of infection in those with known exposure was .20 yr. HCV Genotypes When analyzed by the Okamoto genotyping method, the difference in distribution of genotypes between the HCC1 and HCC2 patients was not significant (Table 2). Eighteen (64%) of the 28 HCC1 patients were infected with type 1b exclusively, whereas 10 (36%) of 28 were coinfected with genotype 1b and another genotype. Twenty-eight (74%) of the 38 HCC2 patients were infected with genotype 1b alone, whereas another six (21%) were coinfected with genotype 1b and another genotype. Table 3 illustrates the distribution of genotypes between the HCC1 and HCC2 groups based on the RFLP method; All 38 samples from the HCC2 group were available for genotyping by this method but, because serum had been depleted for six of the HCC1 group, only 22 of 28 samples from the HCC1 group could be typed by RFLP. As determined by the RFLP method, the distribution of genotypes between the two groups was significantly different (p 5 0.003), based primarily on the divergence in the prevalence of genotype 1a and untypable specimens between the two groups. Of the HCC1 samples 40.9% could not be typed by this method, compared with 13.2% of the HCC2 samples. Genotype 1a was more prevalent in the HCC2 group, whereas specimens that could not be typed were more common in the HCC1 group; however, the prevalence of Table 3. Genotype Analysis of 60 HCV-Infected Patients With or Without HCC by the RFLP Method Genotype
HCC1 (n 5 22); n (%)
HCC2 (n 5 38); n (%)
1a 1b 2a 2b 3a 4 Untypable
2 (9.1) 9 (40.9) 1 (4.6) 1 (4.6) 0 (0) 0 (0) 9 (40.9)
18 (47.4) 9 (23.7) 0 (0) 2 (5.3) 1 (2.6) 3 (7.9) 5 (13.2)
The difference in genotype distribution between the HCC1 and HCC2 groups was significant by Fisher’s exact test (p 5 0.003). RFLP-restriction fragment length polymorphism; other abbreviations are as in Table 1.
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genotype 1b did not differ significantly between the two groups (p 5 0.24). Concordance between the two genotyping methods was poor; only 7 (54%) of 13 HCC1 samples in which both methods could be performed were completely concordant. Partial concordance was shown in another five of 13 samples, in which the RFLP method confirmed one of the two genotypes identified by the Okamoto method. Complete discordance between the two methods was identified in one sample (8%): the genotype was mixed 1b and 2b by the method of Okamoto et al., but 1a by the RFLP method. Concordance could not be determined for the 9 HCC1 samples that were untypable by the RFLP method. Concordance between the Okamoto and RFLP methods of genotyping could be assessed in 29 (76%) of 38 samples in the HCC2 group excluding four that were untypable by the Okamoto method and five by the RFLP method. In the HCC2 group, complete concordance between genotyping methods occurred in only eight (27.5%) of 29 samples, partial concordance in one (3.4%) of 29 samples, and complete discordance in 19 (65.5%) of 29 samples. As shown above (Table 1), the HCC1 and HCC2 groups differed in distribution of age, gender, and race. To determine the impact of these variables on any association between genotype and HCC, we assessed the effect of genotype (1b alone versus non-1b or mixed infection) on the presence of HCC1 or HCC2 with logistic regression models controlling for age, gender and race (white versus nonwhite). No significant association between genotype (1b versus non-1b or mixed infection) and HCC was observed for either genotyping method in the multivariate model either (data not shown). When we compared the impact of type 1b alone versus non-1b or mixed infections on HCC, we found a significant difference in gender (male predominance) in the HCC1 group between genotype 1b and other types for those obtained by the Okamoto method (Table 4). No other patient characteristics distributed unevenly between Okamoto genotype categories for the HCC1 and HCC2 groups, and no patient characteristics distributed unevenly between RFLP genotype categories for the HCC1 and HCC2 groups (Table 5). HCV RNA Level HCV RNA levels were determined by the Chiron bDNA assay in 27 of 28 HCC1 samples and 31 of 38 HCC2 samples for which adequate serum was available for analysis (Fig. 1). Fourteen of the 27 HCC1 and 15 of the 31 HCC2 samples had HCV RNA levels that were below the detection limit of the bDNA assay (2.0 3 105 Eq/ml). The range of bDNA levels was broad in both groups, ranging from below the detection limit to 232.5 3 105 Eq/ml in the HCC2 group, and below the detection limit to 50.3 3 105 Eq/ml in the HCC1 group. For patients with detectable HCV RNA by this assay, the difference in mean bDNA level between the HCC2 group (51 3 105 Eq/ml) and the
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Table 4. Clinical Features and Genotype 1b Distribution by the Method of Okamoto et al. in HCC1 and HCC2 Patients HCC1 (n 5 28)
Mean age (yr)‡ Gender: n (%) Male Female Race: n (%) Caucasian Black Hispanic Asian Route of HCV transmission§ IVDA Transfusion Needle sticks, tattoos Unknown
Type 1b (n 5 18)
Other Types (n 5 10)
59.9
64.9
15 (83.3) 3 (16.7)
3 (30.0) 7 (70.0)
10 (55.6) 1 (5.6) 2 (11.1) 5 (27.8)
4 (40.0) 0 (0) 5 (50.0) 1 (10.0)
1 (5.6) 6 (33.3) 1 (5.6) 10 (55.6)
0 (0) 3 (33.3) 0 (0) 6 (66.7)
HCC2 (n 5 36) p Value 0.21* 0.01†
Type 1b (n 5 26)
Other Types (n 5 10)
48.2
47.6
25 (89.3) 3 (10.7)
9 (90.0) 1 (10.0)
22 (78.6) 1 (3.5) 5 (17.9) 0 (0)
10 (100.0) 0 (0) 0 (0) 0 (0)
5 (17.9) 8 (28.5) 5 (17.9) 10 (35.7)
2 (20.0) 4 (40.0) 1 (10.0) 3 (30.0)
0.16†
p Value 0.89* 1.0† 0.48†
1.00†
0.92†
* Two sample t test; † Fisher’s exact test (two tail). ‡ Age was not known for two patients in the HCC1 group (both had mixed genotypes). § Information regarding route of infection was unavailable for one patient in the HCC1 group, who had mixed genotype. Abbreviations are as in Table 1.
HCC1 group (15.7 3 105 Eq/ml) did not reach statistical significance (p 5 0.06). There was no significant difference in the overall distribution of HCV RNA levels between the HCC1 group and the HCC2 group (p 5 0.60, Mann-Whitney test). Within each group, no significant difference occurred in bDNA levels between patients infected with genotype 1b alone and those infected with other genotypes or 1b plus another genotype as determined by either method (Table 6). In addition, no significant difference in mean bDNA levels occurred between HCC1 and HCC2 patients infected with genotype 1b alone (p 5 0.40). To control for differences in age, race, and gender distribution between the HCC1 and HCC2 groups, we performed a multivariate analysis with bDNA level as the dependent variable. All bDNA values below the detection limit of 2.0 3 105 Eq/ml
were given a value of 0 for this analysis. This multivariate analysis revealed no significant difference in bDNA levels between the HCC1 and HCC2 groups (p 5 0.34) and age was the only predictor for HCC in the model (p 5 0.01). HGV Coinfection Hepatitis G virus coinfection was identified in only three of 66 (4.5%) samples in this study: one (3.6%) of 28 in the HCC1 group and two (5.2%) of 38 in the HCC2 group (p . 0.05). Table 7 summarizes the clinical and HCV genotypic characteristics of each patient. One of the HCC2 patients was infected with genotype 2b as determined by the RFLP method but was untypable by the Okamoto method. This patient acquired HCV infection through blood transfusion.
Table 5. Clinical Features and Genotype 1b Distribution by 59 UTR/RFLP in HCC1 and HCC2 Patients HCC1 (n 5 22)
Mean age (yr) Gender: n (%) Male Female Race: n (%) Caucasian Black Hispanic Asian Route of HCV transmission IVDA Transfusion Needle sticks, tattoos Unknown
Type 1b (n 5 9)
Other Types (n 5 13)
60.9
60.8
7 (77.8) 2 (22.2)
8 (61.5) 5 (38.5)
3 (33.3) 0 (0) 3 (33.3) 3 (33.3)
8 (61.5) 0 (0) 2 (15.4) 3 (23.1)
0 (0) 3 (33.3) 1 (11.1) 5 (55.6)
0 (0) 5 (38.5) 0 (0) 8 (61.5)
HCC2 (n 5 38) p Value 0.97* 0.65†
Type 1b (n 5 9)
Other Types (n 5 29)
52.0
46.9
8 (88.9) 1 (11.1)
26 (89.7) 3 (10.3)
5 (55.6) 1 (11.1) 3 (33.3) 0 (0)
27 (93.1) 0 (0) 2 (6.9) 0 (0)
2 (22.2) 3 (33.3) 3 (33.3) 1 (11.1)
5 (17.2) 3 (10.4) 9 (31.0) 12 (41.4)
0.47†
0.30* 1.00† 0.20†
0.63†
* Two sample t test. † Fisher’s exact test. UTR 5 untranslated region; other abbreviations are as in Table 1.
p Value
0.19†
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Figure 1. HCV bDNA Levels in HCC2 and HCC1 Patients. Mean HCV bDNA determined in patients with HCV bDNA levels above the detection limit of the bDNA assay (2.0 3 105 Eq/ml). There was no statistically significant difference between the mean bDNA levels in the HCC1 and HCC2 groups (p 5 0.06). A two sample t test was used to compare means. There was no statistically significant difference in number of patients in the HCC1 and HCC2 groups with HCV bDNA levels below the detection limit (p 5 1.00 by Fisher’s exact test). SE 5 standard error of the mean; HCV 5 hepatocellular carcinoma; HCV 5 hepatitis C virus.
The other HCC2 patient was infected with mixed genotype 1b and 2a by the Okamoto method but was untypable by the RFLP method. The route of acquisition of HCV infection in this patient was unknown. The HCC1 patient was infected with mixed genotype 1b and 2b by the Okamoto method and genotype 2b by the RFLP. This patient acquired HCV infection through blood transfusion.
DISCUSSION Chronic infection follows acute HCV infection in almost all patients, and among patients with chronic hepatitis C, cir-
rhosis develops in approximately 20% over the first two decades (25), and HCC occurs at an annual rate of approximately 1.5% in cirrhotics with hepatitis C (1). Whether virus and/or host factors contribute to the risk of HCC in cirrhotics with hepatitis C is unknown, but such factors as patient age, duration of infection, level of viremia, and HCV genotype have been considered. Although variability exists in geographic distribution of HCV genotypes, the frequency of HCC in patients with hepatitis C appears to be uniform from country to country. Still, genotype 1b has been associated with more severe liver disease than other genotypes (3, 26), and the possibility has been suggested that genotype
Table 6. Correlation HCV bDNA Levels and Genotype 1b in HCC1 and HCC2 Patients by Genotype as Determined by RFLP HCC1 (n 5 22); n (%) Viremia level (3105 Eq/ml) ,2.0: n (%) $2.0: n (%) Mean†
Genotype 1b (n 5 9)
Other Genotypes (n 5 13)
3 (33) 6 (67) 17.8
8 (62) 5 (38) 15.8
HCC1 (n 5 31); n (%) p Value 0.39* 0.84†
* Fisher’s exact test (two tail). † Determined in patients with HCV RNA levels above the detection limit (2.0 3 105 Eq/ml). ‡ Two sample t test. Abbreviations are as in Table 1.
Genotype 1b (n 5 8)
Other Genotypes (n 5 23)
3 (37.5) 5 (62.5) 43.3
12 (52.2) 11 (47.8) 54.5
p Value 0.69* .78†
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Table 7. Clinical Characteristics of Three Patients With HCV and HGV Coinfection Stage of Disease HCC2 HCC2 HCC1
Genotype by Okamoto
Genotype by RFLP
Mode of Infection
Negative 1b, 2a 1b, 2b
2b Negative 2b
Blood transfusion Unknown Blood transfusion
Abbreviations are as in Table 1.
1b may predispose to HCC. Nousbaum et al. (2) reported that genotype 1b was the most prevalent genotype among the French and Italian patients they studied with chronic hepatitis, cirrhosis, and HCC associated with hepatitis C. In contrast, Silini et al. (4) found in a case-control study of almost 1000 Italian patients, 166 of whom had HCC, that genotype 1b was significantly more common in patients with HCC than in patients without HCC. In a prospective study of Italian cirrhotics with hepatitis C, the same authors showed that the risk of HCC was significantly higher in patients with genotype 1b than in those infected with other HCV genotypes (5) and that genotype was the most determining risk factor. In our study of 28 American cirrhotic patients with chronic hepatitis C and HCC and 38 cirrhotic patients with chronic hepatitis C without HCC, we found no significant difference in HCV genotype distribution between the two groups. Our initial approach was to use the popular Okamoto genotyping method. Although the high prevalence of genotype 1b in these patients was consistent with the observation that genotype 1b is common in the US population (10), the high frequency of mixed genotypes in both the HCC1 (36%) and HCC2 (21%) groups exceeded the 5–12% frequency of mixed genotypes obtained by others with the Okamoto method (2, 10). The sample size and clinical data available did not allow us to assess the contribution of virus, patient, and epidemiological variables associated with mixed genotype infection. To corroborate the genotype results obtained with the Okamoto method, we undertook a second approach, RLFP of the 59 UTR. Again, we could identify no genotypespecific link with HCC; however, our RLFP findings, so discordant with the genotype profiles obtained with the Okamoto method, served to reduce confidence in the conclusions of the many other reports relying on the Okamoto genotyping method. The Okamoto method may overidentify genotype 1b (10, 27), and this potential limitation may account for the unexpectedly high frequency of genotype 1b in our study sample. Because of what appears to be an artifactual overidentification of genotype 1b by the Okamoto method, and because the RFLP method shows a high concordance with other genotyping methods, including slotblot hybridization and line-probe assay (11), our Okamotoderived results in particular and Okamoto-derived results in general may be less reliable. The practical implication of such reduced confidence in the Okamoto method is that many of the studies associating genotype 1b with severe
liver disease relied entirely on the Okamoto genotyping method. Still, whether we relied upon the Okamoto or the RFLP genotyping method, we could not demonstrate a higher prevalence of genotype 1b in patients with HCC, in agreement with the study by Nousbaum et al. (2) of French and Italian patients but in disagreement with the two studies by Silini et al. (4) and Bruno et al. (5) in Italian patients. The reliability of our findings may be limited by our small sample size and by the fact that the genotype in a much larger proportion of the HCC1 group (40%) than the HCC2 group (13%) was untypable by the RFLP method, resulting in a potential underestimation of genotype 1b in the HCC1 population. On the other hand, the fact that genotyping was successful with one of the two methods we used in assigning genotypes to almost all patients in both groups weighs against such an underestimation bias. Alternatively, the difference between the prevalence of genotype 1b in our study of American patients and that in the studies of Silini et al. and Bruno et al. (4, 5) of Italian patients may reflect true geographic differences in genotype distribution and the likelihood of HCC. In this vein, 47% of our HCC2 group were infected with HCV genotype 1a, which contrasts with the much lower frequencies of genotype 1a among HCC2 patients in the studies cited above from Italy and France, 1–11% (2, 4, 5). On the other hand, regardless of whether the frequency of genotype 1a was high in our HCC2 group or low in the HCC2 group of Nousbaum et al. (2) genotype was not associated with HCC in either study. Similarly, although levels of HCV RNA have been reported to correlate proportionately (28, 29) or inversely (9, 20, 30) with the severity of liver disease, and although studies of the relationship between HCV RNA level and HCV genotype remain inconclusive (2, 3, 11, 18 –20, 31, 32), our study supports the conclusion that level of hepatitis C viremia does not correlate either with the severity of liver disease, as reflected by HCC, or with specific genotypes. In our evaluation of potential cofactors predisposing to HCC in patients with HCV infection, we considered the role of HGV coinfection. Originally postulated to cause acute and chronic hepatitis, HGV has not been found, in and of itself, to cause hepatitis or to have an impact on the progression of hepatitis B or C (33–37). The frequency of HGV coinfection in our study cohort (three of 66, 4.5%) was too low to support any conclusions about the role of HGV as a cofactor in the pathogenesis of HCV-associated cirrhosis or HCC. In the United States, HCV genotype 1b is equally prevalent in HCV-infected cirrhotics with or without HCC, and this prevalence is not influenced by age, race, or route of HCV transmission. Despite a number of limitations in our study, our results show that the Okamoto method of HCV genotyping overestimates the prevalence of genotype 1b and support the conclusion that genotyping methods based on RFLP analysis of the highly conserved 59 UTR sequence of the HCV genome are more reliable. Thus, previously re-
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ported associations, based upon Okamoto genotyping, between genotype 1b and features of chronic hepatitis C should be approached with skepticism. In cirrhotic patients with hepatitis C, we found no significant difference between HCC1 and HCC2 groups in the prevalence of genotype 1b, level of viremia, or HGV coinfection. These findings suggest that other, more complex, viral or host factors may influence the development of HCC in patients with HCVassociated cirrhosis.
ACKNOWLEDGMENTS The authors thank YuChiao Chang, Ph.D., of the Medical Practices Evaluation Center, Massachusetts General Hospital for statistical assistance; Philip Davis (supported by National Institutes of Health grant P30DK43351-08) for oligonucleotide preparation; and John Vergalla for performing bDNA assays. This work was supported by National Institutes of Health (NIH) Institutional Training Grant T32DK07191-23 and the Robert Wood Johnson Minority Medical Faculty Development Grant (to A.E.R.); by NIH grant K08AI01210 and an Elsevier Award from the American Digestive Health Foundation (to M.J.K.); by NIH grant AI41219 (to J.Y.N.L.): and by NIH DK-01952 and CA-54525 (to T.J.L.). Additional support for this study was provided by a gift from Mrs. Mouna Al-Ayoub to the Massachusetts General Hospital Hepatitis Research Fund. Reprint requests and correspondence: Andrea E. Reid, M.D., GI Unit GJ724, Massachusetts General Hospital, Fruit Street, Boston, MA 02114. Received July 31, 1998; accepted Jan. 29, 1999.
REFERENCES 1. Fattovich G, Giustina G, Degos F, et al. Morbidity and mortality in compensated cirrhosis type C: A retrospective follow-up study of 384 patients. Gastroenterology 1997;112: 463–72. 2. Nousbaum J-B, Pol S, Nalpas B, et al. Hepatitis C virus type 1b (II) in France and Italy. Ann Intern Med 1995;122:161– 8. 3. Pozzato G, Kaneko S, Moretti M, et al. Different genotypes of hepatitis C virus are associated with different severity of chronic liver disease. J Med Virol 1994;43:291– 6. 4. Silini E, Bottelli R, Asti M, et al. Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: A casecontrol study. Gastroenterology 1996;111:199 –205. 5. Bruno S, Silini E, Crosignani A, et al. Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: A prospective study. Hepatology 1997;25:754 – 8. 6. Simmonds P. Variability of hepatitis C virus. Hepatology 1995;21:570 – 83. 7. Simmonds P, Alberti A, Alter H, et al. A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatology 1994;19(suppl):1321– 4. 8. Okamoto K, Sugiyama Y, Okada S, et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: Application to clinical surveys and tracing infectious sources. J Gen Virol 1992;73:673–9.
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9. Mahaney K, Tedeschi V, Maertens G, et al. Genotyping analysis of hepatitis C virus in American patients. Hepatology 1994;20:1405–11. 10. Lau J, Mizokami M, Kolberg J, et al. Application of six hepatitis C virus genotyping systems to sera from chronic hepatitis C patients in the United States. J Infect Dis 1995; 171:281–9. 11. Lau J, Davis G, Prescott L, et al. Distribution of hepatitis C virus genotypes determined by line probe assay in patients with chronic hepatitis C seen at tertiary referral centers in the United States. Ann Intern Med 1996;124:868 –76. 12. Zein N, Rakela J, Krawitt E, et al. Hepatitis C virus genotypes in the United States: Epidemiology, pathogenicity, and response to interferon therapy. Collaborative Study Group. Ann Intern Med 1996;125:634 –9. 13. Pawlotsky J-M, Tsakiris L, Roudot-Thoraval F, et al. Relationship between hepatitis C virus genotypes and sources of infection in patients with chronic hepatitis C. J Infect Dis 1995;171:1607–l0. 14. Dusheiko G, Schmilovitz-Weiss H, Brown D, et al. Hepatitis C virus genotypes: An investigation of type-specific differences in geographic origin and disease. Hepatology 1994;19: 13–18. 15. Prati D, Capelli C, Zanella A, et al. Influence of different hepatitis C virus genotypes on the course of asymptomatic hepatitis C virus infection. Gastroenterology 1996;110:178 – 83. 16. Pistello M, Maggi F, Vatteroni L, et al. Prevalence of hepatitis C virus genotypes in Italy. J Clin Microbiol 1994;32:232– 4. 17. Silini B, Bono F, Cividini A, et al. Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities. Hepatology 1995;21:285–90. 18. Yamada M, Kakumu S, Yoshioka K, et al. Hepatitis C virus genotypes are not responsible for development of serious liver disease. Dig Dis Sci 1994;39:234 –39. 19. Yotsuyanagi H, Koike K, Yasuda K, et al. Hepatitis C virus genotypes and development of hepatocellular carcinoma. Cancer 1995;76:1352–5. 20. Mita E, Hayashi N, Kanazawa Y, et al. Hepatitis C virus genotype and RNA titer in the progression of type C chronic liver disease. J Hepatol 1994;21:468 –73. 21. Nagayama R, Tsuda F, Okamoto H, et al. Genotype dependence of hepatitis C virus antibodies detectable by the firstgeneration enzyme-linked immunosorbent assay with C100-3 protein. J Clin Invest 1993;92:1529 –33. 22. Simmonds P, McOmish F, Yap P, et al. Sequence variability in the 59 noncoding region of hepatitis C virus: Identification of a new virus type and restrictions on sequence diversity. J Gen Virol 1993;74:661– 8. 23. Collins M, Zayati C, Detmer J, et al. Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. Anal Biochem 1995;226:120 –9. 24. Bhardwaj B, Qian K, Detmer J, et al. Detection of GB virusC/hepatitis G virus RNA in serum by reverse transcription polymerase chain reaction. J Med Virol 1997;52:92– 6. 25. Seeff L, Buskell-Bales Z, Wright E, et al. Long-term mortality after transfusion-associated non-A, non-B hepatitis. N Engl J Med 1992;327:1906 –11. 26. Tanaka K, Ikematsu H, Hirohata T, et al. Hepatitis C virus infection and risk of hepatocellular carcinoma among Japanese: Possible role of type 1b (II) infection. J Natl Cancer Inst 1996;88:742– 6. 27. Lee J, Roth W, Zeuzem S. Evaluation and comparison of different hepatitis C virus genotyping and serotyping assays. J Hepatol 1997;26:1001–9. 28. Lau J, Davis G, Kniffen J, et al. Significance of serum hepatitis
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C virus RNA levels in chronic hepatitis C. Lancet 1993;314: 1501– 4. Gretch D, Corey L, Wilson J, et al. Assessment of hepatitis C virus RNA levels by quantitative competitive RNA polymerase chain reaction: High-titer viremia correlates with advanced stage of disease. J Infect Dis 1994;169:1219 –25. Magrin S, Craxi A, Fabriano C, et al. Hepatitis C viremia in chronic liver disease: Relationship to interferon-a or corticosteroid treatment. Hepatology 1994;19:273–79. Smith D, Davidson F, Yap P, et al. Levels of hepatitis C virus in blood donors infected with different virus genotypes. J Infect Dis 1996;173:727–30. Feray C, Gigou M, Samuel D, et al. Influence of the genotypes of hepatitis C virus on the severity of recurrent liver disease after liver transplantation. Gastroenterology 1995;108:1088–96.
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33. Alter M, Gallagher M, Morris T, et al. Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection. N Engl J Med 1997;336:741– 6. 34. Dickens T, Lemon S. GB virus C, hepatitis G virus, or human orphan flavivirus? Hepatology 1997;25:1285– 6. 35. Alter H, Nakatsuji Y, Melpolder J, et al. The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. N Engl J Med 1997;336:747–54. 36. Tanaka E, Alter H, Nakatsuji Y, et al. Effect of hepatitis G virus infection on chronic hepatitis C. Ann Intern Med 1996; 125:740 –3. 37. Kao J-H, Chen P-J, Lai M-Y, et al. GB Virus-C/Hepatitis G virus infection in an area endemic for viral hepatitis, chronic liver disease, and liver cancer. Gastroenterology 1997;112: 1265–70.
Vol. 94, No. 6, 1999 ISSN 0002-9270/99/$20.00 PII S0002-9270(99)00209-9
Hepatitis C Virus Genotypes and Viremia and Hepatocellular Carcinoma in the United States Andrea E. Reid, M.D., Margaret James Koziel, M.D., Ignasio Aiza, M.D., Lennox Jeffers, M.D., Rajender Reddy, M.D., Eugene Schiff, M.D., Johnson YN Lau, M.D., Jules L. Dienstag, M.D., and T. Jake Liang, M.D. Gastrointestinal Unit and Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; Liver Diseases Section, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; Center for Liver Diseases, University of Miami School of Medicine, Miami, Florida; and Division of Gastroenterology, Hepatology and Nutrition, University of Florida, Gainesville, Florida
OBJECTIVE: Hepatitis C virus (HCV) is a well recognized cause of hepatocellular carcinoma (HCC). The pathogenic significance of HCV genotypes in hepatocarcinogenesis is undefined. The aim of this study was to investigate the genotypic distribution and viremic level of HCV in patients with HCV-associated cirrhosis with or without HCC. METHODS: A total of 28 HCV-infected patients with HCC (HCC1) and 38 patients with HCV-associated cirrhosis without HCC (HCC2) were studied. HCV genotype was assessed by the genotype-specific polymerase chain reaction (PCR) method of Okamoto and restriction fragment length polymorphism (RFLP) of the 59 untranslated region (59 UTR). Hepatitis C viremia was quantitated with the branched-chain DNA (bDNA) assay. RESULTS: Using the Okamoto method, we found genotype 1b in 64% of the HCC1 group and 74% of the HCC2 group, 36% of the HCC1 group and 16% of the HCC2 group were coinfected with a combination of genotype 1b and another genotype. Using the RFLP method, we found genotype 1b in 41% of the HCC1 group and in 24% of the HCC2 group. Other genotypes accounted for 18% of the HCC1 group and 55% of the HCC2 group; no combination genotypes were identified. Poor concordance occurred between the two genotyping methods. Mean bDNA levels were not significantly different between the two groups. CONCLUSIONS: Our study demonstrates that no particular HCV genotypes were associated with HCC and genotype did not appear to influence the development of HCV-associated HCC. (Am J Gastroenterol 1999;94:1619 –1626. © 1999 by Am. Coll. of Gastroenterology)
INTRODUCTION More than 70% of patients infected with HCV progress to chronic hepatitis and, during the first two decades of chronic hepatitis C, cirrhosis develops in at least 20%. Individuals
with HCV-associated cirrhosis have a 1.5% annual risk of developing hepatocellular carcinoma (1). Attempts to identify any clinical and virological factors that may influence the development of cirrhosis and hepatocellular carcinoma (HCC) in individuals infected with HCV have been largely inconclusive, but several studies have suggested a role for HCV genotypes (2–5). Substantial divergence in nucleotide sequences occur among isolates of HCV. Early investigations revealed a $70% overall homology among various isolates of HCV, with genetic heterogeneity in both structural and nonstructural regions (6). Phylogenetic analyses classify HCV into at least six major genotypes on the basis of overall sequence similarity in both coding and noncoding regions of the viral genome (7). One genotyping method was developed by Okamoto et al. (8), which divides HCV isolates into genotypes I, II, III, and IV on the basis of variations in the nucleotide sequences within the core (C) gene. A consensus nomenclature system with six major genotypes and 11 subtypes based on complete or partial genome analyses has been proposed by Simmonds et al. (7). In addition, variation exists in geographic distribution of HCV genotypes. The genotype distribution in the United States appears to be region-dependent; types 1a and 1b occur in almost equal prevalence in some populations (9 –11) but not in others (1a . 1b) (12). The clinical importance of viral genotype remains a subject of considerable debate. Several investigators have reported that genotype 1b is associated with older patient age (2), longer duration of infection (2, 11), acquisition of infection by routes other than injection drug use (13), poor responsiveness to interferon-a therapy (9, 14), higher levels of viremia (10), and more severe liver disease, including cirrhosis and HCC (2– 4, 15–17). In contrast, other investigators have failed to demonstrate any relation between genotype and severity of liver disease, including HCC (18 –20). In this study, we determined the prevalences of various HCV genotypes in patients with HCV-associated HCC in
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the US and investigated the relationship between the levels of hepatitis C viremia and development of HCC.
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Table 1. Clinical Characteristics of 66 Anti-HCV–Positive Patients With or Without HCC
MATERIALS AND METHODS Serum samples from 28 HCV-infected patients with cirrhosis and hepatocellular carcinoma (HCC1 group) were collected from the Massachusetts General Hospital (n 5 13) and the University of Miami (n 5 15). Serum samples collected at the same institutions between 1990 and 1994 from 38 unselected, random patients with HCV infection and histologically proven cirrhosis without HCC were analyzed as controls (HCC2 group). Serum samples were stored at 280° C with minimal freezing and thawing prior to analysis. Analysis of HCV Genotype and Level of Viremia Total RNA was extracted from 100 ml of serum using RNAzol B (Biotecx Laboratories, Houston, TX) or Trizol (Life Technologies, Gaithersburg, MD). The extracted RNA was dissolved in 20 ml of DEPC water and stored at 280° C. Genotyping according to the methods of Okamoto et al. was performed as described (8, 21). PCR products were subjected to electrophoresis and visualized by Southern blot analysis using an HCV cDNA probe containing the core sequence. Negative controls were included at the RNA extraction, reverse transcription, and first and second PCR steps to monitor for contamination. Results were considered valid if all negative controls were negative through the second PCR step. An alternative genotyping method was performed for comparison. In this method, restriction fragment length polymorphism (RFLP) of the 59 untranslated region (59 UTR) PCR product was performed (22) with minor modification (10). As specificity controls, one serum sample from an HCV-seronegative control and one serum sample from a patient previously shown to be positive for anti-HCV and HCV RNA were included for every eight samples tested. The 59UTR PCR amplicons were digested by restriction enzymes HaeIII/Rsal and MvaI/HinfI in order to differentiate HCV into major genotypes 1– 6. Subtypes 1a/c and 1b were further differentiated by restriction with BstUI. Subtypes 2a/c, 2b, 3a, and 3b were further differentiated by digestion with ScrfI. Previous studies have shown that most of the 1a/c samples were 1a, and 2a/c samples were 2c (Lau et al., unpublished data). Hence, these samples were referred to as 1a and 2c, respectively. For selected samples, HCV genotype was confirmed serologically using a commercially available serologic genotyping assay (Provided by Murex, Kent, UK). Previous studies have shown a high concordance of genotypes between the RFLP method and this serotype assay (10). HCV RNA levels were quantitated with a second generation, branched chain DNA (bDNA) assay (Quantiplex HCV RNA 2.0; Chiron, Emeryville, CA) (23) and quantitated as HCV RNA equivalents/ml (Eq/ml).
Mean age (yr) Female: n (%) Race: n (%) Caucasian Black Hispanic Asian Route of HCV transmission IVDA Transfusion Needle sticks, tattoos Unknown Clinical information missing
HCC1 (n 5 28); n (%)
HCC2 (n 5 38); n (%)
61.4 10 (35.7)
48.1 4 (10.5)
14 (50) 1 (3.6) 7 (25) 6 (21.4)
32 (84.2) 1 (2.6) 5 (13.2) 0 (0)
1 (3.7) 9 (33.3) 1 (3.7) 16 (59.3) 1 (3.7)
7 (18.4) 12 (31.6) 6 (15.8) 13 (34.2) 0 (0)
p Value ,0.001* 0.017† ,0.001†
0.076†
* Two sample t test. † Fisher’s exact test. HCC 5 hepatocellular carcinoma; HCV 5 hapatitis C virus; IVDA 5 intravenous drug abuse.
HGV RNA Assay In our evaluation of potential cofactors predisposing to HCC in patients with HCV infection, we considered the role of Hepatitis G virus (HGV) coinfection. HGV is a recently discovered flavivirus-like RNA virus with ,25% homology with HCV. To determine the prevalence of HGV coinfection in our patients, RNA was extracted from serum and resuspended in 15 ml water, and 4 ml was used for the generation of cDNA by random priming. Nested PCR was performed using primers from the 59 untranslated region of the HGV genome (24). The final PCR product was subjected to electrophoretic analysis. Statistical Analysis A two tailed x2 test or Fisher’s exact test was used for statistical analysis of categorical variables. Comparisons were made with a two sample t test when data were normally distributed or with Mann-Whitney test when the distribution of values was not normal. Logistic regression analysis was performed to control for potential confounders (e.g., age, sex, and race) in predicting the relationship between genotype and HCC. All analyses were done using the SAS System (Cary, NC) on a Sparc 20 UNIX Workstation.
RESULTS Patient Characteristics The clinical characteristics of the HCC1 and HCC2 patients are listed in Table 1. Patients with HCC were significantly older and included more women than did the HCC2 patients. The HCC2 group consisted predominately of Caucasians and included no Asians, whereas the HCC1 group, only half of which was Caucasian, included six (21%) Asians. The mode of transmission was identified in only 39% of the HCC1 group and 66% of the HCC2 group. The
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Hepatitis C Genotypes, Viremia, and Hepatocellular Carcinoma
Table 2. Genotype Analysis of 66 HCV-Infected Patients With or Without HCC by the Method of Okamoto et al. Genotype 1b Mixed infection 1b and 1a 1b and 2a 1b and 2b Untypable
HCC1 (n 5 28); n (%)
HCC2 (n 5 38); n (%)
18 (64.3)
28 (73.7)
4 (14.3) 4 (14.3) 2 (7.1) 0 (0)
2 (5.3) 4 (10.5) 0 (0) 4 (10.5)
The difference in genotype distribution between the HCC1 and HCC2 groups was not significant by Fisher’s exact test (p 5 0.08). Abbreviations are as in Table 1.
duration of infection in those with known exposure was .20 yr. HCV Genotypes When analyzed by the Okamoto genotyping method, the difference in distribution of genotypes between the HCC1 and HCC2 patients was not significant (Table 2). Eighteen (64%) of the 28 HCC1 patients were infected with type 1b exclusively, whereas 10 (36%) of 28 were coinfected with genotype 1b and another genotype. Twenty-eight (74%) of the 38 HCC2 patients were infected with genotype 1b alone, whereas another six (21%) were coinfected with genotype 1b and another genotype. Table 3 illustrates the distribution of genotypes between the HCC1 and HCC2 groups based on the RFLP method; All 38 samples from the HCC2 group were available for genotyping by this method but, because serum had been depleted for six of the HCC1 group, only 22 of 28 samples from the HCC1 group could be typed by RFLP. As determined by the RFLP method, the distribution of genotypes between the two groups was significantly different (p 5 0.003), based primarily on the divergence in the prevalence of genotype 1a and untypable specimens between the two groups. Of the HCC1 samples 40.9% could not be typed by this method, compared with 13.2% of the HCC2 samples. Genotype 1a was more prevalent in the HCC2 group, whereas specimens that could not be typed were more common in the HCC1 group; however, the prevalence of Table 3. Genotype Analysis of 60 HCV-Infected Patients With or Without HCC by the RFLP Method Genotype
HCC1 (n 5 22); n (%)
HCC2 (n 5 38); n (%)
1a 1b 2a 2b 3a 4 Untypable
2 (9.1) 9 (40.9) 1 (4.6) 1 (4.6) 0 (0) 0 (0) 9 (40.9)
18 (47.4) 9 (23.7) 0 (0) 2 (5.3) 1 (2.6) 3 (7.9) 5 (13.2)
The difference in genotype distribution between the HCC1 and HCC2 groups was significant by Fisher’s exact test (p 5 0.003). RFLP-restriction fragment length polymorphism; other abbreviations are as in Table 1.
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genotype 1b did not differ significantly between the two groups (p 5 0.24). Concordance between the two genotyping methods was poor; only 7 (54%) of 13 HCC1 samples in which both methods could be performed were completely concordant. Partial concordance was shown in another five of 13 samples, in which the RFLP method confirmed one of the two genotypes identified by the Okamoto method. Complete discordance between the two methods was identified in one sample (8%): the genotype was mixed 1b and 2b by the method of Okamoto et al., but 1a by the RFLP method. Concordance could not be determined for the 9 HCC1 samples that were untypable by the RFLP method. Concordance between the Okamoto and RFLP methods of genotyping could be assessed in 29 (76%) of 38 samples in the HCC2 group excluding four that were untypable by the Okamoto method and five by the RFLP method. In the HCC2 group, complete concordance between genotyping methods occurred in only eight (27.5%) of 29 samples, partial concordance in one (3.4%) of 29 samples, and complete discordance in 19 (65.5%) of 29 samples. As shown above (Table 1), the HCC1 and HCC2 groups differed in distribution of age, gender, and race. To determine the impact of these variables on any association between genotype and HCC, we assessed the effect of genotype (1b alone versus non-1b or mixed infection) on the presence of HCC1 or HCC2 with logistic regression models controlling for age, gender and race (white versus nonwhite). No significant association between genotype (1b versus non-1b or mixed infection) and HCC was observed for either genotyping method in the multivariate model either (data not shown). When we compared the impact of type 1b alone versus non-1b or mixed infections on HCC, we found a significant difference in gender (male predominance) in the HCC1 group between genotype 1b and other types for those obtained by the Okamoto method (Table 4). No other patient characteristics distributed unevenly between Okamoto genotype categories for the HCC1 and HCC2 groups, and no patient characteristics distributed unevenly between RFLP genotype categories for the HCC1 and HCC2 groups (Table 5). HCV RNA Level HCV RNA levels were determined by the Chiron bDNA assay in 27 of 28 HCC1 samples and 31 of 38 HCC2 samples for which adequate serum was available for analysis (Fig. 1). Fourteen of the 27 HCC1 and 15 of the 31 HCC2 samples had HCV RNA levels that were below the detection limit of the bDNA assay (2.0 3 105 Eq/ml). The range of bDNA levels was broad in both groups, ranging from below the detection limit to 232.5 3 105 Eq/ml in the HCC2 group, and below the detection limit to 50.3 3 105 Eq/ml in the HCC1 group. For patients with detectable HCV RNA by this assay, the difference in mean bDNA level between the HCC2 group (51 3 105 Eq/ml) and the
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Table 4. Clinical Features and Genotype 1b Distribution by the Method of Okamoto et al. in HCC1 and HCC2 Patients HCC1 (n 5 28)
Mean age (yr)‡ Gender: n (%) Male Female Race: n (%) Caucasian Black Hispanic Asian Route of HCV transmission§ IVDA Transfusion Needle sticks, tattoos Unknown
Type 1b (n 5 18)
Other Types (n 5 10)
59.9
64.9
15 (83.3) 3 (16.7)
3 (30.0) 7 (70.0)
10 (55.6) 1 (5.6) 2 (11.1) 5 (27.8)
4 (40.0) 0 (0) 5 (50.0) 1 (10.0)
1 (5.6) 6 (33.3) 1 (5.6) 10 (55.6)
0 (0) 3 (33.3) 0 (0) 6 (66.7)
HCC2 (n 5 36) p Value 0.21* 0.01†
Type 1b (n 5 26)
Other Types (n 5 10)
48.2
47.6
25 (89.3) 3 (10.7)
9 (90.0) 1 (10.0)
22 (78.6) 1 (3.5) 5 (17.9) 0 (0)
10 (100.0) 0 (0) 0 (0) 0 (0)
5 (17.9) 8 (28.5) 5 (17.9) 10 (35.7)
2 (20.0) 4 (40.0) 1 (10.0) 3 (30.0)
0.16†
p Value 0.89* 1.0† 0.48†
1.00†
0.92†
* Two sample t test; † Fisher’s exact test (two tail). ‡ Age was not known for two patients in the HCC1 group (both had mixed genotypes). § Information regarding route of infection was unavailable for one patient in the HCC1 group, who had mixed genotype. Abbreviations are as in Table 1.
HCC1 group (15.7 3 105 Eq/ml) did not reach statistical significance (p 5 0.06). There was no significant difference in the overall distribution of HCV RNA levels between the HCC1 group and the HCC2 group (p 5 0.60, Mann-Whitney test). Within each group, no significant difference occurred in bDNA levels between patients infected with genotype 1b alone and those infected with other genotypes or 1b plus another genotype as determined by either method (Table 6). In addition, no significant difference in mean bDNA levels occurred between HCC1 and HCC2 patients infected with genotype 1b alone (p 5 0.40). To control for differences in age, race, and gender distribution between the HCC1 and HCC2 groups, we performed a multivariate analysis with bDNA level as the dependent variable. All bDNA values below the detection limit of 2.0 3 105 Eq/ml
were given a value of 0 for this analysis. This multivariate analysis revealed no significant difference in bDNA levels between the HCC1 and HCC2 groups (p 5 0.34) and age was the only predictor for HCC in the model (p 5 0.01). HGV Coinfection Hepatitis G virus coinfection was identified in only three of 66 (4.5%) samples in this study: one (3.6%) of 28 in the HCC1 group and two (5.2%) of 38 in the HCC2 group (p . 0.05). Table 7 summarizes the clinical and HCV genotypic characteristics of each patient. One of the HCC2 patients was infected with genotype 2b as determined by the RFLP method but was untypable by the Okamoto method. This patient acquired HCV infection through blood transfusion.
Table 5. Clinical Features and Genotype 1b Distribution by 59 UTR/RFLP in HCC1 and HCC2 Patients HCC1 (n 5 22)
Mean age (yr) Gender: n (%) Male Female Race: n (%) Caucasian Black Hispanic Asian Route of HCV transmission IVDA Transfusion Needle sticks, tattoos Unknown
Type 1b (n 5 9)
Other Types (n 5 13)
60.9
60.8
7 (77.8) 2 (22.2)
8 (61.5) 5 (38.5)
3 (33.3) 0 (0) 3 (33.3) 3 (33.3)
8 (61.5) 0 (0) 2 (15.4) 3 (23.1)
0 (0) 3 (33.3) 1 (11.1) 5 (55.6)
0 (0) 5 (38.5) 0 (0) 8 (61.5)
HCC2 (n 5 38) p Value 0.97* 0.65†
Type 1b (n 5 9)
Other Types (n 5 29)
52.0
46.9
8 (88.9) 1 (11.1)
26 (89.7) 3 (10.3)
5 (55.6) 1 (11.1) 3 (33.3) 0 (0)
27 (93.1) 0 (0) 2 (6.9) 0 (0)
2 (22.2) 3 (33.3) 3 (33.3) 1 (11.1)
5 (17.2) 3 (10.4) 9 (31.0) 12 (41.4)
0.47†
0.30* 1.00† 0.20†
0.63†
* Two sample t test. † Fisher’s exact test. UTR 5 untranslated region; other abbreviations are as in Table 1.
p Value
0.19†
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Figure 1. HCV bDNA Levels in HCC2 and HCC1 Patients. Mean HCV bDNA determined in patients with HCV bDNA levels above the detection limit of the bDNA assay (2.0 3 105 Eq/ml). There was no statistically significant difference between the mean bDNA levels in the HCC1 and HCC2 groups (p 5 0.06). A two sample t test was used to compare means. There was no statistically significant difference in number of patients in the HCC1 and HCC2 groups with HCV bDNA levels below the detection limit (p 5 1.00 by Fisher’s exact test). SE 5 standard error of the mean; HCV 5 hepatocellular carcinoma; HCV 5 hepatitis C virus.
The other HCC2 patient was infected with mixed genotype 1b and 2a by the Okamoto method but was untypable by the RFLP method. The route of acquisition of HCV infection in this patient was unknown. The HCC1 patient was infected with mixed genotype 1b and 2b by the Okamoto method and genotype 2b by the RFLP. This patient acquired HCV infection through blood transfusion.
DISCUSSION Chronic infection follows acute HCV infection in almost all patients, and among patients with chronic hepatitis C, cir-
rhosis develops in approximately 20% over the first two decades (25), and HCC occurs at an annual rate of approximately 1.5% in cirrhotics with hepatitis C (1). Whether virus and/or host factors contribute to the risk of HCC in cirrhotics with hepatitis C is unknown, but such factors as patient age, duration of infection, level of viremia, and HCV genotype have been considered. Although variability exists in geographic distribution of HCV genotypes, the frequency of HCC in patients with hepatitis C appears to be uniform from country to country. Still, genotype 1b has been associated with more severe liver disease than other genotypes (3, 26), and the possibility has been suggested that genotype
Table 6. Correlation HCV bDNA Levels and Genotype 1b in HCC1 and HCC2 Patients by Genotype as Determined by RFLP HCC1 (n 5 22); n (%) Viremia level (3105 Eq/ml) ,2.0: n (%) $2.0: n (%) Mean†
Genotype 1b (n 5 9)
Other Genotypes (n 5 13)
3 (33) 6 (67) 17.8
8 (62) 5 (38) 15.8
HCC1 (n 5 31); n (%) p Value 0.39* 0.84†
* Fisher’s exact test (two tail). † Determined in patients with HCV RNA levels above the detection limit (2.0 3 105 Eq/ml). ‡ Two sample t test. Abbreviations are as in Table 1.
Genotype 1b (n 5 8)
Other Genotypes (n 5 23)
3 (37.5) 5 (62.5) 43.3
12 (52.2) 11 (47.8) 54.5
p Value 0.69* .78†
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Table 7. Clinical Characteristics of Three Patients With HCV and HGV Coinfection Stage of Disease HCC2 HCC2 HCC1
Genotype by Okamoto
Genotype by RFLP
Mode of Infection
Negative 1b, 2a 1b, 2b
2b Negative 2b
Blood transfusion Unknown Blood transfusion
Abbreviations are as in Table 1.
1b may predispose to HCC. Nousbaum et al. (2) reported that genotype 1b was the most prevalent genotype among the French and Italian patients they studied with chronic hepatitis, cirrhosis, and HCC associated with hepatitis C. In contrast, Silini et al. (4) found in a case-control study of almost 1000 Italian patients, 166 of whom had HCC, that genotype 1b was significantly more common in patients with HCC than in patients without HCC. In a prospective study of Italian cirrhotics with hepatitis C, the same authors showed that the risk of HCC was significantly higher in patients with genotype 1b than in those infected with other HCV genotypes (5) and that genotype was the most determining risk factor. In our study of 28 American cirrhotic patients with chronic hepatitis C and HCC and 38 cirrhotic patients with chronic hepatitis C without HCC, we found no significant difference in HCV genotype distribution between the two groups. Our initial approach was to use the popular Okamoto genotyping method. Although the high prevalence of genotype 1b in these patients was consistent with the observation that genotype 1b is common in the US population (10), the high frequency of mixed genotypes in both the HCC1 (36%) and HCC2 (21%) groups exceeded the 5–12% frequency of mixed genotypes obtained by others with the Okamoto method (2, 10). The sample size and clinical data available did not allow us to assess the contribution of virus, patient, and epidemiological variables associated with mixed genotype infection. To corroborate the genotype results obtained with the Okamoto method, we undertook a second approach, RLFP of the 59 UTR. Again, we could identify no genotypespecific link with HCC; however, our RLFP findings, so discordant with the genotype profiles obtained with the Okamoto method, served to reduce confidence in the conclusions of the many other reports relying on the Okamoto genotyping method. The Okamoto method may overidentify genotype 1b (10, 27), and this potential limitation may account for the unexpectedly high frequency of genotype 1b in our study sample. Because of what appears to be an artifactual overidentification of genotype 1b by the Okamoto method, and because the RFLP method shows a high concordance with other genotyping methods, including slotblot hybridization and line-probe assay (11), our Okamotoderived results in particular and Okamoto-derived results in general may be less reliable. The practical implication of such reduced confidence in the Okamoto method is that many of the studies associating genotype 1b with severe
liver disease relied entirely on the Okamoto genotyping method. Still, whether we relied upon the Okamoto or the RFLP genotyping method, we could not demonstrate a higher prevalence of genotype 1b in patients with HCC, in agreement with the study by Nousbaum et al. (2) of French and Italian patients but in disagreement with the two studies by Silini et al. (4) and Bruno et al. (5) in Italian patients. The reliability of our findings may be limited by our small sample size and by the fact that the genotype in a much larger proportion of the HCC1 group (40%) than the HCC2 group (13%) was untypable by the RFLP method, resulting in a potential underestimation of genotype 1b in the HCC1 population. On the other hand, the fact that genotyping was successful with one of the two methods we used in assigning genotypes to almost all patients in both groups weighs against such an underestimation bias. Alternatively, the difference between the prevalence of genotype 1b in our study of American patients and that in the studies of Silini et al. and Bruno et al. (4, 5) of Italian patients may reflect true geographic differences in genotype distribution and the likelihood of HCC. In this vein, 47% of our HCC2 group were infected with HCV genotype 1a, which contrasts with the much lower frequencies of genotype 1a among HCC2 patients in the studies cited above from Italy and France, 1–11% (2, 4, 5). On the other hand, regardless of whether the frequency of genotype 1a was high in our HCC2 group or low in the HCC2 group of Nousbaum et al. (2) genotype was not associated with HCC in either study. Similarly, although levels of HCV RNA have been reported to correlate proportionately (28, 29) or inversely (9, 20, 30) with the severity of liver disease, and although studies of the relationship between HCV RNA level and HCV genotype remain inconclusive (2, 3, 11, 18 –20, 31, 32), our study supports the conclusion that level of hepatitis C viremia does not correlate either with the severity of liver disease, as reflected by HCC, or with specific genotypes. In our evaluation of potential cofactors predisposing to HCC in patients with HCV infection, we considered the role of HGV coinfection. Originally postulated to cause acute and chronic hepatitis, HGV has not been found, in and of itself, to cause hepatitis or to have an impact on the progression of hepatitis B or C (33–37). The frequency of HGV coinfection in our study cohort (three of 66, 4.5%) was too low to support any conclusions about the role of HGV as a cofactor in the pathogenesis of HCV-associated cirrhosis or HCC. In the United States, HCV genotype 1b is equally prevalent in HCV-infected cirrhotics with or without HCC, and this prevalence is not influenced by age, race, or route of HCV transmission. Despite a number of limitations in our study, our results show that the Okamoto method of HCV genotyping overestimates the prevalence of genotype 1b and support the conclusion that genotyping methods based on RFLP analysis of the highly conserved 59 UTR sequence of the HCV genome are more reliable. Thus, previously re-
AJG – June, 1999
ported associations, based upon Okamoto genotyping, between genotype 1b and features of chronic hepatitis C should be approached with skepticism. In cirrhotic patients with hepatitis C, we found no significant difference between HCC1 and HCC2 groups in the prevalence of genotype 1b, level of viremia, or HGV coinfection. These findings suggest that other, more complex, viral or host factors may influence the development of HCC in patients with HCVassociated cirrhosis.
ACKNOWLEDGMENTS The authors thank YuChiao Chang, Ph.D., of the Medical Practices Evaluation Center, Massachusetts General Hospital for statistical assistance; Philip Davis (supported by National Institutes of Health grant P30DK43351-08) for oligonucleotide preparation; and John Vergalla for performing bDNA assays. This work was supported by National Institutes of Health (NIH) Institutional Training Grant T32DK07191-23 and the Robert Wood Johnson Minority Medical Faculty Development Grant (to A.E.R.); by NIH grant K08AI01210 and an Elsevier Award from the American Digestive Health Foundation (to M.J.K.); by NIH grant AI41219 (to J.Y.N.L.): and by NIH DK-01952 and CA-54525 (to T.J.L.). Additional support for this study was provided by a gift from Mrs. Mouna Al-Ayoub to the Massachusetts General Hospital Hepatitis Research Fund. Reprint requests and correspondence: Andrea E. Reid, M.D., GI Unit GJ724, Massachusetts General Hospital, Fruit Street, Boston, MA 02114. Received July 31, 1998; accepted Jan. 29, 1999.
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