Genotypes and titers of hepatitis C virus for predicting response to interferon in patients with chronic hepatitis C

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Interferon induces remission in about 50% of patients with chronic hepatitis C, but it is difficult to predict which patients will respond. Host and viral factors were evaluated for correlation with response to interferon in patients with chronic
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  Journal of Medical Virology 42299405 (1994) Genotypes and Titers of Hepatitis C Virus for Predicting Response to Interferon in Patients With Chronic Hepatitis C Kunihiko Hino, Shigehiko Sainokami, Kazumi Shimoda, Shiro Iino, Yu Wang, Hiroaki Okamoto, Yuzo Miyakawa, and Makoto Mayumi Second Department of Internal Medicine, National Defense Medical College, Saitama-Ken K.H.,S.S.,K.S.), Institute of Medical Science, St. Marianna University School of Medicine, Kanagawa-Ken S.I.j, Immunology Division, Jichi Medical School, Tochigi-Ken H.O.,M.M.), and Mita Institute, Tokyo Y.M.), Japan; Institute o Hepatology, Beijing Medical University, Beijing, China (Y.W.) Interferon induces remission in about 50% of pa- tients with chronic hepatitis C, but it is difficult to predict which patients will respond. Host and vi- ral factors were evaluated for correlation with response to interferon in patients with chronic hepatitis C. Recombinant interferon alpha-2b with a total dose of 480-560 million units was given to 136 patients, of whom 74 (54%) re- sponded. Genotypes of hepatitis C virus (HCV) n sera, I, II, 111 IV, and V, were determined by poly- merase chain reaction (PCR) with type-specific primers. In 72 patients, pretreatment levels of HCV RNA were titrated by PCR in serial tenfold dilutions of RNA extracted from serum. Re- sponse to interferon occurred in 34 (40%) of 85 patients infected with HCV of genotype II less frequently than in 22 (85%) of 26 with genotype 111 P < 0.001 or in 7 (70%) of 10 with genotype IV. Of 51 patients with genotype II HCV, 6 of (75%) with HCV RNA titers <lo6 responded, more fre- quently than 4 of 43 (9%) with titers lo6 P < 0.001 1 Responders were younger than non-re- sponders (45.7 2 11.7 vs. 50.3 2 9.6 yr) and had received transfusions less frequently (26174 or 35% vs. 37/62 or 6O%, < 0.01). Response to interferon correlated inversely with the severity of liver histopathology. These results indicate that response to interferon is influenced by HCV genotypes and pretreatment levels of HCV RNA in Serum. 1994 Wiley-Liss, nc. KEY WORDS: HCV genotypes, polymerase chain reaction, host and viral factors of non-A, non-B NANB) hepatitis cases worldwide [Choo et al., 1989; Kuo et al., 1989; Alter et al., 19891. Enzyme immunoassays (EIA) for antibodies to HCV (anti-HCV) of the first and second generations have been useful for excluding blood units contaminated with HCV and efficient in preventing post-transfusion hepatitis C [Alter et al., 1989; Aach et al., 1991; Dona- hue et al, 19921. HCV is known for its propensity to cause chronic disease. Hepatitis persists in approximately 50% of pa- tients after primary infection with HCV, and some of them develop cirrhosis and hepatocellular carcinoma [Dienstag, 1983; Alter et al., 19891. This background stresses the need for effective treatment of patients with persistent HCV infection accompanied by clinical disease, especially of those with chronic hepatitis C. Serological diagnosis of HCV infection opened the way for randomized, placebo-controlled trials of inter- feron for the treatment of patients with chronic hepati- tis c. Interferon has beneficial effects with short-term and long-term response rates averaging at 50% and 25%, respectively, as estimated by the normalization or decrease of alanine aminotransferase (ALT) levels in sera [Davis et al., 1989; Di Bisceglie et al., 1989; Davis, 1990; Tine' et al., 19911. It has been difficult, however, to predict which patients will respond to interferon. Based on the sequence similarity of HCV isolates for which the entire nucleotide sequence is determined, at least four genotypes of HCV have been recognized, which differ from each other in >21% of -9,400 nucle- otides (nt) [Choo et al., 1991; Takamizawa et al., 1991; Okamoto et al., 1991, 1992al. These are provisionally designated genotypes I, 11,111, and IV [Okamoto et al., 1992a,cl, and appear to have a distinct epidemiologic distribution that might be associated with different dis- INTRODUCTION ~ ~~~ ~~~~~ Accepted for publication July 14,1993. The discovery Of hepatitis virus (HCV) and serolog- Address reprint to Dr, M, Mayumi, Immunology Divi- ical assays for HCV infection have enabled the diagno- sis of hepatitis C, which is responsible for the majority sion, 329-04, Japan. Medical School, Minamikawachi-Machi, Tochigi-Ken 994 WILEY-LISS, INC.  300 Hino et al. replaced by #299 with a sequence of 5’-ACCCAACAC- TACTCGGCTAG-3’ representing nt 250-269; nucleo- tides were numbered from the putative 5’-end of the HCV genome [Okamoto et al., 1992bl. The modified PCR amplified a product of 225 base pairs (bp) with a sensitivity comparable with that of the srcinal method. Titer of HCV RNA was expressed by the recip- rocal of the highest tenfold dilution of RNA extracted from 0.1 ml of serum, in which HCV RNA was detect- able after amplification by PCR. Genotypes of HCV Genotypes of HCV, provisionally designated I, 11,111, IV, and V [Mori et al., 1992; Okamoto et al., 1992a,c, 19931, were determined by amplification by PCR with type-specific primers. Briefly, a part of the HCV core gene spanning nt 480-751 (272 bp) was amplified on HCV cDNA with universal primers. A portion of the product was then amplified by PCR with two universal primers (sense) and the mixture of five primers (an- tisense) deduced from sequences of the HCV core gene, each of which was specific for genotype I, 11,111, IV or V [Okamoto et al., 1992c, 19931. Genotypes were distin- guished from each other by the size of products: 49 bp for I; 144 bp for 11; 174 bp for 111; 123 bp for IV; and 88 bp for V. ease-inducing activity. A different typing method is proposed [Chan et al., 1992; Simmonds et al., 19931 which classifies HCV isolates into three major types (1, 2, and 3) on a phylogenetic basis, with each type being divided into distinct subtypes. Subtypes la and lb in this classification correspond to genotypes I and 11, re- spectively; 2a and 2b to I11 and IV; and 3a and 3b to V and VI [Mori et al., 1992; Okamoto et al., 19931. In another classification [Houghton et al., 1991; Cha et al., 19921, group I corresponds to genotype I (la), group I1 to genotype I1 (lb), and group I11 includes genotypes I11 (2a) and IV (2b), while group IV encompasses genotype V (3a) and group V includes a herd of variants reported exclusively from South Africa so far. Genotypes of HCV were determined, in terms of I, 11, 111, IV, and V, on genomic RNA in sera from 136 pa- tients with chronic hepatitis C or cirrhosis who received treatment with recombinant alpha-2b interferon. In 72, HCV RNA was titrated by PCR in serial tenfold dilu- tions of RNA extracts from sera. The response to inter- feron was evaluated for correlation with HCV geno- types and pretreatment titers of HCV RNA in sera as well as with various host factors. MATERIALS AND METHODS Patients One hundred thirty-six patients with type C chronic hepatitis or cirrhosis were studied. The sera were posi- tive for anti-HCV by EIA-I1 and with HCV RNA after amplification by PCR, but negative for hepatitis B sur- face antigen. They continued to have serum levels of alanine aminotransferase (ALT) greater than 2.5 times the normal value (>113 IU/liter) during 12 months or longer before they received interferon. Classification of liver disease was based on the histopathology in liver biopsy [de Groote et al., 19681. There were 8 patients with chronic persistent hepatitis (CPH), 75 with chronic active hepatitis of the 2A category (CAHZA), 37 with CAH2B (more severe than CAHBA), and 17 with cirrhosis (LC). Serum samples were collected at one-to- four-week intervals before, during, and longer than 6 months after the completion of therapy for monitoring biochemical and virological markers. They were freshly frozen and stored at -80°C until tests for HCV RNA and determination of HCV genotypes. Serological Tests Anti-HCV was determined by EIA-I1 (Abbott HCV EIA 2nd Generation, Dainabot, Tokyo, Japan), and hepatitis B surface antigen by reversed passive hemag- glutination with commercial kits (MyCell, Institute of Immunology Co., Ltd., Tokyo, Japan). Amplification of HCV RNA by PCR On a template of RNA extracted from 0.1 ml of se- rum, HCV RNA was reverse-transcribed and amplified by PCR with nested primers deduced from the 5’-non- coding region of the HCV genome [Okamoto et al., 19901. Antisense primer in the original method (#36), for cDNA synthesis and the first stage of PCR, was Treatment Patients received recombinant interferon alpha-2b (Intron-A, Schering-Plough, Kenilworth, NJ [distrib- uted by Yamanouchi Pharmaceutical Co., Tokyo, Ja- pan]) in a total dose of 480-560 million units (MU). Fifty patients received 6 MU daily for 2 weeks intra- muscularly and then 6 MU three times a week for 22 weeks (total dose: 480 MU), 69 patients 10 MU daily for 2 weeks and 10 MU three times a week for 12 weeks (500 MU), and the remaining 17 patients 10 MU daily for 8 weeks (560 MU). There were no appreciable differ- ences in age, sex, history of transfusion of patients or genotypes of HCV infecting them among the three groups. Patients were monitored for serum levels of ALT, total protein, albumin, and bilirubin, as well as red cell, white cell, and thrombocyte counts. Responses to Interferon Complete response was defined by the normalization of ALT levels (~45 U/liter) during the therapy, which was maintained for 6 months or longer after interferon was withdrawn. Partial response was defined by the de- crease of ALT to levels within twice the normal limit (~90 U/liter) that continued for 6 months or longer after withdrawal. Patients with either complete or partial re- sponse were considered as responders, and those meet- ing with neither of these criteria as non-responders. Statistical Analysis Differences in dichotomous variables between groups were compared by the x or Fisher’s exact test. Contin- uous variables were compared by the Student’s t-test.  Genotypes/Titers of HCV and Response to Interferon 301 TABLE I. Comparison of Demographic, Clinicopathological, and Virological Features of Patients Who Did or Did Not Respond to Interferon Features Responders Non-responders Differences P) Number of patients 74 62 Age (yr) 45.7 11.7 50.3 ? 9.6 <0.01 Male 51 (69 ) 34 (55%) History of transfusion 26 (35 ) 37 (60 ) <0.01 Alanine aminotransferase (IUlliter) 135 129 148 k 78 Liver histopathologyb Chronic persistent hepatitis 6 (8 ) 2 (3 ) Liver cirrhosis 3 (4 ) 14 (23 ) <0.01 I 0 (0%) 0 (0%) I1 34 (46 ) 51 (82 ) <0.001 I11 22 (30 ) 4 (6 ) <0.001 IV 7 (9 ) 3 (5 ) V 0 (0%) 0 (0 ) Mixedd 8 (11 ) 3 (5 ) Unknowne 3 (4 ) 1(2 ) Chronic active hepatitis 2A 47 (64 ) 27 (44 ) C0.05 Chronic active hepatitis 2B 18 (24 ) 19 (31 ) Genotypes of HCV Normal values are 5 to 45 IU per liter. bClassified by the criteria of de Groote et al. [1968]. Determined by the compatibility of type-specific primers in the amplification by PCR. dIncluded wo cases with genotypes I and 11; three with I1 and 111; two with I1 and IV; three with 111 and IV; one with 11,111, and IV. With low HCV RNA titers <lo2). RESULTS Comparison of Host and Viral Features in Patients With Chronic Hepatitis C Who Did or Did Not Respond to Interferon Among 136 patients who received recombinant al- pha-2b interferon in a total dose of 480-560 MU, 74 (54 ) responded; complete response occurred in 57 (42%) and partial response in 17 (12%). There were no appreciable differences in the response rate to inter- feron among the three groups of patients on slightly different regimens. Table I compares various host and viral features between 74 responders and 62 non-re- sponders. Responders were younger than non-respond- ers (45.7 ? 11.7 vs. 50.3 * 9.6, P < 0.01). The frequency of CAH2A was higher (64 vs. 44 , P < 0.05), while that of cirrhosis was lower (4 vs. 23%, P < 0.01), in responders than in non-responders. Responders had received blood transfusion less fre- quently than non-responders (35 vs. 60 , P < 0.01). History of transfusion was reported by 12 (70 ) of 17 patients with LC, more often than by 4 (50%) of 8 with CPH, 35 (47 ) of 74 with CAHBA, or 12 (32 ) of 37 with CAH2B P < 0.05). Of 63 patients with a history of transfusion, 26 (41 ) esponded to interferon, less fre- quently than 48 (66 ) of 73 without transfusion P < 0.01). Genotypes of HCV in Responders and Non-Responders to Interferon Marked differences were noted in the proportion of HCV genotypes between responders and non-respond- ers. Genotype I1 was found less frequently (46 vs. 82%, P < 0.001), while genotype I11 more frequently (30 vs. 6 , P < 0.001), in responders than in non- responders. Table I1 compares the response to interferon in refer- ence to HCV genotypes and liver histopathology. Of 85 patients who had HCV of genotype 11, 34 (40 ) re- sponded, less frequently than 22 (85%) of 26 patients with genotype I11 P < 0.001), or 7 (70 ) of 10 with genotype IV. The response was inversely correlated with the severity of liver disease, regardless of HCV genotypes; it occurred less frequently in patients with cirrhosis (3117 or 18 ) han in those with CPH (6/8 or 75 , P < 0.051, CAH2A (47174 or 64 , P < 0.01) or CAH2B (18/37 or 49 ). Patients on interferon were monitored for HCV RNA in sera detectable after amplification by PCR (Table 111). The rate of disappearance of HCV RNA from serum was higher in responders than in non-responders at the completion of therapy, and at 6 or 12 months thereafter. Responders lost HCV RNA more frequently than non- responders at 6 and 12 months after the withdrawal of interferon. Rates of HCV RNA disappearance in re- sponders at 6 months (51 ) was similar to that at twelve months (54 ), hereby indicating few relapsers among responders who did not have detectable HCV RNA in sera at six months after the completion of inter- feron therapy. HCV RNA Titers in Pretreatment Sera From Responders and Non-Responders Figure 1 shows HCV RNA titers in pretreatment sera from randomly selected 72 patients with various liver disease (CPH, 6; CAHBA, 34; CAH2B, 19; LC, 13), in- cluding 29 responders and 43 non-responders. Sera  302 TABLE 11. Response to Interferon in Patients Stratified by Hepatitis C Virus GenotvDes and Liver HistoDatho1op.v Hino et al. HCV Liver histopathologya genotypes CPH CAH2A CAH2B LC Total I1 214 19139 10127 3/15 34/85 (50%) (49%) (37%) (20%) (40%) I11 212 14/16 616 012 22/26 IV (100%) (88%) (100%) (0 ) (85%) 111 619 010 010 7/10 (100%) (67%) (70%) Mixed 111 617 113 010 811 1 (100%) (86%) (33%) (73%) Unknown' 010 213 111 010 314 (67%) (100%) (75%) Total 618 47174 1813 7 3/17 741136 (75%) (64%) (49%) (18%) (51%) CPH, chronic persistent hepatitis; CAH, chronic active hepatitis; LC, liver cirrhosis. bGenotypes were not determined due to low HCV RNA titers. TABLE 111. Disappearance of Hepatitis C Virus RNA From Serum in Responders and Non-Responders to Interferon Time after interferon therapy Responders Non-responders Differences PI At completion 51/71 (72%) 32/62 (52%) Six months 36/71 (51%) 2162 (3%) <0.001 Twelve months 30156 (54%) 2/49 (4 ) <0.001 10 *Viral RNA was determined by PCR in sera from patients who did or did not respond to interferon at the completion of therapy as well as 6 and 12 months thereafter. 108 with genotype I1 HCV had HCV RNA titers signifi- cantly higher than those with genotype I11 (the loga- rithmic mean: 6.9 * 1.2 vs. 5.0 1.3, P < 0.001). Overall, the response to interferon occurred in 16 (29%) of 55 patients with HCV RNA titers 3106, less frequently than in 13 (76%) of 17 with titers <lo6 P < 0.01). Among patients with HCV RNA titers ranging from lo5 to lo7, 10 of 33 (30%) with genotype I1 HCV responded, less often than 6 of 7 (86%) with genotype I11 (P < 0.05). Of 51 patients with genotype I1 HCV, 8 (19%) of 42 with HCV RNA titers 3106 responded, less frequently than 6 (75%) of 8 with titers <lo6 P < 0.01). An inverse correlation between pretreatment HCV RNA titers and response to interferon was observed in patients controlled for liver histopathology. Response rates in patients with HCV RNA titers 2106 and those with titers <lo6 were 1/3 (33%) vs. 3/3 (100%) for CPH; 11/28 (39%) vs. 5/6 (83%) or CAH2A; 4/16 (25%) vs. 313 (100%) for CAHBB; and 1/9 (11 ) s. 1/4 (25%) for LC. Of 53 patients with CAHBA or CAHBB, 15 (34%) of 44 with HCV RNA titers 310' responded, significantly less frequently than 8 (89%) of 9 with titers <lo6 P < 0.01). Response to Interferon in Patients With HCV of Mixed Genotypes There were 11 patients who were multiply infected with more than one (mixed) genotypes of HCV (Table IV). Nine of them carried two HCV strains of different a 107 zl RESPONDERS o NON-RESPONDERS . .. . 0 . In3I o . 1021 I1 Ill IV MIXED (n = 51) (n = 12) (n = 6 (n = 3) GENOTYPES OF HEPATITIS C VIRUS Fig. 1. Pretreatment titers of hepatitis C irus RNA and genotypes in responders and non-responders to interferon therapy. Titers of HCV RNA and HCV genotypes are shown for 72 patients, of whom 29 responded to interferon and 43 did not.  GenotypesITiters of HCV and Response to Interferon 303 TABLE IV. Response to Interferon in Patients Who Were Infected With Hepatitis C Virus of Mixed Genotypes Case no. 1 2 3 4 5 6 7 8 9 10 11 Age and sex 38 F 61 F 30 M 42 M 57 M 56 F 30 M 41 M 49 M 52 M 65 M HCV Liver genotypes histopathology I, I1 CAH2A I, I1 CAH2B 11, I11 CAH2A 11, 111 CAH2A 11, 111 CAH2B 11, 111, IV CAH2B 11, IV CAH2A 11, IV CPH 111, IV CAHXA 111, IV CAH2A 111. IV CAH2A History of Response to transfusion interferon No No Yes Yes No Yes Yes Yes Yes Yes Yes CPH, chronic persistent hepatitis; CAH, chronic active hepatitis. genotypes, and the remaining one those of three dis- tinct genotypes. None of them had liver cirrhosis. Only three (27%) of them had history of transfusion, and eight (73%) responded to interferon. DISCUSSION Therapeutic trials of interferon for patients with NANB hepatitis were started several years ago, when the diagnosis of the disease was based on exclusion criteria [Hoofnagle et al., 1986; Jacyna et al., 1989; Kakumu et al., 19891. Introduction of EIA-I with the recombinant C100-3 protein to the serological diagno- sis of hepatitis C [Kuo et al., 19891 has made the evalu- ation of interferon therapy more reliable. Beneficial effects of interferon have been reported in a number of randomized, placebo-controlled trials on patients posi- tive for EIA-I. The efficacy in these trials, however, varies rather widely from 40 to 70% of patients who responded with complete or partial remission; relapse is frequent, occurring in 25% to 80% of responders within 6 months after withdrawal of interferon [Davis et al., 1989; Di Bisceglie et al., 1989; Davis, 1990; Tine' et al., 19911. In the present study, the therapeutic effect of recom- binant alpha-2b interferon with a total dose of 480-560 MU was evaluated on patients with chronic hepatitis C or cirrhosis and positive for HCV RNA in sera, in an attempt to sort out host or viral factors influencing the response to interferon. Of the 136 patients studied, 74 (54%) esponded while the remaining 62 (46%) failed to do so. There were some host factors influencing the response to interferon. Responders were younger than non-responders (45.7 11.7 vs. 50.3 ? 9.6 yr), and had received transfusion less frequently (26174 or 35 vs. 37162 or 60%, P < 0.01). Response was more common in patients with precirrhotic disease than in those with cirrhosis. A viral factor predictive of the response to interferon was the HCV genotype. The response was less frequent in patients infected with genotype I1 HCV (34185 or 40%) than in those with genotype I11 (22126 or 85%, P < 0.001) or genotype IV (7110 or 70%). The influence of HCV genotypes on response to interferon was observed in patients controlled for the severity of liver histopa- thology (Table 11). Genotype-dependent responses to in- terferon have been reported in smaller series of pa- tients [Takada et al., 1992; Yoshioka et al., 19921. Another viral factor predictive of the response was the pretreatment level of HCV RNA in serum. Among patients carrying HCV of genotype 11, response to inter- feron was less frequent in those with HCV RNA titers a106 han in those with titers <lo6 (8142 or 19% vs. 618 or 75%, P < 0.01). An inverse correlation between pre- treatment HCV RNA titers and response was applica- ble to patients infected with HCV of the other geno- types also. Overall, 16 (29%) of 55 patients with HCV RNA titers 3 O6 responded, less often than 13 (76%) of 17 with titers <lo6 P < 0.001). Although HCV titers in sera of patients may fluctuate, the obtained results in- dicate that HCV RNA levels, representing viral repli- cation in the liver, would be a factor influencing the response to interferon. A similar view is also proposed in previous reports [Kaneko et al., 1992; Takada et al., 19921. Some association was noted between HCV genotypes and titers of HCV RNA. The pretreatment mean log titer of HCV RNA in sera with HCV of genotype I1 was higher than that in those with genotype I11 (6.9 * 1.2 vs. 5.0 ? 1.3, P < 0.001), indicating that HCV of geno- type I1 would replicate more actively than HCV of geno- type I11 in hosts. The influence of genotypes on HCV RNA levels in sera has also been observed by others [Yoshioka et al., 19911. The resistance to interferon of patients with HCV of genotype 11, therefore, would have been relevant to a high replicative activity associ- ated with HCV of this genotype. The ultimate goal of interferon therapy is the eradi- cation of HCV from the liver. This is not easily verified, but can be reflected by the disappearance of HCV from circulation. The response to interferon, therefore, may be monitored more directly and precisely by HCV RNA than by ALT levels [Shindo et al., 1991; Chayama et al.,
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