AJ.208 AASLD ABSTRACTS GASTROENTEROLOGY, VoL 108, NO. 4

• SIGNIFICANCE OF HCV-RNA LEVEL AND RELATIONSHIP TO VIRAL GENOTYPE IN PATIENTS WITH CHRONIC HEPATITIS C IN THE U.S.N.N. Zein, J.B. Gross, Jr., J.J. Potefucha, A.A. Gossard, J. Germer, D.H. Persing. Mayo Clinic and Foundation, Rochester, MN 55905

Recently the branched-DNA technique has made it possible to quantitate HCV-RNA levels in patients with chronic hepatitis C. AIMS: 1) To determine the clinical, biochemical, and histological significance of HCV-RNA levels. 2) To determine the relationship between HCV-RNA level and HCV genotype. METHODS: Sera of 60 HCV-infected patients were collected prior to interferon treatment. Pre-treatment liver biopsies were reviewed. HCV-RNA levels were quantitated using the Chiron DNA single amplification assay. HCV genotypes were determined by direct automated sequencing of desalted amplification products using primers specific for the NS5 region (Simmonds classification). RESULTS:

HCV Genotvnes l_~a lb. 2_.bb 3a

N=60 37 (61%) 11 (19%) 8 (13%3 4 (7%) bDNA 100 76 14 67 (xl05~a/ml'~

The mean HCV-RNA level in patients with mild hepatitis (defined as ALT <3x normal and the absence of cirrhosis) was 74 x 105 compared to 96 x 10 5 in patients with severe hepatitis. There was no correlation between the serum HCV-RNA level and the degree of inflammation in the liver biopsy specimen. The pre-treatment serum HCV-RNA level did not predict the response to interferon at the end of 6 months of therapy. SUMMARY: 1) Infection with HCV genotype 2b was associated with significantly lower HCV-RNA levels than infection with genotypes la, lb, or 3a. 2) Patients with mild hepatitis tended to have lower HCV-RNA levels then those with severe hepatitis. 3) The pre-treatment HCV-RNA level did not predict biochemical response to interferon at the end of 6 months of therapy. CONCLUSION: Although HCV-RNA levels were lower in patients infected with genotype 2b, a genotype previously shown to be interferon-responsive, HCV-RNA levels did not correlate with response to treatment in the group as a whole. Therefore, the clinical utility of performing quantitative HCV-RNA measurements in • addition to genotyping remains to be investigated.

• SIGNIFICANCE OF SERUM IRON, IRON SATURATION, AND FERRITIN IN PATIENTS INFECTED WITH DIFFERENT HEPATITIS C GENOTYPES. N.N. Zein, J.B. Gross, Jr., J.J. Poterucha. A.A. Gossard. N.K. Wendt, P.S. Mitchell, D.H. Persing. Mayo Clinic & Foundation. Rochester, MN

Hepatic iron overload can lead to hepatic fibrosis and cirrhosis. Our previous data have shown that certain HCV genotypes, e.g., lb, lead to more severe liver disease and are less responsive to interferon therapy. AIMS: 1) To study serum iron indices in patients infected with different HCV genotypes. 2) To evaluate the ability of serum iron indices to predict the response to interferon therapy in patients with chronic hepatitis C. METHODS: Pre-treatment sera were available from 39 patients with chronic hepatitis C who had had complete iron studies performed. RNA was extracted from serum using chaotropic lysis and isopropanol precipitation. Direct automated sequencing was performed on the purified PCR products. The Simmonds system was used for HCV genotyping of the NS5 region. RESULTS: Mean levels of serum iron, transferrin saturation, and ferritin are shown according to HCV genotype in the table below.

N Iron Saturation Ferritin ot~/mi) (%) (~t~)

la 22 143 45 195 lb 10 153 49 412" 2b 5 119 35 218 3a 2 165 43 160 *p<0.05 The mean serum ferritin level in patients with genotype lb was significantly higher than in patients with other genotypes (p<0.05), There was a trend toward lower serum iron levels and transferrin saturation in patients with genotype 2b (4 out of 5 of whom responded to interferon). Among the 10 patients who had a biochemical remission at the end of 6 months of interferon therapy, the serum ferritin was lower than in the 29 unresponsive patients (187 ~tg/L vs. 283), while there were no differences in serum iron (135 vs. 148 ~tg/ml) or transferrin saturation (43 vs. 50%). CONCLUSIONS: 1) Patients infected with genotype lb have significantly higher serum ferritin levels than patients with other genotypes. 2) Lower pre-treatment serum ferritin may be associated with a higher probability of response to interferon therapy. Whether the assoeation of higher serum ferritin to a more pathogenic HCV genotype and poorer response to therapy is related to increased iron deposition in the liver or to some other important predictive factor has yet to be determined.

THE SELEC'q~ON OF INTERFERON-RESISTANT HCV-GENOTYPES FOLLOWING SIX MONTHS 'IT-IERAPY. N.N: Zein. J.J. Poterucha, J.B. Gross, A.A. Gossard, J.J. Germer, N.K. Wendt, P.S. Mitchel, N. Ramzan, D.H: Persing. Mayo Clinic, Rochester, MN.

HCV, like other RNA viruses, exist as a group of quasispecies and genotypes in the infected host. Therapy with interferon alpha has shown to induce biochemical and virological response in a propo~on of patients with chronic hepatitis C infection. However, most of them will relapse. HYPOTHESIS: In patients with mixed infections, treatment with interferon alpha (INF) may eliminate interferon-sensitive species, therefore allowing the INF-resistant strain to become predominant. METHODS: Pre- and six months post-interferon treatment sera were obtained from 22 patients with chronic hepatitis C infection. The HCV genotype was determined using automated direct sequencing of NS 5 amplification products. RESULTS: Nine of 22 patients (41%) had complete biochemical response at the end of six months therapy (defined as normalization of ALT); seven relapsed after therapy was stopped. Thirteen patients (59%) did not respond. Two patients were long-term responders and remained PCR negative after therapy. The HCV genotype distribution in each group, pre- and post-treatment is illustrated in the table. Three of four patients whose HCV genotypes switched were genotype 2b prior to treatment. All converted to HCV genotype la which is associated with resistance to INF. One patient who initially responded to treatment and then relapsed harbored HCV genotype la prior to therapy, and HCV genotype lb after therapy; the latter may also be relatively more resistant to INF.

Relapses = 7 Nouresponders = 13 Pre-Rx Post-Rx Pre-Rx Post-Rx

la 2 3 7 8 lb 1 2 3 3 2b 3 1 1 0 3a 1 1 2 2

CONCLUSIONS: In all four cases in which genotype sw ~d, the predominant HCV genotypes went from relatively more sensitive to more resistant subtypes after interferon therapy. The use of interferon, analogous to antibiotic selection of resistant bacterial strains, may enhance the selection of more resistant and potentially more pathogenic genotypes of hepatitis C virus.

VIRAL GENOTYPES AND THE BREAKTHROUGH PHENOMENON DURING INTERFERON TREATMENT OF CHRONIC HEPATITIS C. NN Zein. S. Jonnalagadda, JB Gross, Jr, DR Reddy, B Price, LJ Jeffers, JJ Poterucha, ER Schiff, and DH Persing. Mayo Clinic and Foundation, Rochester, MN; U Miami School of Medicine, Miami, FL.

Some patients undergoing interferon treatment for chronic hepatitis C exhibit a "breakthrough" phenomenon consisting of a recurrent rise in aminotranferases during therapy, after an initial normalization. AIM: We were interested to know whether the breakthrough phenomenon was explained by a change to a more resistant HCV genotype. METHODS: A breakthrough was defined as two Successively normal ALT values at least one month apart during interferon therapy, followed by at least one abnormal ALT value while still on treatment. We identified 9 patients who exhibited this phenomenon. HCV genotypes were determined prior to therapy and at the time of breakthrough. In addition, genotypes were determined in serum samples which were available from 4 patients approximately one month after breakthrough. Direct automated sequencing of NS 5 amplification products was performed with genotype assignment according to the Simmondsclassification. RESULTS: The distribution of HCV genotypes was as follows: genotype la, 4 patients; genotype lb, 2 patients; genotype 2a, 1 patient; genotype 2b, 1 patient; and genotype 3a, l patient. Genotypes at the time of breakthrough were identical to pre-treatment genotypes in all nine patients~ Furthermore, no genotype switch was Seen in the four samples available one month after therapy. CONCLUSION: The breakthrough phenomenon during interferon treatment of chronic hepatitis C is not associated with a change in HCV genotype. Further studies of virologic, immunologic, and pharmacologic factors in patients with chronic hepatitis C will be necessary to establish the mechanism of the breakthrough phenomenon.