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Discrepancy between biochemical and virological responses to interferon-α in chronic hepatitis C
Serum samples were prepared within 4 h of collection and stored 20°C. They were thawed only once before testing for HCV RNA. This preparative procedure affects serum HCV RNA levels minimally and therefore should reflect the actual level of HCV viraemia in vitro.4 Serum HCV RNA was amplified by reversetranscription nested polymerase chain reaction (RT-PCR) with primers from the highly conserved 5’untranslated region and quantified by the branched DNA signal amplification (bDNA) assay.s HCV genotypes were identified in paired serum samples from before and after IFNa by primer-specific RT-PCR based on the HCV core region.6 Paired (before and after IFNa) liver biopsy samples (formalin-fixed, paraffin-embedded) from 14 patients 7 were also available for HCV RNA testing. 4 of 5 patients who showed no biochemical response to at -
Discrepancy between biochemical and virological responses to interferon-&agr; in chronic hepatitis C
Serial serum samples and pretreatment and post-treatment liver tissue from patients with chronic hepatitis C virus (HCV) infection were tested for HCV RNA by reverse-transcription polymerase chain reaction and branched DNA signal amplification assays. At the end of treatment with interferon-a OFNot), 4 of 5 patients showing no biochemical response (in alanine aminotransferase activity), 4 of 5 with transient responses, and 1 of 5 showing complete and sustained responses had HCV RNA detectable in serum. The corresponding numbers for liver tissue were 5, 5, and 0 (of 4). However, all 5 complete responders had virological relapses within 6 months. Biochemical response may not reflect virological profile during lFN&agr; treatment of HCV. Lancet
1993; 342: 1208-09
Interferon-a (IFNa) therapy is effective in lowering serum alanine aminotransferase (ALT) activity to normal values in 40% of patients with chronic hepatitis C virus (HCV) infection.1,2 However, at least half of the responders relapse when IFNa is discontinued. Definitions of response to IFNcx have been based on improvement in serum ALT. Although a previous study suggested that patients whose serum ALT falls to normal on IFNa are likely to become negative for HCV RNA in serum,3it is not clear whether a complete and sustained biochemical response to treatment is accompanied by sustained disappearance of HCV virus. We studied 15 patients with chronic HCV infection who took part in a randomised IFN&agr; therapy trial (table). They represented three groups of patients defined by their biochemical response to IFNot therapy. 5 patients had persistently abnormal ALT activity (above 50 IU/L) during and after treatment (no response), 5 had normal serum ALT during IFNA therapy but a rise shortly after IFNot was discontinued (complete response and relapse), and 5 had normal serum ALT during IFNa therapy that persisted for at least 6 months after discontinuation of the drug (complete response). Patient selection criteria and treatment protocol have been
reported previously.’1
IFNa
positive for HCV RNA in serum throughout (figure); the other patient was only transiently negative. HCV RNA was detected in liver biopsy samples from before and after IFNa in all 5 patients. 3 of 5 patients with complete response and relapse were persistently positive for HCV RNA in serum. In the other 2, HCV RNA became undetectable during treatment but reappeared before the end of therapy in 1. HCV RNA was detected in all liver biopsy samples from the 5 patients. In the 5 patients with complete and sustained were
treatment
biochemical responses, HCV RNA was undetectable after 12 weeks of IFNa therapy. However, in 1 patient HCV reappeared before the end of therapy (figure). All 5 patients had virological relapse within 6 months. In 3, HCV RNA was detectable within 12 weeks of IFNa withdrawal. Serum HCV RNA remained at low values (bDNA up to 0 52 x 106 Eq/mL) in 3 patients during follow-up of 1-25 and 3-0 years (ALT still normal at last follow-up). 2 other patients had persistently higher HCV RNA values (range 0-5-17-7 x 106 Eq/mL), despite having normal ALT for 1-5 and 2-5 years before late biochemical relapse. HCV RNA was detected in all 5 liver biopsy samples from before IFNa therapy but in none of the 4 available after IFNa therapy. In all 15 patients HCV genotypes were the same before IFNa therapy and at the time of relapse (table). Biochemical response to IFNa therapy in chronic HCV infection does not correlate with eradication of HCV. This finding raises important questions about the definition of response to IFNa therapy. If we are treating liver disease, the biochemical definition (better still with histological confirmation) is most appropriate. However, persistence of HCV in these patients suggests that even complete biochemical responders are susceptible to relapse. Thus, long-term follow-up is essential even for these patients. It is not clear why patients with complete and sustained responses showed no biochemical evidence of disease when they had levels of HCV viraemia similar to those before
i ime (WK) Serum HCV RNA values to biochemical response to IFNa therapy Figure: according A= no biochemical response; B= complete response with relapse; C= complete and sustained response. *Patients with late biochemical relapses.
1208
7
8
Diamond DA, Davis GL, KP Qian, Lau JYN. Detection of hepatitis C viral sequences in formalin-fixed, paraffin-embedded liver tissue: effect of interferon-alpha therapy. J Med Virol (in press). Qian C, Camps J, Maluenda MD, et al. Replication of peripheral blood mononuclear cells: effect of alpha-interferon therapy. J Hepatol 1992; 16: 380-83.
Hepatobiliary Diseases, Department of Medicine, University of Florida, PO Box 100214 JHMHC, Gainesville, Florida 32610, USA (J Y N Lau MD, D A Diamond BA, J Kniffen RN, G L Davis MD); and Second Department of Medicine, Nagoya City University Medical School, Japan (M Mizokami MD, T Ohno MD) Correspondence to: Dr J Y N Lau
Section of
*By enzyme immunoassay 2. tBy RT-PCR. tbDNA assay; unit is genome equivalents per mL. Table : Clinical, biochemical, and virological details of study
patients is that IFNa level that is able to cause liver damage. Our inability to detect HCV RNA in liver tissue at the end of therapy may be due simply to the insensitivity of the current RT-PCR. However, the similarity of HCV RNA values before and after IFNa treatment, despite normal serum ALT, argues against this explanation. A second possibility is that IFNa selects non-cytopathic HCV subtypes, but the identity of HCV genotypes before and after IFNa therapy makes this explanation unlikely. It is possible, however, that our subtyping method did not include the IFNa-sensitive elements and that some IFNaresistant strains that are not differentiated by the current subtyping method escaped. Thirdly, relapse could be due to HCV derived from an extrahepatic reservoir. HCV RNA has been detected in peripheral-blood mononuclear cells.s If this hypothesis were true, HCV RNA would not be present in the liver in complete and sustained responders with virological relapse. A final possibility is that IFNa therapy may alter the host-HCV interaction, rendering the infection less harmful to the host and hampering the development of liver disease. In that case, IFNcx could be considered as both an antiviral and a disease-modifying agent in the treatment of chronic HCV infection.
treatment.
The
most
suppresses HCV below
a
References
2
3
4 5 6
men
likely explanation
We thank Schering-Plough (Kenilworth, New Jersey, USA) for funding the interferon-a treatment trial and the Chiron Corporation for providing the bDNA assay. This study was supported by grants DSR-D-15, DSR-RDA-1-15 from Division of Sponsored Research, University of Florida, Gainesville, American Liver Association Hans Popper Liver Scholar Award, and Glaxo Institute of Digestive Health Clinical Investigator Award (to JYNL). The Clinical Research Center in the University of Florida was supported by a grant from the National Institutes of Health (5MOIRROO082).
1
Plasma concentrations of phyto-oestrogens in Japanese
Davis GL, Balart LA, Schiff ER, et al. Treatment of chronic hepatitis C with recombinant interferon alpha: a multicentre randomized, controlled trial. N Engl J Med 1989; 321: 1501-06. Di Bisceglie AM, Martin P, Kassianides C, et al. Recombinant interferon alpha therapy for chronic hepatitis C. A randomized, double blinded, placebo-controlled trial. N Engl J Med 1989; 321: 1506-10. Shindo M, Di Bisceglie AM, Cheung L, et al. Decrease in serum hepatitis. C virus RNA during alpha-interferon therapy for chronic hepatitis C. Ann Intern Med 1991; 115: 700-04. Lau JYN, Davis GL, Kniffen J, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993; 341: 1501-04. Lau JYN, Davis GL, Brunson ME, et al. Hepatitis C virus infection in renal transplant recipients. Hepatology (in press). Okamoto H, Shgiyama Y, Okada S, et al. Typing hepatitis C virus by polymerase chain reaction with type specific primers: application to clinical survey and tracing infectious sources. J Gen Virol 1992; 73: 673-79.
A low mortality from prostatic cancer is found in Japanese men consuming a low-fat diet with high content of soy products, a rich source of isoflavonoids. We therefore assayed four isoflavonoids in plasma of 14 Japanese and 14 Finnish men. The geometric mean plasma total individual isoflavonoid levels were 7 to 110 times higher in the Japanese than in the Finnish men. Genistein, a tyrosine kinase inhibitor, occurred in the highest concentration (geometric mean 276 nmol/L). We hypothesise that these high phyto-oestrogen levels may inhibit the growth of prostatic cancer in Japanese men, which may explain the low mortality from prostatic cancer in that country. Lancet 1993; 342: 1209-10
probably the most important environmental factor influencing cancer risk and mortality. In Japan and some other Asian countries mortality from prostatic cancer is low, despite the same incidence of latent and small or non-infiltrative prostatic carcinomas as in Western countries.1 Decreased prostatic cancer risk has been found in Adventist menwho eat a lot of beans, lentils, peas, and some dried fruits (all sources of flavonoids) and in men of Japanese ancestry in Hawaii3 who eat rice and tofu, a soybean product containing isoflavonoids in great quantities.4 Could the low incidence and mortality of prostatic cancer in Japan be due to phyto-oestrogens, especially isoflavonoids, inhibiting the growth of latent cancers?S Soy is protective in prostatic dysplasia in mice,6 and genistein and its precursor, biochanin A, inhibit the growth of androgen-dependent and androgen-independent prostatic cancer cells in culture.7 The therapeutic effect of oestrogens in prostatic cancer suggests that phytooestrogens may inhibit prostatic cancer cell growth during the promotional phase of the disease, or may influence
Diet is
differentiation.
sufficiently sensitive isotope-dilution gas-chromatographic/ mass-spectrometric method (ID-GC/MS) now allows assay of four isoflavonoids in human plasma.8 The method is based on solid-phase extraction after adding triethylamine sulphate to the samples to liberate protein-bound sulphates. The extract is fractionated into an unconjugated (free) plus sulphate fraction and a glucuronide fraction containing monoglucuronides, diglucuronides, and sulphoglucuronides. After solvolysis of the sulphates, but before their enzymatic hydrolysis, deuterated A
internal standards for all compounds
are
added
to correct
for all
1209
Discrepancy between biochemical and virological responses to interferon-&agr; in chronic hepatitis C
Serial serum samples and pretreatment and post-treatment liver tissue from patients with chronic hepatitis C virus (HCV) infection were tested for HCV RNA by reverse-transcription polymerase chain reaction and branched DNA signal amplification assays. At the end of treatment with interferon-a OFNot), 4 of 5 patients showing no biochemical response (in alanine aminotransferase activity), 4 of 5 with transient responses, and 1 of 5 showing complete and sustained responses had HCV RNA detectable in serum. The corresponding numbers for liver tissue were 5, 5, and 0 (of 4). However, all 5 complete responders had virological relapses within 6 months. Biochemical response may not reflect virological profile during lFN&agr; treatment of HCV. Lancet
1993; 342: 1208-09
Interferon-a (IFNa) therapy is effective in lowering serum alanine aminotransferase (ALT) activity to normal values in 40% of patients with chronic hepatitis C virus (HCV) infection.1,2 However, at least half of the responders relapse when IFNa is discontinued. Definitions of response to IFNcx have been based on improvement in serum ALT. Although a previous study suggested that patients whose serum ALT falls to normal on IFNa are likely to become negative for HCV RNA in serum,3it is not clear whether a complete and sustained biochemical response to treatment is accompanied by sustained disappearance of HCV virus. We studied 15 patients with chronic HCV infection who took part in a randomised IFN&agr; therapy trial (table). They represented three groups of patients defined by their biochemical response to IFNot therapy. 5 patients had persistently abnormal ALT activity (above 50 IU/L) during and after treatment (no response), 5 had normal serum ALT during IFNA therapy but a rise shortly after IFNot was discontinued (complete response and relapse), and 5 had normal serum ALT during IFNa therapy that persisted for at least 6 months after discontinuation of the drug (complete response). Patient selection criteria and treatment protocol have been
reported previously.’1
IFNa
positive for HCV RNA in serum throughout (figure); the other patient was only transiently negative. HCV RNA was detected in liver biopsy samples from before and after IFNa in all 5 patients. 3 of 5 patients with complete response and relapse were persistently positive for HCV RNA in serum. In the other 2, HCV RNA became undetectable during treatment but reappeared before the end of therapy in 1. HCV RNA was detected in all liver biopsy samples from the 5 patients. In the 5 patients with complete and sustained were
treatment
biochemical responses, HCV RNA was undetectable after 12 weeks of IFNa therapy. However, in 1 patient HCV reappeared before the end of therapy (figure). All 5 patients had virological relapse within 6 months. In 3, HCV RNA was detectable within 12 weeks of IFNa withdrawal. Serum HCV RNA remained at low values (bDNA up to 0 52 x 106 Eq/mL) in 3 patients during follow-up of 1-25 and 3-0 years (ALT still normal at last follow-up). 2 other patients had persistently higher HCV RNA values (range 0-5-17-7 x 106 Eq/mL), despite having normal ALT for 1-5 and 2-5 years before late biochemical relapse. HCV RNA was detected in all 5 liver biopsy samples from before IFNa therapy but in none of the 4 available after IFNa therapy. In all 15 patients HCV genotypes were the same before IFNa therapy and at the time of relapse (table). Biochemical response to IFNa therapy in chronic HCV infection does not correlate with eradication of HCV. This finding raises important questions about the definition of response to IFNa therapy. If we are treating liver disease, the biochemical definition (better still with histological confirmation) is most appropriate. However, persistence of HCV in these patients suggests that even complete biochemical responders are susceptible to relapse. Thus, long-term follow-up is essential even for these patients. It is not clear why patients with complete and sustained responses showed no biochemical evidence of disease when they had levels of HCV viraemia similar to those before
i ime (WK) Serum HCV RNA values to biochemical response to IFNa therapy Figure: according A= no biochemical response; B= complete response with relapse; C= complete and sustained response. *Patients with late biochemical relapses.
1208
7
8
Diamond DA, Davis GL, KP Qian, Lau JYN. Detection of hepatitis C viral sequences in formalin-fixed, paraffin-embedded liver tissue: effect of interferon-alpha therapy. J Med Virol (in press). Qian C, Camps J, Maluenda MD, et al. Replication of peripheral blood mononuclear cells: effect of alpha-interferon therapy. J Hepatol 1992; 16: 380-83.
Hepatobiliary Diseases, Department of Medicine, University of Florida, PO Box 100214 JHMHC, Gainesville, Florida 32610, USA (J Y N Lau MD, D A Diamond BA, J Kniffen RN, G L Davis MD); and Second Department of Medicine, Nagoya City University Medical School, Japan (M Mizokami MD, T Ohno MD) Correspondence to: Dr J Y N Lau
Section of
*By enzyme immunoassay 2. tBy RT-PCR. tbDNA assay; unit is genome equivalents per mL. Table : Clinical, biochemical, and virological details of study
patients is that IFNa level that is able to cause liver damage. Our inability to detect HCV RNA in liver tissue at the end of therapy may be due simply to the insensitivity of the current RT-PCR. However, the similarity of HCV RNA values before and after IFNa treatment, despite normal serum ALT, argues against this explanation. A second possibility is that IFNa selects non-cytopathic HCV subtypes, but the identity of HCV genotypes before and after IFNa therapy makes this explanation unlikely. It is possible, however, that our subtyping method did not include the IFNa-sensitive elements and that some IFNaresistant strains that are not differentiated by the current subtyping method escaped. Thirdly, relapse could be due to HCV derived from an extrahepatic reservoir. HCV RNA has been detected in peripheral-blood mononuclear cells.s If this hypothesis were true, HCV RNA would not be present in the liver in complete and sustained responders with virological relapse. A final possibility is that IFNa therapy may alter the host-HCV interaction, rendering the infection less harmful to the host and hampering the development of liver disease. In that case, IFNcx could be considered as both an antiviral and a disease-modifying agent in the treatment of chronic HCV infection.
treatment.
The
most
suppresses HCV below
a
References
2
3
4 5 6
men
likely explanation
We thank Schering-Plough (Kenilworth, New Jersey, USA) for funding the interferon-a treatment trial and the Chiron Corporation for providing the bDNA assay. This study was supported by grants DSR-D-15, DSR-RDA-1-15 from Division of Sponsored Research, University of Florida, Gainesville, American Liver Association Hans Popper Liver Scholar Award, and Glaxo Institute of Digestive Health Clinical Investigator Award (to JYNL). The Clinical Research Center in the University of Florida was supported by a grant from the National Institutes of Health (5MOIRROO082).
1
Plasma concentrations of phyto-oestrogens in Japanese
Davis GL, Balart LA, Schiff ER, et al. Treatment of chronic hepatitis C with recombinant interferon alpha: a multicentre randomized, controlled trial. N Engl J Med 1989; 321: 1501-06. Di Bisceglie AM, Martin P, Kassianides C, et al. Recombinant interferon alpha therapy for chronic hepatitis C. A randomized, double blinded, placebo-controlled trial. N Engl J Med 1989; 321: 1506-10. Shindo M, Di Bisceglie AM, Cheung L, et al. Decrease in serum hepatitis. C virus RNA during alpha-interferon therapy for chronic hepatitis C. Ann Intern Med 1991; 115: 700-04. Lau JYN, Davis GL, Kniffen J, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993; 341: 1501-04. Lau JYN, Davis GL, Brunson ME, et al. Hepatitis C virus infection in renal transplant recipients. Hepatology (in press). Okamoto H, Shgiyama Y, Okada S, et al. Typing hepatitis C virus by polymerase chain reaction with type specific primers: application to clinical survey and tracing infectious sources. J Gen Virol 1992; 73: 673-79.
A low mortality from prostatic cancer is found in Japanese men consuming a low-fat diet with high content of soy products, a rich source of isoflavonoids. We therefore assayed four isoflavonoids in plasma of 14 Japanese and 14 Finnish men. The geometric mean plasma total individual isoflavonoid levels were 7 to 110 times higher in the Japanese than in the Finnish men. Genistein, a tyrosine kinase inhibitor, occurred in the highest concentration (geometric mean 276 nmol/L). We hypothesise that these high phyto-oestrogen levels may inhibit the growth of prostatic cancer in Japanese men, which may explain the low mortality from prostatic cancer in that country. Lancet 1993; 342: 1209-10
probably the most important environmental factor influencing cancer risk and mortality. In Japan and some other Asian countries mortality from prostatic cancer is low, despite the same incidence of latent and small or non-infiltrative prostatic carcinomas as in Western countries.1 Decreased prostatic cancer risk has been found in Adventist menwho eat a lot of beans, lentils, peas, and some dried fruits (all sources of flavonoids) and in men of Japanese ancestry in Hawaii3 who eat rice and tofu, a soybean product containing isoflavonoids in great quantities.4 Could the low incidence and mortality of prostatic cancer in Japan be due to phyto-oestrogens, especially isoflavonoids, inhibiting the growth of latent cancers?S Soy is protective in prostatic dysplasia in mice,6 and genistein and its precursor, biochanin A, inhibit the growth of androgen-dependent and androgen-independent prostatic cancer cells in culture.7 The therapeutic effect of oestrogens in prostatic cancer suggests that phytooestrogens may inhibit prostatic cancer cell growth during the promotional phase of the disease, or may influence
Diet is
differentiation.
sufficiently sensitive isotope-dilution gas-chromatographic/ mass-spectrometric method (ID-GC/MS) now allows assay of four isoflavonoids in human plasma.8 The method is based on solid-phase extraction after adding triethylamine sulphate to the samples to liberate protein-bound sulphates. The extract is fractionated into an unconjugated (free) plus sulphate fraction and a glucuronide fraction containing monoglucuronides, diglucuronides, and sulphoglucuronides. After solvolysis of the sulphates, but before their enzymatic hydrolysis, deuterated A
internal standards for all compounds
are
added
to correct
for all
1209