LETTERS
406
A
12345678
B
Fig. 1. PCR products of HCV cDNA using the d94, d95, Nl and N2 set of primers (A) and the NCR set of primers (B) on gel electrophoresis. Lane 1, molecular weight marker; lane 2, positive control; lane 3, negative control; lane 4, sample from patient with proven hepatitis B infection: and lanes 5-8, samples from patients 1, 2, 3 and 4. respectively.
round. Using the d94, d95, Nl, N2 set of primers (2) we performed the first round of PCR consisting of 35 cycles of denaturation for 80 s at 95 “C, annealing for 40 s at 58 “C and extension for 60 s at 72 “C. Ten ~1 of amplified material were subjected to the second round of PCR consisting of 25 cycles of denaturation for 60 s at 95 “C, annealing for 60 s at 46 “C and extension for 60 s
TO THE EDITOR
72 “C. Using the NCR 1,2,3 and 4 set of primers (3) we performed the first PCR round consisting of 35 cycles of denaturation for 60 s at 95 “C, annealing for 60 s at 50 “C and extension for 60 s at 72 “C. The second PCR round consisted of 30 cycles of denaturation for 60 s at 94 “C, annealing for 60 s at 46 “C and extension for 60 s at 72 “C. The last cycle of each PCR round was followed by an extension of 7 min at 72 “C. Reagents without template and RNA isolated from a liver with proven hepatitis B were used as negative controls. As a positive control we used RNA isolated from the liver of a patient who had a partial hepatectomy because of a hepatocellular carcinoma and a nonalcoholic cirrhosis. The patient’s serum had HCV antibodies, tested by an enzyme immunoassay (Abbott Wiesbaden). By using the first set of primers we amplified HCV cDNA sequences in three of six cases (Fig. lA), while by using the second set of primers we amplified HCV cDNA in four of six cases (Fig. 1B). The use of this method to detect HCV RNA in material from liver needle biopsies may help in the definite diagnosis of HCV. Since only part of the material is enough for the detection of HCV RNA, this could complement the diagnosis of HCV based upon morphological criteria. at
PCR
References 1 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9. 2 Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, et al.
Ioannis D. Diamantis’, Christine E. McGandy’, Irmgard Pult’, Jean-Jacques Widmann2, Fred Gudat’ and Leonardo Bianchi’ ‘Institute of Pathology, University Hospital, Schdnbeinstrasse 40, CH-4003, Base1 and ‘Institute of Pathology, University Hospital, CMU, CH-1200 Geneva, Switzerland
Detection of hepatitis C viral sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. 3 Garson JA, Ring C, Tuke P, Tedder RS. Enhanced detection by PCR of hepatitis C virus RNA. Lancet 1990; 336:878
HEPAT 00976
Hormonal treatment of hepatocellular In response to the letter of Farinati et al. (1) about hormonal manipulation of hepatocellular carcinoma (HCC), we would like to correct one small omission. In their discussion of anti-androgen therapy, they stated that they were aware of three studies. We would like to draw their attention to a fourth, in which the progestogenie anti-androgen cyproterone acetate was administered to 25 cirrhotic patients with HCC (23 male) (1).
carcinoma
There was an objective response to therapy in five patients, and furthermore, the response appeared to be correlated with a fall in free levels of the potent androgen Sa-dihydrotestosterone. In this study, while the compounding variables or ‘biases’ referred to by Farinati et al. were not entirely eliminated, the aetiology of cirrhosis, Gkuda grade and, in most patients, the pre- and post-treatment hormonal
LETTERS
TO THE EDITOR
status were clearly recorded. If normal criteria for c motherapy of solid tumours are adopted, the trial showed a modest, but useful 20% response rate. If we adopt the criteria of Farinati et al., that the only response that can be expected is stable disease, then 12/25 (48%) had a measurable response to cyproterone acetate. However, we do not believe that this is a reasonable approach and prefer to assess response in a conventional manner. Furthermore, in published studies the response of HCC to sex hormones is not limited to anti-androgens and anti-oestrogens. Progestogens may also be important. The early study of Friedman et al. (3), which Farinati et al. suggested showed a response to tamoxifen, in fact used a megesterol acetateimedroxy progesterone combination, and two further studies have shown a reeferences 1 Farinati F, De Maria M, Chiaramonte M, Fagiuoli S. Salvagnani M, Naccarato R. Hormonal treatment of hepatocellular carcinoma. J Hepatol 1991; 12: 402. 2 Forbes A, Wilkinson ML, Iqbal MJ, Johnson PJ, Williams R. Response to cyproterone acetate treatment in primary hepatocellular carcinoma is related to fall in free Sa-dihydrotestosterone. Eur J Cancer Clin Oncol 1987; 23: 1659-64.
407 sponse when tamoxifen was combined with norethisterone (4) but not when it was used alone (5). Therefore, while agreeing with Farinati et al. that manipulation of steroid hormones may have some part to play in prevention or treatment of hepatocellular carcinoma, we do not believe, in the absence of data to demonstrate it, that the tamoxifen effect demonstrated in their own studies published in the Journal in 1990, necessarily demonstrates an anti-oestrogenic effect. Tamoxifen is well known to have cytotoxic effects separate from its anti-oestrogenic effects. Mark L. Wilkinson and Alastair Forbes Char@ Cross Hospital, Fulham Palace Road, London W6, United Kingdom
3 Friedman MA, Demanes DJ. Hoffman PG. Hepatoma: hormone receptors and therapy. Am J Med 1982; 73: 362-5. 4 Thinchet J-C, Roudil F, Vaysse J, Beaugrand M. Effet dune association Tamoxifene-norethisterone chez 16 malades atteints du carcinome hepatocellulaire. Gastroenterol Clin Biol 1985; 9: 455. 5 Paliard P, Clement G, Saez S, Cheval J, Partensky C. Traitement du carcinome hepatocellulaire par le tamoxifine. Gastroenterol Clin Biol 1984; 8: 680-l.
HEPAT 00981
ronic Cnronic hepatitis D virus (HDV) infection is known to cause severe and rapidly progressive liver disease, and interferon-a has been reported recently to be effective in a proportion of these patients (l-3). In the U.K., we encountered a number of problems in treating patients with chronic HDV infection. Firstly, most of our patients were intravenous drug users or ex-addicts which was in accord to our recent survey in Southeast London (4). Four out of ten patients were found to be poorly compliant and the intermittent use of interferon-a increased the incidence and severity of side-effects from the drug. Secondly, the necessity to inject interferon-a presented particular psychological stresses to those who have overcome a past addiction. In addition, the malaise and depression caused by interferon-a led to intermittent re-use References Bonino F, Negro F, Baldi M, et al. The natural history of chronic delta hepatitis. In: Rizzetto M. Germ JL. Purcell RI-I. eds. The Hepatitis Delta Virus and Its Infection. New York: Alan R Liss, 1987; 145-52. Rosina F, Pintus C, Rizzetto M, et al. Long-term interferon treatment of chronic hepatitis D: a multicentre Italian study. J
of addictive drugs in three out of eight ex-addicts. Ihirdly, five out of 10 patients treated had advanced liver disease and with low platelet count (range:50144.109/l). In two patients, interferon-a
therapy induced
a further drop in the platelet count to less than 20.109/l which precluded further interferon-a injections. These problems indicate the difficulty of treating Thronic HDV infection in drug users, and also highlights the need for greater attention to the psychological needs of these patients undergoing interferon-a therapy.
Ruth King, Johnson Y.N. Lau and Roger Williams institute of Liiw Studies, King’s College Scizool of Medicine and Dentistry, London SES, United Kingdom
Hepatol 1990; 11: S149-50. 3 Di Bisceglie AM, Martin P. Lisker-Melman M. et al. Therapy of chronic delta hepatitis with interferon alpha-2b. J Hepatol 1990: 11: s151-4. 4 Smith HM, Alexander G.IM, Webbs G, McManus T, McFarlane IG, Williams R. Hepatitis B and delta infection among ‘at risk’ populations in South East London. J Epidemiol Community Health 1991; in press.
406
A
12345678
B
Fig. 1. PCR products of HCV cDNA using the d94, d95, Nl and N2 set of primers (A) and the NCR set of primers (B) on gel electrophoresis. Lane 1, molecular weight marker; lane 2, positive control; lane 3, negative control; lane 4, sample from patient with proven hepatitis B infection: and lanes 5-8, samples from patients 1, 2, 3 and 4. respectively.
round. Using the d94, d95, Nl, N2 set of primers (2) we performed the first round of PCR consisting of 35 cycles of denaturation for 80 s at 95 “C, annealing for 40 s at 58 “C and extension for 60 s at 72 “C. Ten ~1 of amplified material were subjected to the second round of PCR consisting of 25 cycles of denaturation for 60 s at 95 “C, annealing for 60 s at 46 “C and extension for 60 s
TO THE EDITOR
72 “C. Using the NCR 1,2,3 and 4 set of primers (3) we performed the first PCR round consisting of 35 cycles of denaturation for 60 s at 95 “C, annealing for 60 s at 50 “C and extension for 60 s at 72 “C. The second PCR round consisted of 30 cycles of denaturation for 60 s at 94 “C, annealing for 60 s at 46 “C and extension for 60 s at 72 “C. The last cycle of each PCR round was followed by an extension of 7 min at 72 “C. Reagents without template and RNA isolated from a liver with proven hepatitis B were used as negative controls. As a positive control we used RNA isolated from the liver of a patient who had a partial hepatectomy because of a hepatocellular carcinoma and a nonalcoholic cirrhosis. The patient’s serum had HCV antibodies, tested by an enzyme immunoassay (Abbott Wiesbaden). By using the first set of primers we amplified HCV cDNA sequences in three of six cases (Fig. lA), while by using the second set of primers we amplified HCV cDNA in four of six cases (Fig. 1B). The use of this method to detect HCV RNA in material from liver needle biopsies may help in the definite diagnosis of HCV. Since only part of the material is enough for the detection of HCV RNA, this could complement the diagnosis of HCV based upon morphological criteria. at
PCR
References 1 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9. 2 Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, et al.
Ioannis D. Diamantis’, Christine E. McGandy’, Irmgard Pult’, Jean-Jacques Widmann2, Fred Gudat’ and Leonardo Bianchi’ ‘Institute of Pathology, University Hospital, Schdnbeinstrasse 40, CH-4003, Base1 and ‘Institute of Pathology, University Hospital, CMU, CH-1200 Geneva, Switzerland
Detection of hepatitis C viral sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. 3 Garson JA, Ring C, Tuke P, Tedder RS. Enhanced detection by PCR of hepatitis C virus RNA. Lancet 1990; 336:878
HEPAT 00976
Hormonal treatment of hepatocellular In response to the letter of Farinati et al. (1) about hormonal manipulation of hepatocellular carcinoma (HCC), we would like to correct one small omission. In their discussion of anti-androgen therapy, they stated that they were aware of three studies. We would like to draw their attention to a fourth, in which the progestogenie anti-androgen cyproterone acetate was administered to 25 cirrhotic patients with HCC (23 male) (1).
carcinoma
There was an objective response to therapy in five patients, and furthermore, the response appeared to be correlated with a fall in free levels of the potent androgen Sa-dihydrotestosterone. In this study, while the compounding variables or ‘biases’ referred to by Farinati et al. were not entirely eliminated, the aetiology of cirrhosis, Gkuda grade and, in most patients, the pre- and post-treatment hormonal
LETTERS
TO THE EDITOR
status were clearly recorded. If normal criteria for c motherapy of solid tumours are adopted, the trial showed a modest, but useful 20% response rate. If we adopt the criteria of Farinati et al., that the only response that can be expected is stable disease, then 12/25 (48%) had a measurable response to cyproterone acetate. However, we do not believe that this is a reasonable approach and prefer to assess response in a conventional manner. Furthermore, in published studies the response of HCC to sex hormones is not limited to anti-androgens and anti-oestrogens. Progestogens may also be important. The early study of Friedman et al. (3), which Farinati et al. suggested showed a response to tamoxifen, in fact used a megesterol acetateimedroxy progesterone combination, and two further studies have shown a reeferences 1 Farinati F, De Maria M, Chiaramonte M, Fagiuoli S. Salvagnani M, Naccarato R. Hormonal treatment of hepatocellular carcinoma. J Hepatol 1991; 12: 402. 2 Forbes A, Wilkinson ML, Iqbal MJ, Johnson PJ, Williams R. Response to cyproterone acetate treatment in primary hepatocellular carcinoma is related to fall in free Sa-dihydrotestosterone. Eur J Cancer Clin Oncol 1987; 23: 1659-64.
407 sponse when tamoxifen was combined with norethisterone (4) but not when it was used alone (5). Therefore, while agreeing with Farinati et al. that manipulation of steroid hormones may have some part to play in prevention or treatment of hepatocellular carcinoma, we do not believe, in the absence of data to demonstrate it, that the tamoxifen effect demonstrated in their own studies published in the Journal in 1990, necessarily demonstrates an anti-oestrogenic effect. Tamoxifen is well known to have cytotoxic effects separate from its anti-oestrogenic effects. Mark L. Wilkinson and Alastair Forbes Char@ Cross Hospital, Fulham Palace Road, London W6, United Kingdom
3 Friedman MA, Demanes DJ. Hoffman PG. Hepatoma: hormone receptors and therapy. Am J Med 1982; 73: 362-5. 4 Thinchet J-C, Roudil F, Vaysse J, Beaugrand M. Effet dune association Tamoxifene-norethisterone chez 16 malades atteints du carcinome hepatocellulaire. Gastroenterol Clin Biol 1985; 9: 455. 5 Paliard P, Clement G, Saez S, Cheval J, Partensky C. Traitement du carcinome hepatocellulaire par le tamoxifine. Gastroenterol Clin Biol 1984; 8: 680-l.
HEPAT 00981
ronic Cnronic hepatitis D virus (HDV) infection is known to cause severe and rapidly progressive liver disease, and interferon-a has been reported recently to be effective in a proportion of these patients (l-3). In the U.K., we encountered a number of problems in treating patients with chronic HDV infection. Firstly, most of our patients were intravenous drug users or ex-addicts which was in accord to our recent survey in Southeast London (4). Four out of ten patients were found to be poorly compliant and the intermittent use of interferon-a increased the incidence and severity of side-effects from the drug. Secondly, the necessity to inject interferon-a presented particular psychological stresses to those who have overcome a past addiction. In addition, the malaise and depression caused by interferon-a led to intermittent re-use References Bonino F, Negro F, Baldi M, et al. The natural history of chronic delta hepatitis. In: Rizzetto M. Germ JL. Purcell RI-I. eds. The Hepatitis Delta Virus and Its Infection. New York: Alan R Liss, 1987; 145-52. Rosina F, Pintus C, Rizzetto M, et al. Long-term interferon treatment of chronic hepatitis D: a multicentre Italian study. J
of addictive drugs in three out of eight ex-addicts. Ihirdly, five out of 10 patients treated had advanced liver disease and with low platelet count (range:50144.109/l). In two patients, interferon-a
therapy induced
a further drop in the platelet count to less than 20.109/l which precluded further interferon-a injections. These problems indicate the difficulty of treating Thronic HDV infection in drug users, and also highlights the need for greater attention to the psychological needs of these patients undergoing interferon-a therapy.
Ruth King, Johnson Y.N. Lau and Roger Williams institute of Liiw Studies, King’s College Scizool of Medicine and Dentistry, London SES, United Kingdom
Hepatol 1990; 11: S149-50. 3 Di Bisceglie AM, Martin P. Lisker-Melman M. et al. Therapy of chronic delta hepatitis with interferon alpha-2b. J Hepatol 1990: 11: s151-4. 4 Smith HM, Alexander G.IM, Webbs G, McManus T, McFarlane IG, Williams R. Hepatitis B and delta infection among ‘at risk’ populations in South East London. J Epidemiol Community Health 1991; in press.
