quantitative analysis of hepatitis c virus rna in liver biopsies by competitive reverse...

Journal of Hepatology 1995; 23: 403411 Printed in Denmark . AN rights reserved

Copyright 0 Journal of Hepatology 1995

Journal of Hepatology

ISSN 0168-8278

Quantitative analysis of hepatitis C virus RNA in liver biopsies by competitive reverse transcription and polymerase chain reaction

Gabriele Grassi’, Gabriele Pozzato2, Michele Moretti2 and Mauro Giacca’

‘International Cemtre for Genetic Engineering and Biotechnology, AREA Science Park, Padriciano, Trieste and 21nstitute of Patologia Medica, University of Trieste Medical School, Trieste. Italy

Background/Aims: The determination of HCVRNA concentration in liver samples is likely to provide interesting insights for the study of disease progression and for evaluation of the efficacy of anti-viral therapy. Methods: A procedure was developed for the precise quantification of HCV-RNA in liver biopsies, based on the competitive reverse transcription-polymerase chain reaction technology. This competitive assay con- sists of the co-amplification of the target RNA with known amounts of a competitor RNA molecule containing the same sequence as the target plus an insertion in the middle, allowing resolution of the two amplification products by gel electrophoresis. Results: The amounts of HCV-genomic-RNA and @-actin mRNA (the latter being used as an internal standard to overcome the problem of reproducibility of quantitative RNA extraction) were evaluated in liver biopsies of 15 patients affected by hepatitis C

T E DEVELOPMENT of laboratory assays for the detection of serum antibodies against the hepatitis

C virus (HCV), the major causative agent of non-A non-B hepatitis, has facilitated the diagnosis of viral infection. The presence of serum reactivity, however, does not allow discrimination between present (active) and past (resolved) infection; for this purpose, only the direct detection of viral RNA proves current viral replication. Since the amount of HCV-RNA in serum is very low, its detection requires the extraordinary sensitivity of the reverse transcription-polymerase chain reaction (RTPCR) technique. In fact, by its utilization, it could be demonstrated that the majority of anti-HCV antibody positive patients are indeed carriers of HCV,

Received 2 August 1994; revised 4 January; accepted 6 March 199s

Correspondence: Dr. Mauro Giacca, ICGEB, Padriciano, 99, 34012 Trieste, Italy

virus-positive chronic liver disease at the time of diagnosis. All the patients underwent a-interferon therapy for 6 months and were subsequently followed for at least 1 further year after the end of treatment. Viral RNA concentration (which ranged from 2 to 2.7 X 105 HCV-RNA molecules per 106 pactin molecules) directly correlated with the efficacy of treatment, indicating that low levels of viral replication in the liver are associated with a poor response to therapy. Conclusions: This study suggests that the determination of viral load in the liver is an important prognostic tool for the prediction of the efficacy of a-interferon therapy.

Key words: Chronic liver disease; Competitive reverse transcription-polymerase chain reaction; Hepatitis C virus; Interferon therapy. 0 Journal of Hepatology.

and that carriers of HCV-RNA could also be found among anti-HCV negative patients (1).

Due to its extraordinary sensitivity, RTPCR is the method of choice for the detection of low amounts of nucleic acids. However, since the yield of the amplification reaction is poorly reproducible, it cannot provide quantitative information. Precise quantitation of HCV-RNA concentration in serum and tissues, on the contrary, is needed for monitoring disease progression and response to therapy. It should be considered, in fact, that there are compelling reasons to devise means to identify predictive factors on the long-term outcome of the disease, since a relevant fraction (from 60 to 80% (2,3)) of patients infected by HCV develop chronic hepatitis, and most of them progress to cirrhosis, por- tal hypertension, hepatic failure and, often, hepatocellular carcinoma. The most reliable approaches to quantitative RT-PCR are those based on reverse-transcription and amplification of the sample, together

403

G. Grassi et al.

with a reference template sharing almost the totality of the target sequence, and thus the two templates are subjected to the same experimental variables in both reactions (competitive RTPCR (4)). Since the competitor is added in known amounts, the concentration of the unknown target RNA can easily be derived. Ac- cording to this method, we have developed a competitive RTPCR procedure for the quantitation of HCV- RNA by the construction and utilization of RNA competitors containing the target viral sequence plus 20 additional nucleotides in the middle, in order to allow easy resolution of the two molecular species (target and competitor) simply by gel electrophoresis (5).

Several authors have quantitatively determined the amount of HCV-RNA in serum (6-8); these studies, however, suffer from the poor quantitative and qualita- tive reproducibility of the RNA extraction procedures from biological samples (especially because of the possibility of RNA degradation, even in the most con- trolled experimental conditions (9)) in the absence of other reference RNA standards within the extracted sample. Furthermore, since HCV infection, although systemic, mostly affects the liver, it appears that the determination of HCV-RNA concentration in liver samples could provide a better tool for the search for prognostic markers for disease progression and antiviral therapy evaluation. In this study, we analyze the amount of HCV-RNA in liver biopsies, utilizing the amount /I-actin mRNA as an internal control for RNA concentration and integrity. The HCV-RNA concentration is then correlated with the histological findings and with the outcome of a-interferon therapy.

Materials and Methods Patient selection Fifteen consecutive patients with HCV chronic hepatitis, referred to the Trieste Medical School, were en- rolled in this study between January 1992 and July 1992, from a group of 154 potential candidates; in- formed consent was obtained from each patient. In- clusion criteria were: 1) persistent elevation of serum ALT (at least twice the upper normal limit) for more than 12 months; 2) presence of anti-HCV antibodies, as evaluated with a second generation, enzyme-linked immunosorbent assay kit (ORTHO-HCV, Ortho Diag- nostic System, Raritan, NJ, USA); positive samples were confirmed by immunoblotting (RIBA, Chiron Corp., Emeryville, CA, USA); 3) availability of a liver biopsy performed within 1 year from enrollment in the study, with histological findings compatible with chronic persistent or active hepatitis (with or without cirrhosis). Exclusion criteria were: 1) any positive marker of HBV infection; 2) history of daily alcoholic intake >40 g; 3) intravenous drug abuse within 6 months; 4) serological evidence for autoimmune hepatitis (antinuclear, anti-smooth muscle and/or anti- LKM antibodies); and 5) positivity of anti-human immunodeficiency virus (HIV) antibodies. Before entry into the study, hemochromatosis, Wilson’s disease, hemophilia, and a- 1 -antitrypsin deficiency were ex- cluded by conventional tests. None of the patients had a history of ascites, variceal bleeding (as assessed by gastroscopy) or encephalopathy. Women of childbear- ing age used contraceptive methods during treatment.

A history of parenteral exposure was obtained in

TABLE 1

Clinical, biochemical, and histological tindings of patients. The ALT, AST, and GGT serum activities are expressed as U/l. CAH: chronic active hepatitis; CPH: chronic persistent hepatitis; C: cirrhosis

Patient Age Sex Source of Histology ALT AST infection (NV -W (NV -QW ET<*21

1 64 F unknown CAH+C 97 15 99 2 30 F iv. drugs CAH 457 216 59 3 34 M i.v. drugs CPH 15 61 57 4 68 M unknown CPH 63 52 19 5 22 M i.v. drugs CPH 170 87 27 6 69 F unknown CPH 13 47 11 I 60 F unknown CAH 350 266 85 8 68 M unknown CAH 155 160 58 9 69 M transfusion CAH+C 82 15 16

10 70 M unknown CAH 109 91 119 11 29 M unknown CAH 202 82 27 12 59 M unknown CPH 314 204 607 13 29 F unknown CPH 98 15 59 14 24 M unknown CPH 169 65 31 15 65 M unknown CPH 135 18 12

Mean?s.d. 51%19 1705116 108277 862142

404

less than half of the patients. The clinical, biochemical, and histological findings of patients are given in Table 1.

Study design Since all previous experiences showed that patients only rarely recovered without therapy, the selected patients were not randomized and were treated in an open manner. All the subjects received cr2a-recombinant interferon, 6 million units subcutaneously three times a week for 4 weeks, followed by 3 million units three times a week for 5 months. After cessation of treatment, each patient was followed for a further 12 months.

Blood samples were obtained before the beginning, every 2 weeks during the first month of therapy, every 4 weeks during the subsequent 5 months of treatment and the 12 months of the follow-up period after cessation of treatment. Blood samples were analyzed for erythrocyte, leukocyte and thrombocyte count determination, and measurement of serum urea, creatinine, ALT, AST, bilirubin, gamma-glutamyl-transpeptidase, and alkaline phosphatase concentrations. HIV serol- ogy was performed every 6 months.

Histological evaluation of the liver In each patient, liver biopsy was performed using a Menghini-like needle with an internal diameter of 1.8 mm. The specimens were cut into two equal parts: one half, to be utilized for HCV-RNA titration, was im- mediately frozen in liquid nitrogen and subsequently conserved at -80°C; the other half was fixed in 10% buffer formalin and stained with hematoxylin/eosin, and, for reticulum, with Gomori stain. Liver biopsies were coded and graded blindly by two experienced pathologists.

RNA isolation Frozen liver biopsy samples (mean weight 27 mg, see Table 2) were homogenized in a 500 ~1 Dounce tissue grinder (Wheaton Scientific, Millville, NJ) in 4 M guanidine thiocinate, 25 mM sodium citrate, pH 7.0, 0.5% Sarcosyl, 0.1 M P-mercaptoethanol. Total RNA was extracted according to the method of Chomczyn- ski & Sacchi (lo), with the following minor modifi- cations: RNA was extracted once with phenol-chloroform and precipitated with isopropanol; the pellet was washed in 70% ethanol, resuspended in 60 ~1 of TE (Tris 10 mM, pH 8 and EDTA 0.1 mM) and digested with 1 U of an Rnase-free DNase I (Promega, Madi- son, WI) for 15’ at 37°C.

HCV-RNA quantitation in liver biopsy

TABLE 2

Response to therapy and HCV titration in liver biopsies. The amount of /Sactin and HCV RNA molecules detected in each biopsy is indicated. According to the response to a-IFN therapy - see text for the criteria adopted -, patients are classified as Responders (R), Non- Responders (NR), or Relapsers (REL). ma.: not amplifiable

Patients Response b-actin HCV Weight of to a-IFN (molecules (molecules biopsy

x 106) X103) (mg)

1 NR 100 750 27.2 2 R 12 180 27 3 REL 100 2000 26.6 4 R 100 2500 46.1 5 NR 1000 2 34.1 6 NR 198 1080 28.6 7 NR 140 1260 16 8 NR 140 350 29 9 NR 1050 10500 15

10 REL 105 315 20 11 R 23 6300 30 12 R 10 630 22 13 R 420 n.a. 30 14 NR 23 315 30 15 R 315 3500 30

Mean+s.d. 2492334 212022955 27.4k7.5

Construction and quant@cation of competitor RNA Competitor RNA fragments for quantification of /?- actin mRNA and HCV-RNA from liver biopsies were constructed by a modification of an already described protocol (5,ll) utilizing the recombinant PCR method- ology (12,13). The procedure for competitor RNA construction is schematically shown in Fig. 1. For each RNA, five primers were synthesized (generically named A, T7-A, taill-D, tail2-C and B in Fig. 1 panel A). Primers A and B are used for PCR amplification from the cDNAs, the latter being complementary to the RNA and thus used also for RT priming.

Primer T7-A contains at its 5’ end the recognition sequence for T7 RNA polymerase. Primers taill-D, tail2-C are composed of a 3’-portion corresponding to contiguous DNA sequences on opposite strands of the cDNA (C and D respectively) and of two 5’-tails bear- ing two 20 nt sequences unrelated to the target and complementing each other (tail2 and tail1 respectively). The sequences and PCR amplification parameters for these primers are given in Fig. 2. The procedure for competitor construction is schematically shown in Fig. 1 panel B. After cDNA synthesis obtained by priming with oligo B, two separate amplifi- cations were carried out, with oligos T7-A and tail2- C, and with oligos B and taill-D. The amplification products obtained were resolved by gel electrophoresis, eluted from the gel, denatured, mixed together and re- annealed by virtue of the complementary tails tail1 and tai12. The annealed product was amplified with primers

405

G. Grassi et al.

A

) RNA

B

c3 cDNA synthesis b!j priming with dig0 B

PCR amplification with primen T7-A and tail2-C

PCR ampli@don with primers B and tail&D

PCR amplification with primers T7-A and B

Fig. I. Schematic representation of the method utilized for the construction of RNA competitors for competitive RT- PCR. Panel A. Localization of primers for RT-PCR and competitor construction. The actual DNA sequences for the generically namedprimers A, B, C, D, and the 5’ tails taill, tai12, and l7 are reported in Fig. 2 for both the Bactin and HCV ampltfication sets. Panel B. Procedure for the construction of competitor RNAs. A detailed description of the method is reported in the Methods section.

T7-A and B. The DNA fragment obtained by this procedure corresponds to the original cDNA plus 20 bp in the middle and a T7-RNA polymerase recognition sequence at the 5’ end of the coding strand. Forty nan- ograms of this fragment were used as a template for in vitro transcription with T7-RNA polymerase using a commercial in vitro transcription kit (Promega). One microliter of [32)]-UTP (Amersham, UK; 3000 Ci/ mmol; 10 mCi/ml) was included in the in vitro transcription assay, corresponding to 7 X lo6 cpm, as exper- imentally evaluated by Cerenkov counting in a j?- counter. After the transcription reaction, the template DNA was removed either by DNase I digestion or by resolution of the synthesized RNA by denaturing gel electrophoresis (14) and elution from the gel. The radioactivity of an aliquot of the purified competitor RNA preparation was measured, and its concentration was evaluated from the final specific activity of the

T7: S’-CGGGATCCGGATCCL4ATACGACTCA~ATAGGGAGA-3 bill: S-ACCTGCAGGGATCCGTCGAC-3 tail2: S-GTCGACGGATCCCTGCAGGT-3’

Primer set: &a&in exon 2 exon 3

x ‘-, h

BAI BA4

Pmdwtdz+x 225bp PCR cyck: 94’C-30”R6°C-30”n2’C-30”

primerA: BAI: S-CATGTGCAAGGCCGGClTCG-3 B: BA4: SGAAGGTGTGGTGCCAGP3 c: BAk tail2 + S-CTGGTGCCEGGGCGCCCCA-3 D: BAl+: tail1 + S-GGCGTGATGGTGGGCATGGG-3

1279-1260 1414-1433

Primer set: HCV

c B 8 P -9 b

-+- Cl c2

Product s&x: 2.55 bp PCR cycle: 94”C-3CJV56”C-30”~2°C-u)”

primerA: Cl: 5’ GCCA’EGCG~AGTATGAGT-3 poailiax 73-92 B: C2: 5’ -CACGGTcTAcoAGACCl’CCC-3 328-309 c: c2+: taila+S-C~ AGACCACIAT-3 15~131 D: Cl+: tail1 + S-TGAGTACACCGGAA’ITGCCG-3 151-170

Fig. 2. Sequence and localization of the oligonucleotides utilized in this work and PCR parameters. For each amplification set (p-actin and HCV) the sequences of the generically named primers A and B of Fig. I are reported. These primers were used for amplification of the sample RNAs. Primers C and D contain the specific sequence shown for each set plus a 5’ tail, as indicated. The locations of the primers on the RNA sequence are schematically indicated; numbering is relative to file humaccybb of GenBank for #I- actin and to the HCV sequence reported in ref (16).

labelled UTP and the number of nucleotides incorpor- ated per molecule.

The actual oligonucleotide sequences for the T7, taill, and tail2 tails, and for primers A, B, C, and D for both amplification sets are reported in Fig. 2. Primers for HCV amplification were chosen in the conserved 5’-non-coding region of the HCV genome (15,16). Primers for /3-actin amplification were from exons 2 and 3 of the /Sactin gene, in order to avoid amplification from contaminating genomic DNA.

Quantitation of HCV and p-actin RNAs in liver

biopsies

RNA from liver biopsies was submitted to competitive RT’-PCR experiments using the j?-actin and HCV competitor RNAs. In different reactions, 3 ~1 of each competitor, containing increasing amounts of molecules, were mixed with 1 ,ul of RNA sample (corresponding to l/35 to l/60 of the total RNA extracted from each biopsy) and reverse transcribed in 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgC12, 10 mM DTT, 20 U RNasin (Promega), 1 mM each dNTP 25 pmol of primer and 200 U MuLV reverse transcriptase (BRL,

406


Gaithersburg, MD; final reaction volume: 20 ~1). RNA and primers were annealed in reaction buffer and RNasin for 8 min at 65°C and then cooled on ice before addition of nucleotides and enzyme. The reaction mixture was allowed to proceed for 45 min at 37°C and then stopped by heating at 95°C for 5 min. Four microliters of the RTreaction were added to 76 ~1 of a solution containing 250 ,uM each dNTP, 45 pmol of both antisense and sense primers, 2.5 U of Tug polymerase (Amplitaq, Perkin Elmer Cetus, Norwalk, CT), 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgC&, 0.01% gelatin. The reaction mixtures (80 ~1) were subjected to 40 cycles of amplification in a programmable thermal cycler (Perkin Elmer Cetus) using the cycle profiles indicated in Fig. 2. The PCR products were then resolved on an 8% polyacrylamide gel, stained with ethidium bromide, photographed, and the different intensities of the bands (competitor and cDNA) were estimated by densitometric scanning.

Statistical analysis

Statistical analysis was performed with the SPSS pack- age (17). For continuous variables, one-way analysis of variance between two groups was calculated to pro- duce the Snedecor’s F-factor. Categorical variables were analyzed with a Pearson X2. Discriminant analysis was performed by using the Mann-Whitney and Wilcoxon rank sum tests. Partial associations were determined using the hierarchical log-linear analysis in a multiway cross tabulation.

Results Fifteen patients affected by HCV-positive chronic liver disease were analyzed in this study, in order to investi- gate the correlation between the amount of HCV-RNA in the liver, the severity of the disease, and the effectiveness of a-interferon therapy.

The clinical, biochemical, and histological findings for the patients are shown in Table 1. Liver biopsies were obtained at the time of diagnosis, before cr-interferon therapy; histological examination revealed a variable degree of chronic liver disease, ranging from mild persistent hepatitis to severe chronic active hepatitis complicated by the presence of cirrhosis.

All these patients received a2a-recombinant interferon therapy, according to the protocol reported in the Methods section. The response to treatment was classified according to the evolution of serum ALT levels. Patients who did not normalize serum ALT were defined as “Non-responders”; patients with normaliza- tion of ALT during therapy lasting for at least 12 months after discontinuation of treatment were classified as “Responders”; patients who responded initially

but showed an increase of serum ALT activity after cessation of therapy were classified as “Relapsers”.

At the end of the follow-up period, only five (35.7%) normalized the aminotransferases serum levels perma- nently and were classified as Responders, while the majority were Non-responders (50%) or Relapsers (14.3%, Table 2).

Quantitation of the amount of HCV-RNA in liver biopsies was obtained by a competitive RTPCR procedure (18,19), consisting in the co-amplification of the extracted RNA samples with known amounts of competitor RNA molecules containing the same sequence as the target molecule, except for a small insertion of 20 nucleotides in the middle. By this procedure, the two RNA species compete for reverse-transcription and PCR amplification; as a consequence, any predictable and unpredictable variable affecting either reaction has the same effect on both templates. At the end of the reaction, the amplification products for the two species are resolved by gel electrophoresis and the ratio between the amounts of the amplified products exactly reflects the ratio between the amounts of the two RNA species initially present in the reaction (5,20-23).

The amounts of HCV RNA and /?-actin mRNA molecules were evaluated in the RNA samples extracted from each biopsy. Quantitation of the p-actin transcript was utilized to evaluate the efficiency of the extraction procedure and obtain a measure of RNA concentration, assuming that this transcript is expressed at constant levels in different cell types and conditions. Two RNA competitors were constructed, corresponding to regions in exons 2 and 3 of the p- actin gene and to the 5’-non-coding region of the HCV genome (see Fig. 2 for primer localization; the procedure utilized for competitor construction is schematically shown in Fig. 1 and detailed in the Methods section). The primers utilized for HCV amplification cor- respond to nucleotide boxes which are very conserved in different HCV types (24,25); in fact, using these primers, only in one patient was it not possible to de- tect any amplification product (patient 13, Table 2).

An example of the competitive RT’-PCR procedure adopted is shown in Fig. 3. Different amounts of competitor RNAs for HCV and /I-actin (panels A and B, respectively) were added to a fixed amount of each sample in two independent quantitation experiments, and submitted to reverse-transcription and PCR amplification. The ratio between the two amplification products, as evaluated by densitometric scanning of the ethidium bromide-stained gels, was then plotted against the amount of competitor molecules initially added to the sample. As expected, the points are fitted by a straight line; the equivalence point (i.e. the con-

407

G. Grassi et al.

competitor (molecules)

Fig. 3. Competitive RT-PCR. An example of a competitive RT-PCR experiment is shown for the quantitation of HCV RNA (panel A) and /Sactin mRNA (panel B) molecules for the RNA sample of patient 4 (see Table I). A3xed amount of RNA sample was reverse-transcribed and co-amplified with increasing amounts of competitor RNA molecules, as indicated on top of the gels, and the ampltjkation products resolved by polyacrylamide gel electrophoresis, stained with ethidium bromide and photographed. The ratio between the amplification products for the two species (as evaluated by densitometric scanning) was then plotted against the amounts of competitor added (lower part of each panel). The data areJitted by a straight line; the point corresponding to a I:1 ratio between the two products indicates the amount of RNA molecules initially present in the sample.

centration of competitor giving rise to a 1: 1 competitor:virus ratio) exactly corresponds to the number of viral RNA molecules initially present in the sample. Routinely, two quantification experiments were carried out, the first one with ten-fold dilutions of competitor in order to estimate roughly the amount of target in the sample, and the second one with two-fold dilutions in the detected range for a more precise estimation.

The results of the quantitations are shown in Table 2 for /I-actin and HCV RNAs. For both RNAs, the absolute number of molecules detected is very variable from biopsy to biopsy (ranging from 59 to 7X lo5 molecules per mg of tissue for HCV, patients 5 and 9, respectively, and from 4X lo5 to 7X 10’ for @-actin, patients 2 and 9, respectively). These values reflect the different amounts of viral replication, but also the different cellular contents of each biopsy, and suggest that the absolute determination of HCV concentration in the absence of an internal reference mRNA such as /3- actin is potentially misleading.

In one patient (number 13, Table 2) it was not possible to obtain amplification with the HCV primers.

This patient is positive for the presence of anti-HCV antibodies and for the RTPCR amplification of the core region performed according to Okamoto and co- workers (26). Therefore, the lack of amplification for the 5’-non coding region is probably attributable to sequence variability in the primer recognition sites.

The statistical analysis on HCV quantitation was performed taking into account the following parameters: age, sex, source of infection, ALT, histology, response to antiviral therapy, absolute HCV amounts in liver biopsies, HCV concentration expressed as number of copies per gram of liver tissue and HCV concentration expressed as number of HCV copies per number of p-actin copies. Non-responders and Relapsers, as well as patients with chronic active hepatitis (CAH, Table 1) and chronic active hepatitis plus cirrhosis (CAH+C), were lumped together for statistical analysis. The analysis of the variance did not show any correlation between the different parameters, with the exclusion of the response to a-interferon therapy and the ratio between HCV copies and D-actin copies, which yielded a Snedecor’s F of 3.49, near to statistical sig-

408

nificance (F probability 0.08). The discriminant analy- knowledge of the course of HCV viremia. Additionally, sis showed that the response to antiviral therapy was it should be considered that quantitation of viral nuc- directly associated with the ratio between HCV and /I- leic acids in plasma samples is prone to experimental actin, with a W of 57, Z of -2,6 and a two-tailed p of variability, due to the absence of any available internal 0.0093. The hierarchical log linear analysis did not find reference standard to assess the efficiency of the RNA clearly clustering variables. extraction procedure.

These data suggest that the amount of viral RNA in the liver is inversely correlated to the effectiveness of a- interferon therapy: patients responding to therapy have higher liver titers as compared to patients not responding to therapy. The HCV titer, expressed as the amount of HCV RNA molecules per lo6 /Sactin molecules, is shown in Fig. 4, where the patients are grouped according to the response to therapy.

Discussion Several authors have utilized quantitative techniques to determine the viral load in serum samples of HCV-infected patients with liver disease of different severity. The results of these studies are quite contradictory For example, Hagiwara and co-workers (8) could not find any difference in the serum HCV-RNA concentration at different stages of chronic liver diseases, whereas Kato et al. (7) showed higher levels of HCV-RNA in patients with advanced disease (cirrhosis and hepatocellular carcinoma). The conflicting results obtained in these studies are probably attributable to our still poor

Since the most relevant site for virus-induced disease is the liver, it appears of particular relevance to determine the viral load in hepatic biopsies, trying to correlate the amount of viral replication with prognostic markers for disease progression and for monitoring the efficacy of antiviral therapy. For this purpose, we have utilized a competitive RTPCR procedure involving the simultaneous determination of the amount of HCV and /Sactin RNAs in liver biopsies by co-amplification of the sample with two easily differentiable competitor RNA fragments. This procedure: 1) monitors the efficacy of RNA extraction, as measured by the number of /3-actin mRNA molecules (assuming that the cellular concentration of this transcript does not vary signifi- cantly in different pathophysiological conditions of chronic liver disease); 2) is independent from all the experimental variables affecting either the reverse-transcription or the amplification steps, since these variables do not influence the ratio between the two amplification products; 3) is independent of the total reaction yield; 4) does not require a further restriction en-

270000

260000 60000


patients: 11 12 4 2 15 , 3 10, 14 9 7 1 6 8 5 1 I I I

Responders Relapsers Non responders

Fig. 4. Viral load in the liver and response to a-interferon therapy. The number of HCV RNA molecules detected in liver biopsies are shown for each patient, numbered according to Table I, expressed per million p-actin mRNA molecules in the same samples. Patients are grouped according to the response to therapy, as indicated.

409

G. Grassi et al.

zyme digestion step after amplification, thus avoiding the problem of partial digestions and heteroduplex for- mation (27).

The results obtained by the quantitation of viral RNA in liver biopsies of 14 patients receiving a-interferon therapy clearly indicate that liver histology, age, sex, source of infection, and pre-treatment ALT do not correlate with the levels of viral load. On the contrary, there is a direct correlation between the response to antiviral treatment and the HCV genome level. It should be observed that this correlation is evident only if the absolute number of HCV copies detected in each biopsy is expressed as a ratio of the amount of p-actin molecules, again stressing the importance of an internal reference control to monitor the efficacy of RNA extraction.

These results appear to be in contrast to previous observations indicating higher HCV serum levels in patients with advanced liver disease or not responding to therapy (7). This contrast, however, is only apparent, as few conclusive reports are available about HCV titration in the liver, and a relationship between serum and liver levels of HCV has not been formally estab- lished so far. It should also be considered that the higher levels of HCV in serum of patients with severe liver disease could also be explained by the presence of extra-hepatic sites for HCV replication, since the infection of blood mononuclear cells (B- and T lymphocytes and monocytes) can lead to viral produc- tion from bone marrow and lymphoid organs. Accord- ingly, it has been reported recently that only 1 to 10% of hepatocytes appear to be infected, even in patients with very advanced liver disease (28).

Although the possibility cannot be ruled out that the lower amounts of HCV RNA in patients not responding to therapy could result from different cellular populations .in their liver biopsies (prevalence of non- infected fibroblasts or lymphocytes, expressing the /I- actin mRNA), the most likely explanation for the correlation between high amount of virus and response to therapy is that the efficacy of a-interferon is higher when the virus replicates at a high rate. In this respect, it should be interesting to evaluate the amount of negative strand molecules in the same tissues as a better tool to evaluate HCV replication (work in progress).

In conclusion, this study suggests that a low amount of HCV-RNA in the liver is associated with a poor response to antiviral therapy. Further studies are obvi- ously needed to extend this observation to a larger number of patients, and to compare directly the results of quantitation of the levels of HCV viremia to viral load in the liver within the same population of patients. The results of such studies will be of major im-

410

portance for the understanding of the pathogenetic mechanism of disease and for the clinical management of patients.

Acknowledgements G.G. is supported by toral fellowship.

an S.I.S.S.A. (Trieste) pre-doc-

References 1.

2.

3.

4.

5.

6.

I.

8.

9.

10.

11.

12.

13.

Hagiwara H, Hayashi N, Mita E, Hiramatsu N, Ueda K, Takehara T, Yuki N, Kasahara A, Fuaamoto H, Kamada T. Detection of hepatitis C virus RNA in chronic non-A, non- B liver disease. Gastroenterology 1992; 102: 692-4. Kiyosawa K, Sodeyama T, Tanaka E, Gibo Y, Yoshizawa K, Nakano Y, Furuta S, Akahane Y, Nishioka K, Purcell RH, Alter HJ. Interrelationship of blood transfusion, non-A, non- B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990; 12: 671-5. Hopf U, Moller B, Kuther D, Stemerowicz R, Lobeck H, Ludtke-Handjery A, Walter E, Blum HE, Roggendorf M, Deinhardt F. Long-term follow-up of posttransfusion and sporadic chronic hepatitis non-A, non-B and frequency of circulating antibodies to hepatitis C virus. J Hepatol 1990; 10: 69-16. Ferre E Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl 1992; 2: l-9. Diviacco S, Norio P Zentilin L, Menzo S, Clementi M, Bi- amonti G, Riva S, Falaschi A, Giacca M. A novel procedure for quantitative polymerase chain reaction by coamplifi- cation of competitive templates. Gene 1992; 122: 313-20. Hagiwara H, Hayashi N, Mita E, Naito M, Kasahara A, Fusamoto H, Kamada T. Quantitation of hepatitis C virus RNA in serum of asymptomatic blood donors and patients with type C chronic liver disease. Hepatology 1993; 17: 545- 50. Kato N, Yokosuka 0, Hosoda K, Ito Y, Ohto M, Gmata M. Quantification of hepatitis C virus by competitive reverse transcription-polymerase chain reaction: increase of the virus in advanced liver disease. Hepatology 1993; 18: 1620. Hagiwara H, Hayashi N, Mita E, Naito M, Kasahara A, Fusamoto H, Kamada T. Quantitative analysis of hepatitis C virus RNA in serum during interferon alfa therapy. Gas- troenterology 1993; 104: 877-83. Bartolome J, Castillo I, Quiroga JA, Navas S, Carreilo V Detection of hepatitis C virus RNA in serum and peripheral blood mononuclear cells. J Hepatol 1993; 17 Suppl. 3: S90- s93. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9. Grassi G, Zentilin L, Tafuro S, Diviacco S, Ventura A, Giac- ca M. A rapid procedure for the quantitation of low abun- dance mRNAs by competitive RTPCR. Nucleic Acids Res 1994; 22: 4547-9. Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 1988; 16: 7351-67. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-


directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77: 51-9.

14. Sambrook J, Fritsch EF, Maniatis T Molecular Cloning. A Laboratory Manual, 2nd Edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

15. Okamoto H, Okada S, Sigiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H, Tsuda F, Miyakawa Y, Mayumi M. The S- terminal sequence of the hepatitis C virus genome. Jpn J Exp Med 1990; 60: 167-77.

16. Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J, Onishi E, Andoh T, Yoshida I, Okayama H. Struc- ture and organization of the hepatitis C virus genome iso- lated from human carriers. J Virol 1991; 65: 1105-13.

17. Norusis MJ. Advanced Statistics SPSS/PC. Chicago: SPSS Inc., 1986: 183-204.

18. Gilliland G, Perrin S, Blanchard K, Bunn HE Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Nat1 Acad Sci USA 1990; 87: 2725-9.

19. Wang AM, Doyle MV, Mark DFM. Quantitation of mRNA by the polymerase chain reaction. Proc Nat1 Acad Sci USA 1989; 86: 9717-21.

20. Bagnarelli P Menzo S, Valenza A, Manzin A, Giacca M, Ancarani F, Scalise G, Varaldo PE, Clementi M. Molecular profile of human immunodeficiency virus type-l infection in symptomless patients and in patients with AIDS. J Virol 1992; 66: 7328-35.

21. Menzo S, Bagnarelli P Giacca M, Manzin A, Varalda PE, Clementi M. Absolute quantitation of viremia in HIV-infected asymptomatic subjects by competitive reverse-transcription and polymerase chain reaction. J Clin Microbial 1992; 30: 1752-7.

22. Sestini R, Orlando C, Zentilin L, Gelmini S, Pinzani P, Giac- ca M, Pazzagli M. Measuring c -erbB-2 oncogene amplification in fresh and paraffin-embedded tumors by competitive polymerase chain reaction. Clin Chem 1994; 40: 630-6.

23. Giacca M, Zentilin L, Norio P Diviacco S, Dimitrova D, Contreas G, Biamonti G, Perini G, Weighardt E Riva S, Fal- aschi A. Fine mapping of a replication origin of human DNA. Proc Nat1 Acad Sci USA 1994; 91: 7119-23.

24. Ogata N, Alter HJ, Miller RH, Purcell RH. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Nat1 Acad Sci USA 1991; 88: 33926.

25. Han JH, Shyamala V, Richman KH, Brauer MJ, Irvine B, Urdea MS, Tekamp-Olson P Kuo G, Choo Q-L, Houghton M. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5’ untranslated region and poly(A) tails at the 3’ end. Proc Nat1 Acad Sci USA 1991; 88: 171 l-5.

26. Okamoto H, Sigiyama Y, Okada S, Kurai K, Akahane Y, Sugai Y, Tanaka T, Sato K, Tsuda F, Miyakawa Y, Mayumi M. 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: 673-9.

27. Becker-Andre M, Hahlbrock K. Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res 1989; 17: 9437-99.

28. Gonzalez-Peralta RP, Fang JWS, Davis GL, Gish R, Tsuki- yama-Kohara K, Kohara M, Mondelli MU, Lesniewski R, Phillips MI, Mozokami M, Lau JYN. Optimization for the detection of hepatitis C virus antigens in the liver. J Hepatol 1994: 20: 143-7.

411

quantitative analysis of hepatitis c virus rna in liver biopsies by competitive reverse...

Documents