Interferon treatment of chronic hepatitis C after bone marrow ... - Nature

Bone Marrow Transplantation, (1997) 20, 767–772  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

a-Interferon treatment of chronic hepatitis C after bone marrow transplantation for homozygous b-thalassemia C Giardini1, M Galimberti1, G Lucarelli1, P Polchi1, E Angelucci1, D Baronciani1, B Erer1, D Gaziev1, A Piga2, F Di Gregorio3, MA Romeo3, A Mangiagli4 , E Petrelli5 and P Muretto6 1

Divisione di Ematologia e Centro Trapianto di Midollo Osseo di Muraglia, Azienda Ospedale di Pesaro; 2Istituto di Clinico Pediatrica, Centro Microcitemie, Universita` Torino; 3Divisione Pediatria, Universita` di Catania; 4Divisione Pediatria, Ospedale Umberto I, Siracusa; 5Divisione Malattie Infettive, Azienda Ospedale di Pesaro; and 6Servizio di Anatomia Patologica, Azienda Ospedale di Pesaro, Italy

Summary: No experience has been reported to date in treating chronic hepatitis C virus (HCV) infection with interferon (IFN) therapy after BMT, mainly due to concerns related to the impact of an immunomodulatory drug in patients who are immunologic and haematologic chimeras. However, chronic inflammatory activity related to HCV infection results in a chronic fibrogenous mechanism potentially leading to liver cirrhosis and hepatocellular carcinoma. Moreover, patients transplanted for b-thalassemia could be at greater risk because of concomitant iron overload and pre-existing fibrous liver damage. Eleven patients with serological, biochemical, histological and molecular biological evidence of HCV infection were included in the study and treated for 6– 12 months with recombinant IFN 24–65 months following BMT. The serum alanine aminotransferase (ALT) was persistently elevated (range 85–1242 U/l; mean 416) for at least 1 year prior to IFN treatment. Ten patients completed the protocol; five were considered as responders to treatment. In these five patients the liver histology showed an overall reduction of inflammation and necrosis: histological inflammatory activity improved from chronic active hepatitis (CAH) to chronic persistent hepatitis (three patients) or minimal residual inflammatory activity (two patients). The Knodell total activity score varied from 5.4 (range 3– 9) to 1.4 (range 1–2; P = 0.05). All responding patients revealed negativization of serum HCV-RNA, that has been persistent in four (follow-up 1–3 years). ALT level fell to 15–80 U/l (mean 52; P = 0.0027). No major complications occurred during the therapy and no influence on marrow engraftment parameters were noted. We conclude that IFN therapy does not adversely interfere with engraftment and that it is a feasible therapy for treatment of chronic hepatitis C virus after BMT. Keywords: interferon; HCV; thalassemia; BMT

Correspondence: Dr C Giardini, Divisione di Ematologia e Centro Trapianto di Midollo Osseo di Muraglia, Azienda Ospedale di Pesaro, 61100 Pesaro, Italy Received 21 March 1997; accepted 12 June 1997

Hepatitis C virus (HCV) is responsible for the majority of cases of post-transfusion non-A non-B (NANB) hepatitis. We have previously reported a high prevalence of chronic hepatitis C virus infection in b-thalassemia patients who are candidates for BMT.1 HCV infection has also been reported not to influence transplant outcome and indeed does not constitute a contraindication for bone marrow transplant.1–3 In immunocompetent patients, persistent HCV infection is associated with the development of chronic hepatitis, cirrhosis, and hepatocellular carcinoma.4 The effect of post-transplant transient immunosuppression on the expression and evolution of HCV-related liver disease remains unclear.5,6 IFN therapy has been shown to be effective in the normalization of serum transaminase levels, with improvement in the histological activity of hepatitis, and in the clearing of HCV-RNA from serum in immunocompetent patients with chronic hepatitis C.7–9 Nothing is known about the efficacy, safety and tolerability of this drug in treating chronic HCV infection after BMT, even though some preliminary reports have appeared in organ transplant literature.10,11 A major concern for the use of IFN in BMT recipients is the risk of inducing or precipitating GVHD12 or allograft rejection, which could be related to the induction of class II histocompatibility antigen expression.13 That IFN has been occasionally suspected in precipitating renal and bone marrow allograft rejection among patients shortly after transplantation also adds to the reluctance in using IFN in these patients.14,15 However, IFN has already a well established and interesting role in the early phase after BMT to prevent or treat post-transplant recurrent malignancy.16,17 In the group of patients reported, in view of the evidence of severe liver inflammatory activity, the risk of developing cirrhosis and liver failure within the next few years was, in our opinion, too high to deny them the possible benefits of therapy. IFN therapy for chronic HCV infection was therefore adopted in an open pilot trial for thalassemiamajor patients 24–65 months (mean 40) post-transplant.

IFN therapy of chronic hepatitis C after BMT for thalassemia C Giardini et al

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Patients and methods Criteria for eligibility Eleven patients were included in the study. Ten patients (four males, six females) tolerated the treatment and completed the protocol. Ages ranged from 10 to 20 years (mean 14.5). All patients had been regularly transfused pre-transplant, with the transfusion load ranging from 70 to 404 (mean 188) units of blood. Four patients had undergone splenectomy; all had received desferrioxamine before transplant; however, a variable degree of liver iron load was documented. Liver fibrosis was also present in various degrees of severity in all patients (Table 1). The conditioning regimen for BMT included BU 14 mg/kg and CY 120–200 mg/kg. All patients were negative for antibody to human immunodeficiency virus (HIV) and for hepatitis B antigen, although two patients were positive for hepatitis B core antibody. Eight patients were anti-HBs positive as a result of HBV vaccination. All patients were HCV-Ab and HCVRNA positive in the serum and had mild (two patients), moderate (four patients) or severe (two patients) chronic active hepatitis (CAH) or active post-hepatitic cirrhosis (two patients) on liver biopsy. The serum ALT was persistently elevated (range 85–1242 U/l; mean 416; median 207) in all patients for at least 1 year prior to IFN treatment. Using standard methods the following investigations were carried out during the study period: serum ferritin, liver function tests, oral glucose tolerance test, virology status (hepatitis B and HIV serology, anti-HCV, antiCMV), thyroid function (serum T4, TSH) and immunological tests (autoantibodies, rheumatoid factor, anti-smooth

Table 1 Pre-treatment clinical, laboratory and histological features of patients

muscle antibody, anti-mitochondrial antibody, anti-gastric parietal cell antibody and anti-nuclear factor), serum immunoglobulins and lymphocyte subsets. Serum HCV was assessed by the second–third generation enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostic, Raritan, NJ, USA). Serum HCV-RNA was tested using the PCR technique with the Amplicor Hepatitis C virus (HCV) test (Hoffmann-LaRoche, Basel, Switzerland), which can detect as few as 1000 copies of HCV-RNA per ml of sample. Exclusion criteria The presence of one of the following conditions was considered as a criterion for excluding the patient from the protocol: (1) time after transplant ,2 years; (2) documentation of mixed chimerism with residual host cells on bone marrow (studied by DNA analysis of restriction fragment length polymorphism of variable number tandem repeats (RFLP-VNTR) and, for sex-mismatched pairs, with fluorescence in situ hybridization (FISH) for the Y chromosome); (3) the presence of active chronic GVHD; (4) concomitant immunosuppressive therapy (cyclosporine, azathioprine, steroids, etc); and (5) the presence of hypergammaglobulinemia or autoantibodies. Study protocol Informed consent was obtained from patients following full disclosure of potential risks and benefits of IFN therapy. Recombinant aIFN 3 million units (MU)/m2 administered by subcutaneous injection three times a week was given to all patients. Blood count, renal function, and serum level of liver enzymes were monitored at 1–2 weeks intervals. Therapy was continued for 6–12 months. Follow-up continued for 1–3 years after the discontinuation of treatment.

Mean (range)

Histology Mean age (years) Sex (M/F) Time after transplant (months) Serum alanine aminotransferase (U/l) Ferritin No. of blood transfusions Liver histology CAH mild moderate severe Active cirrhosis Liver siderosis mild moderate severe Liver iron concentration (mg/g) Liver fibrosis mild moderate severe cirrhosis

14.5 (10–20) 4/6 40 (24–65) 416 (85–1242) 3110 (500–9059) 188 (70–404) 2 4 2 2 2 7 1 8.3 (3.8–14.1) 3 4 1 2

The normal reference range for alanine aminotransferase is 2 to 41 U/l; for serum ferritin 20–300 ng/ml; for liver iron concentration ,1 mg/g of dry tissue.

Percutaneous liver biopsy was performed in all patients prior to treatment, 6–12 months after the end of therapy and then yearly. Biopsy specimens were processed for histology by routine methods. Liver biopsy specimens were assessed by a pathologist who was not aware of any therapeutic data. Histological inflammatory activity was assessed using conventional histologic criteria.18 All biopsies were allocated into the following diagnostic categories: (1) normal liver or minimal inflammatory activity (MIA); (2) chronic persistent hepatitis (CPH); (3) mild chronic active hepatitis (CAH); (4) moderate CAH; (5) severe CAH; and (6) active cirrhosis (AC). Biopsy specimens were also semi-quantitatively evaluated according to Knodell’s scoring system, which grades four types of hepatic lesions: (1) periportal/bridging necrosis; (2) intralobular degeneration and focal hepatocellular necrosis; (3) portal inflammation; and (4) fibrosis.19 Dry weight iron content was measured using a modification of the method originally described by Barry and Sherloch.20

IFN therapy of chronic hepatitis C after BMT for thalassemia C Giardini et al

Response criteria Histologic improvement was defined as a decrease of at least one point in the score for necroinflammatory activity (see below) when the final result constituted a score not higher than 2 (ie MIA or CPH). A pre-treatment liver biopsy was compared to a biopsy performed 12–24 months later. Complete response was defined when histologic improvement was associated with a significant decrease in serum transaminases (not higher than twice the normal value, ie ,82 U/l) and clearance of serum HCV-RNA (at least two negative results at 6 months to 1 year interval). Complete normalization of serum transaminases was not judged to be a criterion because of the concomitant variable degree of iron overload that was present in all treated patients. All data are reported as mean values (range), unless otherwise specified. Statistical comparisons were made utilizing the Wilcoxon test for paired data and Mann–Whitney U test. The level of statistical significance (P) was set at 0.05. Results One patient discontinued IFN at 3 weeks because of persistent fever and myalgia. Among 10 subjects completing the protocol, five reached the criteria established for a complete response at the end of therapy. Liver histology showed an overall reduction of inflammation and necrosis in responding patients: from chronic active hepatitis (CAH) to chronic persistent hepatitis (three patients) or minimal inflammatory activity (two patients) (Figure 1 and Table 2). Despite the established histologic criteria for response, two patients revealed a decrease of two points in the score for necroinflammatory activity at the end of the therapy and two a decrease of three points. The last responder (No. 4 in Figure 1), who had a decrease of one point at the end

6

AC

Pt Pt Pt Pt

sev

5 C

4 A mod

Pt Pt Pt Pt Pt

H 3

mild

2 CPH 1

1R 2 3C 4R 5R 6R 7C 8 9R

Pt 10

MIA Pre

Post 1

Post 2

Post 3

Figure 1 Interferon therapy: modification of histologic/inflammatory parameters. Pre: evaluation before starting therapy; Post 1, Post 2, Post 3: 1 year interval follow-up liver biopsies after completing therapy. AC: active cirrhosis; CAH: chronic active hepatitis (mild, moderate, severe); CPH: chronic persistent hepatitis; MIA: normal liver or minimal residual inflammatory activity; R: patients responders to treatment; C: patients with histological diagnosis of cirrhosis.

of therapy (from mild CAH to CPH), revealed an additional decrease of necro-inflammatory activity at the following histologic check-up (ie minimal inflammatory activity). The non-responding patients had no changes in the necroinflammatory score (one patient with moderate CAH) or minimal changes (one patient improved from severe to mild CAH and one patient from moderate to mild CAH). This last patient (No. 2 in Figure 1) has now substantially improved his histologic diagnosis (chronic persistent hepatitis on the last histologic control) while on a phlebotomy program. Fibrosis or cirrhosis scores did not differ significantly. The two patients with a pre-treatment diagnosis of cirrhosis did not respond to treatment, maintaining the HCVRNA positivity and revealing no substantial changes in serum transaminases. Four of the responders and three non-responders received additional one to two follow-up liver biopsies at 1 year intervals (results shown in Figure 1). The semiquantitative (Knodell) scores for histological activity and fibrosis are given in Table 2. Individual components of the histological inflammatory activity scores were comparable in the responders and non-responders. The fibrosis score was lower in responders than in nonresponders (1.6 vs 3.0), although this was not statistically significant. The histology score showed an overall improvement in responding patients. There was a significant improvement in periportal/bridging necrosis (I)), total activity score (I + II + III) and total score (I + II + III + IV) between preand post-therapy biopsies (P = 0.05 for each). Intralobular degeneration (II) and portal inflammation (III) did not show a statistically significant decrease; fibrosis (IV) showed only a minimal change. In contrast, no significant changes have been observed in non-responding patients in any of the parameters considered (Table 2). Anti-HCV tests remained positive after IFN therapy in all subjects throughout the study. The results of HCV-PCR testing are given in Table 3. All responding patients developed a negative serum HCV-RNA (at least two consecutive results at 6 months to 1 year intervals), which has persisted in four patients. One patient (No. 1 in Table 3) became positive for HCVRNA test after two consecutive negative results, more than 2 years after interrupting IFN therapy. The histologic finding at the same time remained unchanged (chronic persistent hepatitis). The patients who responded to interferon had an ALT value before treatment ranging from 122 to 1242 U/l (mean 562; median 469), as compared with 85 to 833 (mean 270; median 85) among those who did not respond. Serum ALT levels improved in all except one patient treated at the end of the therapy. In the five responding patients the ALT fell to 15–80 U/l (mean 52; median 59; P = 0.05) compared with 44 to 470 (mean 198; median 60; P = NS) in the non-responders. This finding has been maintained during follow-up (Figure 2). Initial serum ferritin level and liver iron concentration (LIC) in the liver have not been noted as influencing response to IFN. Patients responding to treatment had a mean LIC (expressed as mg/g of dry tissue) of 9.1 (range

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Table 2

Changes in histological diagnoses and liver biopsy Knodell index Responders (n = 5) Before

Diagnosis Minimal inflammatory activity CPH Mild CAH Moderate CAH Severe CAH Active cirrhosis

Non-responders (n = 5)

After

Before

After

2 3 2 2 1

Knodell I Periportal/Bridging necrosis II Intralobular generation III Portal inflammation IV Fibrosis Total activity score (I + II + III) Total score (I + II + III + IV)

2 1

2 1 2

1.4 1.4 2.6 1.6 5.4 7.0

(1–3) (1–3) (1–3) (0–3) (3–9) (5–12)

0 *(0–0) 0.4 (0–1) 1 (1–1) 1.2 (0–3) 1.4 *(1–2) 2.6 *(2–4)

1.8 1.8 1.8 3.0 5.4 8.4

2 (1–3) (1–3) (1–3) (1–4) (3–9) (4–12)

1.6 1.4 2.2 3.0 5.2 8.2

(0–3) (1–3) (1–3) (1–4) (2–9) (6–12)

*P = 0.05

Table 3 Patient No. 1 2 3 4 5 6 7 8 9 10

HCV-RNA (PCR method) before and after interferon therapy HCV-RNA (Basal)

Post-1

Post-2

Post-3

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

Neg Neg Pos Neg Neg Neg Pos Pos Neg Pos

Neg Pos Pos Neg Neg Neg Pos Pos Neg Pos

Pos Pos Pos Neg

Post 1, Post 2, Post 3: indicate yearly follow-up controls.

3.82–12.22), compared with 7.5 mg/g of non-responding patients (range 5.25–14.07, P = NS). The five patients responding had a mean serum ferritin level of 3297 ng/ml (range 716 to 9059) compared to 2923 ng/ml (range 500 to 8421) of non-responders (P = NS). Also, there was no correlation between CD3 and CD4 lymphocyte levels and response to therapy: responding patients had a value of CD3+/CD4+ lymphocytes ranging from 1809 to 3271 and 1097 to 1963 (mean 2790 and 1609, respectively), compared with 1389–7011 and 387–4407 (mean 4182 and 2151, respectively; P = NS) of nonresponders. Among 10 patients completing the IFN protocol, sideeffects observed were fever and malaise responding to paracetamol pre-medication (five patients) and mild hair loss (one patient). No reduction in RBC, WBC or platelet count was noted during treatment, necessitating dosage reduction or discontinuation of therapy.

ALT 800

Discussion

700 600 500

Responders Non-responders

400 300 200 100 0 normal range Pre

Post 1 Post 2 Post 3

Figure 2 Changes of serum alanine aminotransferase (ALT) of responders and non-responder patients. Normal: ,41 U/l. Values are mean with standard error bars. PRE: pre-treatment values (24–65 months following BMT); Post 1, Post 2, Post 3: indicate follow-up controls after completing treatment at 1 year interval.

The most important observation from this pilot study is that IFN therapy for chronic C viral hepatitis in BMT recipients was safe, effective and well tolerated. In particular, no adverse effects on engraftment or GVHD were observed. The second point is that this is a pediatric patient population, which is not the case in the majority of published works, where IFN therapy was used in adults. A third point is that all patients reported in the present study had some other evidence of liver damage, ie some degree of iron overload and fibrosis, as a consequence of repeated pre-transplant transfusions. In transfusion-dependent thalassemia patients some of these situations, such as a variable degree of iron overload, concomitant hepatitis B or cytomegalovirus infection and pre-existing fibrous liver damage, have been reported to

IFN therapy of chronic hepatitis C after BMT for thalassemia C Giardini et al

have a synergistic effect with chronic HCV infection in accelerating liver damage.21 As a result of these noxious situations, a chronic transaminase alteration is frequent in these patients, even in the absence of HBV/HCV chronic infection.1 That patients with idiopathic hemochromatosis can have liver cirrhosis at diagnosis without any contribution from viral hepatitis also highlights the importance of the iron alone as a potent noxious and fibrogenous agent.22 The influence of iron overload on the response to IFNa treatment has also been reported in several studies, both in normal and in transfusion-dependent thalassemic population.23–25 However, it is not clear if a cut-off value for liver iron burden that would predict a possible response to IFN can be determined. In the study of Olynyk et al23 a LIC .1.1 mg/g predicted a non-response in 88% of patients. The results of this study appear not readily comparable with several reports of IFN therapy in transfusiondependent thalassemia patients in whom the LIC normally exceeds up to 30 times the value of 1 mg/g.25–27 In our patients, we failed to demonstrate a relationship between the degree of residual iron overload (LIC ranging from 3.8 to 14.1 mg/g) and a response to treatment, possibly due to the small number of patients examined. The response rate of post-transfusion chronic active hepatitis C to IFNa therapy is 45–50% for immunocompetent patients, resulting in improvements in transaminasemia, liver histology, and decrease in serum HCV-RNA.7,8 However, the clinical and serological improvements are often short-lived, and relapses are common after cessation of IFN therapy.7 A more prolonged course of therapy has recently proven to increase significantly the response rate.9 It has been postulated that the response rate in multitransfused thalassemia major patients infected with HCV and treated with IFN is similar to that in non-multi-transfused patients.26 Our experience shows that ex-thalassemics with chronic hepatitis C may benefit from IFNa therapy after bone marrow transplantation. However, it is still unclear whether IFN is able to completely eradicate the virus or whether it merely suppresses replication and viral activity. Long-term benefit of the therapy is also uncertain.28 Moreover, in view of the risk of allograft rejection or GVHD, IFN therapy is not advisable for patients with unstable allograft function or chronic GVHD, and for patients in the early post-transplant period while on immunosuppressive therapy. Allogeneic bone marrow transplantation has proven to be curative for b-thalassemic patients with an HLA identical donor;29 however, the evolution of iron overload, liver fibrosis and chronic hepatitis remains one of the major points of concern after transplant, particularly for older and heavily transfused patients.30,31 We have already reported that desferrioxamine and phlebotomies are able to produce a complete clearance of iron deposits after transplant.32,33 IFN therapy seems a promising therapy in the attempt to stop the evolution of viral-related chronic inflammatory activity. If this is true, the objective of arresting the progression of organ damage and possibly of restituting the liver of these patients to a steady-state condition seems not too far from being achieved. A larger, controlled study of IFN therapy in patients who have been enrolled at the end

of complete iron removal therapy is ongoing, which may give important indications for future extensive clinical use. The data reported, however, may be of interest also for non-iron loaded hematology patients with chronic HCV infection after BMT. The multifactorial immunomodulatory and antiviral activity of IFN and the peculiarity of ‘chimeric’ patients after allogeneic BMT suggest a need strictly to maintain the limitations and exclusion criteria already adopted for further studies.

Acknowledgements We thank Ms Aileen Law, a member of our nursing staff, for excellent assistance. This work has been supported by AIL Pesaro and by the Berloni Foundation against Thalassemia.

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