ALBERT

Description: The prevalence of the blood-borne TT virus (TTV) in Italian hemophiliacs treated with different preparations of factor VIII was determined. Of the 178 hemophilic patients (mean age, 29 years), TTV-DNA was found in 123 (69%), in comparison to 22 of 100 (22%) blood donors (P 〈 .0001). Of the 123 patients who tested positive for TTV, significant numbers were also infected with human hepatitis viruses and/or human immunodeficiency virus (HIV): 31% had TTV and hepatitis C virus (HCV), 22% had TTV, and at least 2 of the 4 known human blood-borne viruses tested, whereas 15% had TTV alone. The risk of acquiring TTV alone was only slightly higher in recipients of unmodified plasma factor concentrates (78%, odds ratio, 1.24; 95% confidence interval [CI], 0.27 to 5.79) than in patients treated with virus inactivated concentrates (67%), whereas the risk was significantly lower in recipients of recombinant factors (11%, odds ratio, 0.09; 95% CI, 0.01 to 0.52). Serum alanine aminotransferase (ALT) levels were elevated in 2 of 27 patients (7%) with TTV alone compared with 43 of 56 patients (77%) coinfected with TTV and HCV and compared with 16 of 21 patients (76%) with HCV alone. Taken together, these results indicate that TTV frequently infects Italian hemophiliacs treated with plasma-derived factor VIII concentrates, both unmodified and virus-inactivated. Our results do not suggest a causal effect of TTV on chronic liver disease in these patients.

Description: Abstract 2276 Background: Chronic hepatitis C is the main cause of morbidity and mortality in patients with hemophilia. HCV eradication is the only approach that can halt disease progression and it may be attempted by using antiviral therapy, the standard of care being represented by the combination of pegylated interferon (Peg-IFN) plus ribavirin. Sustained virologic response (SVR) is defined by undetectable HCV-RNA in serum 6 months after therapy completion. The likelihood of a SVR depends on various host and viral factors, as sex, stage of liver fibrosis, HCV genotype and viral kinetics during treatment, being the rapid virologic response (RVR; undetectable HCV-RNA in serum after 4 weeks of therapy) a strong predictor of SVR. Recently, it has been reported that a single-nucleotide polymorphism (SNP) near the IL28B gene, named rs12979860, is a predictor of response to therapy in non-hemophiliacs. This SNP may be genotyped as CC, CT or TT and the CC variant has been associated with higher rates of SVR and with higher rates of spontaneous viral clearance after acute infection. Methods and Results: the SNP was determined by Polymerase Chain Reaction in 189 patients with hemophilia aged 37–54 years (median: 44) infected with HCV. Fifty-five patients (29%) were HIV and 10 (5%) HBV co-infected. HCV genotype 1 was the most prevalent (n=124, 73%) and the IL28B SNP was CC in 70 (37%), CT in 104 (55%) and TT in 15 (8%). The median duration of HCV infection was 34 years (IQR: 28–39) and the median age at antiviral treatment 38 years (IQR: 31–46). Clinical signs of cirrhosis were present in 22 patients (12%). Fifteen patients (8%) had spontaneous clearance of viral infection and 134 (71%) of those with chronic infection underwent antiviral therapy that was Peg-IFN plus ribavirin in 114 (85%). Overall SVR rate was 49% (37% in patients with HCV genotype 1 and 70% in those with HCV genotype 2 or 3). Rapid, early and sustained virologic response were more frequent in patients carrying the CC SNP than in those carrying a non-CC SNP (59 vs 17%, 90 vs 71% and 67 vs 39%, respectively; p

Description: The prevalence of the blood-borne TT virus (TTV) in Italian hemophiliacs treated with different preparations of factor VIII was determined. Of the 178 hemophilic patients (mean age, 29 years), TTV-DNA was found in 123 (69%), in comparison to 22 of 100 (22%) blood donors (P 〈 .0001). Of the 123 patients who tested positive for TTV, significant numbers were also infected with human hepatitis viruses and/or human immunodeficiency virus (HIV): 31% had TTV and hepatitis C virus (HCV), 22% had TTV, and at least 2 of the 4 known human blood-borne viruses tested, whereas 15% had TTV alone. The risk of acquiring TTV alone was only slightly higher in recipients of unmodified plasma factor concentrates (78%, odds ratio, 1.24; 95% confidence interval [CI], 0.27 to 5.79) than in patients treated with virus inactivated concentrates (67%), whereas the risk was significantly lower in recipients of recombinant factors (11%, odds ratio, 0.09; 95% CI, 0.01 to 0.52). Serum alanine aminotransferase (ALT) levels were elevated in 2 of 27 patients (7%) with TTV alone compared with 43 of 56 patients (77%) coinfected with TTV and HCV and compared with 16 of 21 patients (76%) with HCV alone. Taken together, these results indicate that TTV frequently infects Italian hemophiliacs treated with plasma-derived factor VIII concentrates, both unmodified and virus-inactivated. Our results do not suggest a causal effect of TTV on chronic liver disease in these patients.

Description: Anemia is a common side effect of Pegylated Interferon (PegIFN) and Ribavirin (RBV) combined therapy in patients with chronic hepatitis C (HCV). Hemolysis as consequence of RBV concentration in erythrocytes is the most accepted underlying mechanism; however, since the degree of treatment related anemia is highly variable, other pathogenic mechanisms could be hypothesized. The aim of this study was to evaluate the prevalence of hemolysis in patients treated with the combination of PegIFN and RBV compared to patients on monotherapy with PegIFN and to assess other possible mechanisms underlying anaemia. We studied 18 patients with chronic hepatitis C HCV-2 treated with PegIFN-α2a (180 mcg/week) plus RBV (800 mg/day) for 24 weeks and 10 patients with chronic hepatitis B treated with PegIFN-α2a 180 mcg/week monotherapy for 48 weeks. All the patients developed anemia after 24 weeks of treatment, although the mean hemoglobin decrease was higher in HCV patients (2.77 vs 1.82 g/dL at week 24, p=0.03), particularly at week 4 (1.86 vs 0.51 g/dL, p=0.007). Only 3 out of 18 HCV patients (16%) developed hemolytic anemia, documented by a marked increase in reticulocytes (3,4%) and in LDH levels (502 U/L) and by a significant decrease in haptoglobin levels (40,3 mg/dL). These patients showed a sharper and faster decrease of Hb compared to the remaining 15 HCV patients (week 4: 3.40 vs 1.55 g/dL, p=0.01). None of the HBV patients on treatment developed hemolytic anemia. To understand possible mechanisms underlying anemia, we evaluated ex vivo the effect of the therapy on erythropoiesis, through peripheral erythroid progenitors cell coltures and gene expression analysis during treatment. Peripheral blood mononuclear cells were plated in methylcellulose-supporting media at a concentration of 2×105 cells/mL and colony formation (BFUe and CFU-GEMM) was analyzed after 14 day of culture. Expression of γ-globin and GATA2 gene was evaluated with quantitative real-time PCR. HCV patients with hemolysis had an increase in BFUe number at week 4, whereas patients without signs of hemolysis showed a decrease in colonies formation at week 4 (baseline: 15.5 to 7.5 colony / 105 cells). A decrease in BFUe number was also observed in HBV patients along PegIFN treatment period. In all the HCV and HBV patients the reduction in BFUe number was associated with an increase in undifferentiated CFU-GEMM colonies (BFUe: 87 to 76% in HCV, 91 to 64% in HBV; CFU-GEMM: 13 to 24% in HCV, 9 to 36% in HBV). This suggested that the inhibitory effect of PegIFN on erythroid differentiation, supported also by increased expression of primitive erythropoiesis specific genes (γ-globin and GATA2 genes), plays a major role in causing anemia even in combined treatment with RBV. The hemolysis as cause of anemia so far attributed to RBV is apparently restricted to few cases and deserves further investigation.

Description: Hepatocellular carcinoma (HCC) is an increasingly frequent cause of mortality in hemophiliacs with chronic viral hepatitis. Early diagnosis of the tumor at an initial stage is known to improve the outcome of HCC treatment. Because all HCC cases detected in a previous study based upon annual ultrasound (US) surveillance of hemophiliacs with elevated alanine aminotransferase levels were multinodular, this study was designed to evaluate if a more intense surveillance with US and alphafetoprotein (AFP) serum levels of all the patients infected with the hepatitis C virus (HCV) improved the identification of single nodule tumors. A multicenter cohort of 559 HCV-infected hemophiliacs was divided into 2 arms, one followed up at 6-month intervals and one at 12-month intervals depending on the choice and available facilities of each treatment center. During a 6-year surveillance period, HCC was diagnosed in 5 (2.4%) of 210 patients in the 6-month group and in 3 (0.9%) of 349 patients in the 12-month group. The overall incidence rate of HCC was 239 per 100 000 per year (397 per 100 000 per year in the 6-month group and 143 per 100 000 per year in the 12-month group; differences not statistically significant). By multivariate analysis, HCC risk was increased 12.9-fold with alcohol intake more than 80 g/d and 15.2-fold with AFP levels higher than 11 ng/mL. Liver-related death occurred in 8 cases (1.4%), including 3 with HCC. Still alive and tumor free after 24 to 34 months from diagnosis are 3 patients with multinodular tumors treated with repeat chemoembolization followed by orthotopic liver transplantation. In conclusion, 6-month surveillance with US did not increase the chances of detection of single nodule tumors, but it is reasonable to assume that successful treatment of multinodular tumors based upon debulking with chemoembolization and liver transplantation was facilitated by this approach. (Blood. 2003;102:78-82)

Description: Thirty-nine hemophiliac patients, negative for human immunodeficiency virus, with chronic hepatitis C who failed to respond to interferon (IFN) at 3 million units (MU) given subcutaneously thrice weekly for at least 3 months were retreated with 5 MU IFN for 6 months followed by 3 MU IFN in combination with daily oral doses of 1 or 1.2 g ribavirin. Thirty-four patients (87%) completed the study. In 4 patients treatment was discontinued because of treatment-related symptoms; 1 patient dropped out. Dosage reduction was required in 10 patients (26%) because of ribavirin-related anemia or IFN-related side effects. By intention-to-treat analysis, 14 (37%) had a sustained virologic response with preference for those infected by genotypes other than type 1 (43% versus 12%) and with high transaminases levels (168 U/L versus 116 U/L). Thus, IFN and ribavirin combination therapy led to a sustained suppression of hepatitis in one third of hemophiliac patients resistant to conventional monotherapy.

Description: The antiviral drug ribavirin (RBV) is widely used in combination with pegylated interferon (PegIFN) for the treatment of hepatitis C virus (HCV) infection. A major side effect of RBV is a reversible anemia. The mechanism underlying anemia in RBV treated patients remains speculative although hemolysis was suggested. To get further insights, we evaluated in vitro the effect of RBV and PegIFN on proliferation and erythroid differentiation of CD34 pheripheral blood cells. The mononuclear cells derived from pheripheral blood of healthy volunteers were enriched for CD34+ cells by positive selection using anti-CD34-tagged magnetic beads. CD34+ cells were cultured for 14 days with a specific medium containing erythropoietin to induce erythroid differentiation. RBV (0.5mM and 0.1mM) and PegIFN (100U and 1000U) were added alone or in combination at different days of culture: precisely at day 0 corresponding to CD34 stage and at day 7 corresponding to proerythroblast stage. Cells growth and viability were evaluated by tripan blue exclusion; erythroid differentiation was investigated by cytofluorimetric analysis of Glicophorin A (GPA) expression, by morphological analysis on benzidine-May-Grunwald-Giemsa stained smears and by erythroid specific (γ-globin and GATA2) gene expression analysis with real-time PCR. RBV added at day 0 of culture drastically inhibited cell growth and differentiation (RBV 0.1mM: 8.5-fold reduction in cells growth and 1.6-fold reduction in GPA+ cells vs untreated cells at day 14; RBV 0.5mM: 12-fold reduction in cells growth and 30-fold reduction in GPA+ cells vs untreated cells at day 14). Addition of RBV at day 7 of culture had a milder inhibitory effect (RBV 0.1mM: 2.4-fold reduction in cells growth and 0.7-fold reduction in GPA+ cells vs untreated cells at day 14; RBV 0.5mM: 3.6-fold reduction in cells growth and 2.3-fold reduction in GPA+ cells vs untreated cells at day 14). Addition of PegIFN both at day 0 and day 7 of culture delayed erythroid differentiation, with reduction of GPA+ cells, decrease of orthocromatic erythroblasts and increase of more undifferentiated polychromatophilic erythroblasts, and increased expression of primitive erythropoietic genes (tab 1). The combined addition of PegIFN and RBV both at day 0 and day 7 of culture affected cell growth and erythroid differentiation less than RBV alone, with increase of GPA+ cells, decrease of basophilic erythroblasts and increase of more differentiated orthocromatic erythroblasts, and decreased expression of γ-globin gene. However cells treated with the combination of PegIFN and RBV were more undifferentiated than cells treated with PegIFN alone. All these observations confirmed the inhibitory effect of PegIFN on erythroid differentiation and strongly suggested an inhibitory action of RBV on cell growth, particularly on undifferentiated stem cells. These observations provide a model for the pathophysiology of anemia observed with ribavirin treatment. Tab. 1 % GPA+ cells % polychromatophilic e. % orthocromatic e. γ-gene GATA2 gene Untreated cells 61 17 55 1 1 PegIFN 100U 50 37 56 1,75 1,16 PegIFN 1000U 47 57 23 2,39 1,7

Description: The parallel measurement of serum antibodies to the hepatitis G virus (anti-HGV) and of viremia (HGV-RNA) should improve our understanding of HGV transmission by coagulation factor concentrates. The aim of this study was to assess the relationship between HGV, the type of concentrate infused, and liver disease in multitransfused hemophiliacs. To this end, anti-HGV and HGV-RNA were evaluated by an enzyme-linked immunosorbent assay and a nested–polymerase chain reaction assay in patients treated lifelong with nonvirus-inactivated plasma-derived concentrates (n = 128), virus-inactivated concentrates (n = 33), or recombinant factors (n = 7), and in 200 regular blood donors. The prevalence of serum HGV-RNA and anti-HGV was higher in the recipients of nonvirus-inactivated factors than in blood donors (HGV-RNA: 9% v 1.5%, P = .002; anti-HGV: 32% v 5%, P 〈 .0001). In the recipients of virus-inactivated concentrates the prevalences of these markers were similar to those in blood donors (HGV-RNA: 3% v 1.5%; anti-HGV: 15% v 5%). The prevalence of either marker in the recipients of nonvirus-inactivated concentrates was higher than in the recipients of virus-inactivated factors (39% v 18%, P = .04). The former group had serum hepatitis C virus (HCV) RNA or anti-HCV more frequently than the latter group (HCV-RNA: 86% v 15%, P 〈 .0001; anti-HCV: 96% v 18%, P 〈 .0001). Serum alanine aminotransferase was persistently high in 83 (81%) patients with HCV-RNA alone, in 8 (89%) with HCV/HGV coinfection, and in none of the three patients with HGV-RNA only. Thus, HGV infection in hemophiliacs is more common than previous studies of HGV-RNA prevalence have suggested, but it resolved in most cases and caused chronic viremia only in a small number of patients, without biochemical evidence of persistent liver damage.

Description: Abstract 4064 Poster Board III-999 Introduction Liver disease is the second cause of death for thalassemia major (TM) patients, mainly due to HCV infection and transfusional iron overload. Few data are so far available for thalassemia intermedia (TI) patients who are much less transfused but, because of chronic anemia they have an increased iron absorption. Aim the aim of this study was to evaluate the prevalence of liver disease and its progression in adult non-transfusion dependent TI patients. Patients and Methods Seventy adult TI patients (32 female/38 male, aged 42±14 years, range 22-77) regularly cared at Hereditary Anemia Center, University of Milan, were enrolled in this study in 1997 and followed for 10±1 years. Seven patients were lost and 4 died during follow-up. At enrolment (T0) 50 were splenectomized, 51 were occasionally transfused, 46 were irregularly chelated. Twenty-four (34,2%) patients were anti HCV positive of whom 13 (54,1%) were RNA positive. Results Liver transaminases were significantly different (p=0.001) among HCV-RNA positive and negative patients (ALT 59,7±32,1 vs 26,9±20,3 U/L; AST 49,1±22,8 vs 30,6±17,0 U/L respectively). Ferritin levels in the overall group were significantly higher than normal values (734±748 ng/ml). No significant difference in ferritin levels was detected among HCV-RNA positive and negative patients, while overall a correlation (r=0.687, p7.9 kPa (corresponding to fibrosis stage F≥2 of Metavir), and the mean value was 6,7±6,2 kPa; almost all the patients (39/42 – 92,8%) had significant high level of liver iron concentration (LIC measured through MRI T2*≥2 mg/g d.w.) with a correlation between LIC T2* and Fibroscan values (r=0.489, p=0.003). During the follow up 4 patients died: 1 for stroke and 3/4 for liver disease,(one Hepatocarcinoma (HCC) in HCV-RNA positive patient and 2 decompensated cirrhosis). Other two cases of HCC were observed, one in a patient HCV-RNA positive and 1 in an HCV-RNA negative patient; the latter having significant iron overload (LIC through MRI T2* 23,29 mg/g/dw) and a Fibroscan value diagnostic for cirrhosis (43,5 kPa). Conclusions Liver disease is the first cause of death in TI patients; 3 cases of HCC were observed in patient aged 49±1 years old of whom 1 without hepatitis viral infection. The liver damage, detected with high levels of ALT and AST in both HCV-RNA positive and negative patients is mainly related to the parenchymal iron overload and HCV infection. Ferritin, commonly used to monitor iron overload, properly reflects the degree of iron concentration in TM, while is inadequate in TI patients because, even though it correlates with LIC, it underestimates the iron overload. Actually in TI patients iron coming from duodenal absorption is mainly stored in parenchymal liver tissue, while in TM it's primarily distributed in the reticulo-endothelial system that stimulate ferritin production. In conclusion it's mandatory the use of other methods to evaluate LIC, such as MRI T2*, and the introduction of regular chelation therapy in the management of TI patients. Disclosures: No relevant conflicts of interest to declare.

Description: The parallel measurement of serum antibodies to the hepatitis G virus (anti-HGV) and of viremia (HGV-RNA) should improve our understanding of HGV transmission by coagulation factor concentrates. The aim of this study was to assess the relationship between HGV, the type of concentrate infused, and liver disease in multitransfused hemophiliacs. To this end, anti-HGV and HGV-RNA were evaluated by an enzyme-linked immunosorbent assay and a nested–polymerase chain reaction assay in patients treated lifelong with nonvirus-inactivated plasma-derived concentrates (n = 128), virus-inactivated concentrates (n = 33), or recombinant factors (n = 7), and in 200 regular blood donors. The prevalence of serum HGV-RNA and anti-HGV was higher in the recipients of nonvirus-inactivated factors than in blood donors (HGV-RNA: 9% v 1.5%, P = .002; anti-HGV: 32% v 5%, P 〈 .0001). In the recipients of virus-inactivated concentrates the prevalences of these markers were similar to those in blood donors (HGV-RNA: 3% v 1.5%; anti-HGV: 15% v 5%). The prevalence of either marker in the recipients of nonvirus-inactivated concentrates was higher than in the recipients of virus-inactivated factors (39% v 18%, P = .04). The former group had serum hepatitis C virus (HCV) RNA or anti-HCV more frequently than the latter group (HCV-RNA: 86% v 15%, P 〈 .0001; anti-HCV: 96% v 18%, P 〈 .0001). Serum alanine aminotransferase was persistently high in 83 (81%) patients with HCV-RNA alone, in 8 (89%) with HCV/HGV coinfection, and in none of the three patients with HGV-RNA only. Thus, HGV infection in hemophiliacs is more common than previous studies of HGV-RNA prevalence have suggested, but it resolved in most cases and caused chronic viremia only in a small number of patients, without biochemical evidence of persistent liver damage.