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Liver Stiffness Measurements & Fibrosis Detection After Liver Transplant
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"In this study, we prospectively evaluated whether repeated LSM (liver stiffness measurements) during the first year after LT (liver transplant) can identify patients at risk to develop significant fibrosis (F >/=2) or portal hypertension (HVPG >/=6 mmHg) at an early stage......A total of 73 HCV-infected LT recipients underwent repeated LSM at 3, 6, 9, and 12 months and a liver biopsy 1 year after LT (median = 12.3 months). An HVPG measurement was available in 65 patients at the same time......The results show that repeated LSM are able to discriminate between rapid and slow fibrosers during the first year after LT. Our study clearly shows two different speeds of liver fibrosis progression during the first year after LT: slow fibrosers, with fibrosis progression similar to patients without HCV, and rapid fibrosers, with early development of significant fibrosis and portal hypertension.....Liver fibrosis progression is extremely accelerated after LT, and graft cirrhosis develops in a significant proportion of patients within the first years. Early histological damage and increased HVPG values, only 1 year after transplantation, correlate with long-term outcome and identify patients with severe hepatitis C recurrence.....Despite the importance of these results, the main limitation of our study is the number of patients included, especially in the validation group. Nevertheless, it is important to note that our data were obtained using the two current gold standards to assess disease severity: liver biopsy and HVPG measurements.[13][23][38] In addition, an internal validation using a bootstrapping system was performed. Simple scores including bilirubin and LSM, or donor age and LSM at 6 months can accurately predict the risk to develop significant fibrosis or portal hypertension in these patients. This could be relevant to adopt therapeutic decisions at an early stage of HCV recurrence. Although our results need to be validated by other centers, we believe that these models might be widely used in clinical practice."
Liver Stiffness Clue to HCV Recurrence after Liver Transplant
North American Correspondent, MedPage Today
Published: January 05, 2010
Noninvasive liver stiffness measurements can accurately tell which liver transplant patients are having a recurrence of hepatitis C before classical signs are apparent, researchers said.
Six months after transplant, the method -- dubbed transient elastography -- identified patients whose new livers were rapidly developing fibrosis, according to Miquel Navasa, MD, of the Hospital Clinic in Barcelona, and colleagues.
The development of fibrosis of 2 or higher on the Scheuer classification a year after transplant is one sign that the virus has made a severe recurrence, Navasa and colleagues said in the January issue of Hepatology.
In this study, transient elastography also was able to make an early identification of patients with the other sign of recurrence -- portal hypertension a year after transplant, Navasa and colleagues reported.
Early identification of severe recurrence of hepatitis C is important, the researchers said, because the recurrence often results in loss of the grafted organ.
But fibrosis is usually tested with a liver biopsy, and measurements of the hepatic venous pressure gradient to detect portal hypertension are also invasive, the researchers noted.
In transient elastography, on the other hand, a mechanical pulse at the skin is propagated through the liver and its speed is measured with ultrasound. The method can detect fibrosis as well as portal hypertension, another sign of hepatitis C recurrence, the researchers said.
To evaluate the method in the context of liver transplant and hepatitis C, the researchers prospectively evaluated liver stiffness at three, six, nine, and 12 months after transplant in 84 patients with the virus.
Fibrosis in all 84 was measured at 12 months and portal hypertension was measured at the same time in a subset of 74.
Nineteen transplant patients who did not have hepatitis served as controls.
The question, Navasa and colleagues said, was whether repeated liver stiffness measurements would discriminate at an early stage between those at risk of significant fibrosis or portal hypertension.
They defined so-called rapid "fibrosers" as patients with liver fibrosis extending beyond the portal tracts (stages F2 through F4), while slow "fibrosers" were those showing absent or minimal fibrosis (stages F0 through F1).
Postal hypertension was defined as an hepatic venous pressure gradient of six or more millimeters of mercury.
Analysis showed that the median liver stiffness measurements at six, nine, and 12 months (but not at three months) were significantly higher in rapid than in slow fibrosers (at P<0.01 for all time points).
In the 53 slow fibrosers, the researchers found, the median liver stiffness measurement (measured in kilopascals) was 6.9, 6.9, 7.5, and 6.6 at months three, six, nine, and 12, respectively.
There was also no significant increase in the 19 controls.
In contrast, the 31 rapid fibrosers showed a progressive increase over time, with median stiffness of 7.5, 9.9, 9.5, and 12.1 kiloPascals in months three, six, nine, and 12, respectively. The increase was significant at P=0.030, the researchers reported.
In addition, the speed of progression of liver stiffness was significantly greater in rapid than in slow fibrosers, at P<0.001, they found.
The pattern for the hepatic venous pressure gradient was similar, with a significant difference (at P<0.001) in liver stiffness by six months between those at risk for portal hypertension and those not at risk, Navasa and colleagues said.
The researchers cautioned that the number of patients was small and said the study should be replicated at other centers.
Liver stiffness identifies two different patterns of fibrosis progression in patients with hepatitis C virus recurrence after liver transplantation
Hepatology Jan 2010
Jose A. Carrion 1, Ferran Torres 2, Gonzalo Crespo 1, Rosa Miquel 3, Juan-Carlos Garcia-Valdecasas 4, Miquel Navasa 1 *, Xavier Forns 1
1Liver Unit, Institut de Malalties Digestives, Hospital Clinic, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Centro de Investigacion Biomedica en Red de Enfermedades Hep‡ticas y Digestivas (CIBERehd), Barcelona, Spain
2Laboratory of Biostatistics and Epidemiology (Universitat Aut˜noma de Barcelona); Statistics and Methodology Support Unit, Hospital Clinic, IDIBAPS, Barcelona, Spain
3Pathology Unit, Hospital Clinic, IDIBAPS, CIBERehd, Barcelona, Spain
4Liver Transplantation and Hepatic Surgery Department, Hospital Clinic, IDIBAPS, CIBERehd, Barcelona, Spain
email: Miquel Navasa (mnavasa@clinic.ub.es)
Funded by:
Schering and Roche
Fundacion BBVA
Instituto de Salud Carlos III; Grant Number: PI050230, PI080239
Abstract
Significant liver fibrosis (F 2) and portal hypertension (hepatic venous pressure gradient [HVPG] 6 mmHg) at 1 year after liver transplantation (LT) identify patients with severe hepatitis C recurrence. We evaluated whether repeated liver stiffness measurements (LSM) following LT can discriminate between slow and rapid 'fibrosers' (fibrosis stage F2-F4 at 1 year after LT). Eighty-four patients who had undergone LT and who were infected with hepatitis C virus (HCV) and 19 LT controls who were not infected with HCV underwent LSM at 3, 6, 9, and 12 months after LT. All HCV-infected patients underwent liver biopsy 12 months after LT (paired HVPG measurements in 74); 31 (37%) were rapid fibrosers. Median LSM (in kilopascal) at months 6, 9, and 12 were significantly higher in rapid fibrosers (9.9, 9.5, 12.1) than in slow fibrosers (6.9, 7.5, 6.6) (P < 0.01 all time points). The slope of liver stiffness progression (kPa x month) in rapid fibrosers (0.42) was significantly greater than in slow fibrosers (0.05) (P < 0.001), suggesting two different speeds of liver fibrosis progression. Figures were almost identical for patients with HVPG >/=6 mmHg or HVPG < 6 mmHg at 1 year after LT. Multivariate analysis identified donor age, bilirubin level, and LSM as independent predictors of fibrosis progression and portal hypertension in the estimation group (n = 50) and were validated in a second group of 34 patients. The areas under the receiver operating characteristic curve that could identify rapid fibrosers and patients with portal hypertension as early as 6 months after LT were 0.83 and 0.87, respectively, in the estimation group and 0.75 and 0.80, respectively, in the validation group. Conclusion: Early and repeated LSM following hepatitis C recurrence in combination with clinical variables discriminates between rapid and slow fibrosers after LT.
Article Text
Hepatitis C virus (HCV) infection recurs universally after liver transplantation (LT)[1] and graft cirrhosis develops in a significant proportion of patients within the first years after LT.[2-4] As a result of this accelerated course, hepatitis C recurrence is the first cause of graft loss and reduction in patient survival in most liver transplant programs.[5] Thus, identification of patients at risk of severe recurrence at an early stage, in order to adopt therapeutic decisions,[6-8] becomes crucial. It is well known that early histological damage after transplantation correlates with severe hepatitis C recurrence and poor long-term outcome.[9][10] However, the sampling variability of liver biopsy may be a problem in individuals with rapid disease progression.[11][12] Interestingly, the hepatic venous pressure gradient (HVPG) has recently demonstrated to be extremely useful in the transplant setting, being more accurate than liver biopsy at identifying patients at risk of clinical decompensation.[13]
Nevertheless, liver biopsy and HVPG measurement are invasive and expensive methods, particularly if they need to be repeated during follow-up. To date, serological tests[14][15] and direct fibrosis markers[16] have not been fully validated in transplant patients, and diagnostic accuracy of indirect fibrosis markers is significantly lower than in individuals who have not undergone LT.[17-19] The application of these methods in LT recipients is troublesome because some serological markers can be altered by causes not related to fibrosis progression. In contrast, transient elastography, a new noninvasive and reproducible method to identify cirrhosis in HCV-infected patients,[20-22] has been shown to accurately assess liver fibrosis in the transplant setting.[23-25] In a cross-sectional analysis performed in HCV-infected LT recipients, there was a strong relationship between liver stiffness measurements (LSM) and fibrosis stage. More importantly, the correlation between LSM and HVPG was excellent.[23] The latter has recently been confirmed in patients with chronic hepatitis C and cirrhosis.[26][27]
In this study, we prospectively evaluated whether repeated LSM during the first year after LT can identify patients at risk to develop significant fibrosis (F 2) or portal hypertension (HVPG 6 mmHg) at an early stage.
Discussion
This longitudinal study evaluates whether repeated LSM during the first year after LT are useful to identify patients with severe hepatitis C recurrence at an early stage. The results show that repeated LSM are able to discriminate between rapid and slow fibrosers during the first year after LT.
Our study clearly shows two different speeds of liver fibrosis progression during the first year after LT: slow fibrosers, with fibrosis progression similar to patients without HCV, and rapid fibrosers, with early development of significant fibrosis and portal hypertension. In fact, the mathematical mixed model for repeated LSM and the slope of liver stiffness progression in rapid and slow fibrosers, clearly confirmed the different speed of liver stiffness progression in patients with mild and severe recurrence. In a subgroup of patients with cholestatic hepatitis, liver stiffness progression was extremely fast, but the small number of patients does not allow firm conclusions to be drawn. It seems that the events leading to different speeds of fibrosis progression in HCV-infected LT recipients take place very early following transplantation.
Liver fibrosis progression is extremely accelerated after LT, and graft cirrhosis develops in a significant proportion of patients within the first years.[2-4] Early histological damage and increased HVPG values, only 1 year after transplantation, correlate with long-term outcome and identify patients with severe hepatitis C recurrence.[10][13] Patients with significant fibrosis and particularly with portal hypertension 1 year after LT have a high probability of clinical decompensation and graft loss. For this reason, in the current study, patients were classified using both liver biopsy and HVPG, to identify patients with slow or rapid disease progression. In addition, liver stiffness determination has recently been shown to be an excellent noninvasive method to identify patients with significant fibrosis and is even more accurate to diagnose patients with portal hypertension.[23]
In the present study, the diagnostic accuracy of liver stiffness increased with time. The accuracy to identify rapid fibrosers was poor at 3 months, good at 6 and 9 months, and excellent at 12 months after LT, especially in patients with portal hypertension. Actually, we have previously shown that LSM at 1 year after LT is very accurate to identify significant fibrosis and has an excellent correlation with HVPG values to diagnose portal hypertension.[23] On the other hand, the current study shows that LSM at 3 months after LT is not useful in the prediction of the different patterns of HCV recurrence. At this early time, fibrosis deposition is probably too low to affect liver stiffness determination. Moreover, other complications which are frequent during the first months following LT (acute hepatitis, acute rejection, or vascular or biliary problems) might influence liver stiffness independently of the degree of liver fibrosis.[32-35]
Six months appears to be an important time point for LSM for two reasons: first, the accuracy is high enough to discriminate patients with severe recurrence from those with mild recurrence; second, antiviral treatment at this time could probably decrease or even interrupt fibrosis progression in patients with severe hepatitis C recurrence. Therefore, we sought to increase the diagnostic accuracy of liver stiffness by developing fibrosis - and HVPG - scores at this time point. The variables selected in the estimation group by the multivariate regression model were donor age, LSM, and bilirubin at 6 months. Donor age appeared as an important factor influencing HCV recurrence. Several studies have pointed out the importance of this variable in the severity of HCV recurrence.[36][37] In addition, recipients who develop severe HCV-induced graft damage have significantly higher aminotransferases and bilirubin levels than patients with milder forms.[4] The fibrosis score cutoff of -1.99 identified 63% of slow fibrosers with a high NPV of 86%. Interestingly, values higher than -1.27 identified 64%-70% of patients with severe recurrence and 100% of patients with cholestatic hepatitis.
As we have previously shown, LSM are very accurate at identifying patients who underwent liver transplantation who have portal hypertension.[23] This probably also explains the high accuracy of the HVPG score in the current study in which a cutoff of -0.3 identified 89% of patients with normal portal pressure (89% of certainty). In contrast, values higher than 0.15 identified 61% of patients with a risk of developing portal hypertension (92% of certainty). These results reinforce the concept of HVPG determination as a good gold standard for the evaluation of new noninvasive methods.[13][23][38]
These results support the use of noninvasive methods to monitor HCV recurrence in the transplant setting. The fibrosis and/or HVPG score at 6 months may be useful to decide the best therapeutic strategy in these patients. In patients with a HVPG score below -0.3 at 6 months after LT, follow-up with repeated LSM may be appropriate, because 80% (41 of 51) of these patients remain without significant fibrosis at 1 year. In contrast, in patients with a HVPG score higher than 0.15, antiviral treatment should be considered, because 90% (19 of 21) of these patients develop portal hypertension 1 year after LT. Nevertheless, if HCV treatment is indicated, a liver biopsy before treatment initiation is still necessary to exclude other causes of liver dysfunction.[39]
Despite the importance of these results, the main limitation of our study is the number of patients included, especially in the validation group. Nevertheless, it is important to note that our data were obtained using the two current gold standards to assess disease severity: liver biopsy and HVPG measurements.[13][23][38] In addition, an internal validation using a bootstrapping system was performed.
In summary, repeated measurements of liver stiffness in HCV patients after LT allow discrimination between rapid and slow fibrosers. Simple scores including bilirubin and LSM, or donor age and LSM at 6 months can accurately predict the risk to develop significant fibrosis or portal hypertension in these patients. This could be relevant to adopt therapeutic decisions at an early stage of HCV recurrence. Although our results need to be validated by other centers, we believe that these models might be widely used in clinical practice.
Results
Patient Characteristics.
From August 2004 to January 2008, 84 LT recipients with HCV infection and 19 with other etiologies were included. The cause of LT in non-HCV-infected patients was: alcoholic cirrhosis (n = 10), primary biliary cirrhosis (n = 2), Caroli's disease (n = 2), familial amyloid polyneuropathy (n = 2), autoimmune hepatitis (n = 1), and cryptogenetic cirrhosis (n = 2). The baseline characteristics (donors and recipients) of all patients (n = 103), including histological and hemodynamic data 1 year after LT, are summarized in Table 1.
Progression of Liver Stiffness and Fibrosis During the First 12 Months After LT.
All HCV-infected patients showed histological signs of chronic hepatitis C recurrence. Acute rejection was carefully investigated and not detected in any of the liver biopsies performed at 12 months. Liver biopsy in the five patients not infected with HCV showed mild steatosis without Mallory hyaline (n = 1), minimal sinusoidal dilatation (n = 1), and unspecific mononuclear infiltration (n = 3). Of the liver biopsies, 41 (46%) were percutaneous and 48 (54%) were transjugular. The median of total length was 17 mm (8-23 mm) in percutaneous biopsies and 16 mm (6-36 mm) in transjugular biopsies (P = 0.602), with 91% of specimens >/=10 mm, 68% >/=15 mm, and 32% >/=20 mm. We found a good correlation between significant fibrosis and portal hypertension (kappa = 0.62) including liver biopsies < 15 mm (kappa = 0.75) and < 10 mm (kappa = 1.0).
A total of 335 valid LSM were available during the first 12 months after LT. Seventy-seven (19%) LSM were not determined during the study (29 patients underwent three LSM and 24 patients underwent two) for different reasons: 19 patients had a high body mass index (ranging from 28-33), 11 patients with cholestatic hepatitis received antiviral therapy, and 9 control and 14 HCV-infected patients did not strictly comply with the LSM timetable. Progression of LSM after LT was different among the control group, and the slow and rapid fibrosers (Fig. 1A). In all control patients (n = 19), LSM did not significantly increase during the first year after LT. Median LSM at months 3, 6, 9, and 12 were 5.4, 6.2, 6.4, and 5.6 kPa, respectively (P = 0.334). The median LSM of slow fibrosers (n = 53) at months 3, 6, 9, and 12 was 6.9, 6.9, 7.5, and 6.6 kPa, respectively, without a significant increase during follow-up (P = 0.422). By contrast, rapid fibrosers (n = 31) showed a progressive increase over time; the median LSM at months 3, 6, 9, and 12 was 7.5, 9.9, 9.5, and 12.1 kPa, respectively (P = 0.030). LSM differed significantly between rapid and slow fibrosers at months 6 (P < 0.001), 9 (P = 0.002), and 12 (P < 0.001) after LT (Fig. 1A). The figures were almost identical for patients with and without portal hypertension 1 year after LT (Fig. 1B). In patients with cholestatic hepatitis (n = 11), liver biopsy indicated F0 in one patient, F2 in three patients, F3 in two patients, F4 in one patient, and fibrosing cholestatic hepatitis in four patients. All patients with cholestatic hepatitis and HVPG measurements (n = 9) showed portal hypertension and seven had clinically significant portal hypertension (HVPG 10). The mean values of LSM at months 3, 6, and 9 in patients with cholestatic hepatitis were 14.5, 18.2, and 24.5 kPa, respectively (P = 0.050).
The diagnostic accuracy of liver stiffness to identify rapid fibrosers improved over time after LT. The AUROC curve for diagnosis of rapid fibrosers at months 3, 6, 9 and 12 after LT was 0.67, 0.79, 0.77, and 0.92 in the estimation group and 0.47, 0.66, 0.74, and 0.80 in the validation group, respectively (Fig. 2). The sensitivity, specificity, predictive values, and the likelihood ratio of the optimal cutoffs values of liver stiffness at 6 months for predicting significant fibrosis (F >/=2) are summarized in Table 2.
Patients with Discrepancies Between Liver Biopsy and HVPG Values.
Among 74 patients with liver biopsy and HVPG determination, 13 (18%) patients had discrepancies between liver fibrosis and portal pressure. The median length of 'discrepant' biopsies was 17 mm (11-25 mm). There were five patients with F >/=2 and HVPG < 6. These patients (n = 5) had periportal fibrosis (F = 2) and the median LSM was 10.8 kPa (5.9-18 kPa). However, the median HVPG was 4 (3.5-5) mmHg. In contrast, there were 8 patients with F < 2 and HVPG >/=6. The median HVPG was 7 mmHg (6-10 mmHg) and median LSM was 10 kPa (8.4-28 kPa). Liver biopsy showed steatosis >/=60% in one patient, hepatocyte ballooning in three patients, necroinflammatory activity >/=4 in three patients, and sinusoidal fibrosis in four patients.
Correlation Between Liver Stiffness and HVPG Values.
A paired HVPG determination 12 months after LT was available in 74 (88%) HCV-infected patients. The correlation between liver stiffness and HVPG values increased during the first year after LT, being significant after the first 6 months. Differences in LSM between patients who developed portal hypertension (HVPG >/=6 mmHg) (n = 29) and patients with normal portal pressure (HVPG < 6 mmHg) (n = 45) at 1 year after LT were significant at months 6, 9, and 12 (P < 0.001 at all time points; Fig. 1B). The diagnostic accuracy of liver stiffness to identify patients with portal hypertension (HVPG >/=6 mmHg) at 1 year after LT improved over time. The AUROC curves at 3, 6, 9, and 12 months after LT to identify patients with portal hypertension were 0.72, 0.77, 0.80, and 0.92 in the estimation group and 0.58, 0.79, 0.84, and 0.93 in the validation group, respectively (Fig. 2). The comparative performance of liver stiffness cut-off values at 6 months for predicting portal hypertension (HVPG 6) is summarized in Table 2.
Regression Model Using Liver Stiffness Changes/Progression to Recognize Rapid and Slow Fibrosers in the Total Cohort.
In order to demonstrate the existence of different rates of liver fibrosis progression we used the MMRM of liver stiffness determinations. In patients with absent or mild fibrosis (F0-F1) and those with normal portal pressure (HVPG < 6 mmHg) at 1 year after transplantation, liver stiffness did not progress during the first 12 months (P = 0.5). The slope of liver stiffness progression (kPa x month) in these slow fibrosers (0.05) and in patients with normal portal pressure (0.00), was similar to that of controls (-0.02). On the contrary, the slope of liver stiffness progression in patients with significant fibrosis (0.42) or portal hypertension (0.46) at 1 year after LT was significantly higher than that found in controls and slow fibrosers (P < 0.0001) (Fig. 3A). In patients with cholestatic hepatitis the slope (1.54) of liver stiffness progression was significantly higher than that found in rapid fibrosers without cholestatic hepatitis (P < 0.0001) (Fig. 3B).
Predictors of Rapid Fibrosis Progression and/or Portal Hypertension and Estimation of the Model.
Univariate and multivariate analyses were performed in the estimation group (n = 50) to identify the variables associated with the presence of significant fibrosis (F >/=2) at 1 year after LT (Table 3). The univariate analysis identified six variables associated with rapid fibrosis progression: cytomegalovirus infection, alanine aminotransferase level, bilirubin level and HCV viral load (at 3 months), and bilirubin level and LSM (at 6 months). The multivariate analysis showed that only two variables at 6 months were independent predictors of fibrosis: LSM (P = 0.032) and bilirubin level (P = 0.034). We used these variables and their coefficients of regression to construct a predictive model to identify rapid fibrosers (-4.347 + 0.264 x LSM [kPa] 6m + 0.442 x bilirubin [mg/dL] 6m). The diagnostic value of this fibrosis score was assessed in the estimation (area under the curve = 0.83) and validation group (area under the curve = 0.75) (Fig. 4). The results of the internal bootstrap validation gave good estimates for the AUROC curve of 0.840 (0.667-0.960) for fibrosis score. A fibrosis score cutoff of -1.99 identified 63% of slow fibrosers with high certainty (NPV = 86%) in the estimation group. The same cutoff identified 59% of slow fibrosers with 94% of certainty in the validation group (Table 4). Using a higher cutoff of -1.27 we identified 70% of rapid fibrosers in the estimation group (PPV = 70%) and 64% in the validation group (PPV = 58%) (Table 4). This cutoff also identified the 11 patients with cholestatic hepatitis.
Univariate and multivariate analyses were performed in the estimation group (n = 43) to identify the variables associated with the presence of portal hypertension (HVPG >/=6) at 1 year after LT (Table 5). Donor age, cytomegalovirus infection, HCV viral load at 3 months, and LSM at 3 and 6 months were associated with portal hypertension in the univariate analysis. Only two variables were identified as independent predictors of HVPG >/=6 by multivariate analysis: donor age (P = 0.004) and LSM at 6 months (P = 0.003). We used these variables and their coefficients of regression to construct a predictive model to identify patients at risk to develop portal hypertension 6 months after LT (HVPG-score = 0.05 x donor age [years] + 0.26 x LSM [kPa] at 6 months). The diagnostic value of HVPG-score was assessed in the estimation group (area under the curve = 0.87) and in the validation group (0.80) (Fig. 4). The results of the internal bootstrap validation gave good estimates for the AUROC curve of 0.881 (0.708-0.987) for HVPG score. A HVPG score cutoff of -0.3 identified 89% of patients with normal portal pressure with 89% of certainty in the estimation group. The same cutoff identified 85% of patients with HVPG < 6 mmHg (NPV = 85% in the validation group). A cutoff of 0.15 identified 61% of patients with portal hypertension with 92% of certainty in the estimation group and 73% of patients in the validation group (PPV = 90%) (Table 4).
Patients and Methods
Patients.
From August 2004 to January 2008, 132 consecutive patients with HCV recurrence after LT out of a total of 293 patients who underwent transplantation in our institution were considered for the study. Exclusion criteria were: graft or patient survival shorter than 12 months after LT (n = 17); combined kidney and liver transplantation (n = 4); hepatitis B virus or human immunodeficiency virus coinfection (n = 3); presence of ascites (n = 6), body mass index > 33 (n = 2), chronic graft rejection (n = 5), biliary tract complications (n = 8), veno-occlusive disease (n = 1), de novo autoimmune hepatitis (n = 1) and recurrence of hepatocellular carcinoma (n = 1) during the first year after LT. Therefore, the final number of HCV-infected LT recipients included was 84 (64%). Another 19 patients who underwent LT for other etiologies were included as the control group.
Patients were managed according to previously published protocols.[28] Induction immunosuppression was cyclosporine A or tacrolimus and prednisone. Mycophenolate mofetil was added in patients who required cyclosporine or tacrolimus dose reduction or discontinuation. Immunosuppression therapy was recorded throughout the study. Acute rejection episodes were documented by liver histologic analysis and treated with steroid boluses if moderate or severe.
After discharge, patients were visited at the outpatient clinic, monthly for the first 3 months with complete recording of clinical and analytical variables, and every 2 or 3 months thereafter. A total of 73 HCV-infected LT recipients underwent repeated LSM at 3, 6, 9, and 12 months and a liver biopsy 1 year after LT (median = 12.3 months). An HVPG measurement was available in 65 patients at the same time. The remaining 11 patients had cholestatic hepatitis.[29] In these patients, liver biopsy (n = 11) and HVPG (n = 9) were performed when the clinical diagnosis was suspected (median = 6.7 months). LSM before initiation of antiviral treatment were available at 3 and 6 months in eight patients and at months 3, 6, and 9 in three. Another five non-HCV-infected patients with elevated alanine aminotransferase (>/=40 IU/L) underwent a liver biopsy 1 year after LT (median = 13.4 months).
The study was previously approved by the Investigation and Ethics Committee of the Hospital Clinic of Barcelona following the ethical guidelines of the 1975 Declaration of Helsinki. We obtained informed consent from all patients included in the study.
Transient Elastography.
Patients underwent repeated LSM using Fibroscan (Echosens, Paris, France). The operator was a nurse (C.B.) who had been previously trained by a staff member of the Echosens Company and had performed more than 100 LSM in patients with chronic liver disease and LT. Liver stiffness was determined as previously described[20-23] at months 3, 6, 9, and 12 after LT. LSM was determined on the right lobe of the liver. The results were expressed in kilopascals and a median value of 10 acquisitions was considered for analysis, including cases with a success rate lower than 60%.
Liver Biopsies and Paired HVPG Measurements.
Percutaneous or transjugular liver biopsies and HVPG measurements were performed as previously described.[13] Samples were processed at the Pathology Department and stained with hematoxylin and eosin, and Masson's trichrome staining. An expert pathologist (R.M.) who did not know either the HVPG or transient elastography values scored all the histological samples.
Necroinflammatory activity and fibrosis stage were scored using the Scheuer classification, which classifies liver fibrosis as absent (F0), restricted to the portal tract (F1), periportal or portal-portal septa with intact architecture (F2), bridging fibrosis with architectural distortion but no obvious cirrhosis (F3), and cirrhosis (F4).[30][31] The minimal acceptable size of liver biopsy was considered 5 mm.
Study Endpoints and Definitions.
The main endpoint of our study was to evaluate whether repeated LSM, during the first 12 months after LT, was able to discriminate patients at risk to develop significant fibrosis (F >/= 2) or portal hypertension (HVPG >/=6 mmHg) at an early stage.
We defined rapid fibrosers as patients with liver fibrosis extending beyond the portal tracts (F2-F4), while slow fibrosers were those showing absent or minimal fibrosis (F0-F1) at 1 year after LT.
Statistical Analysis.
Quantitative variables were expressed as medians (range). Differences between qualitative variables were assessed with the Fisher exact test. Differences between quantitative variables were analyzed with a nonparametric test (Mann-Whitney or Kruskal-Wallis for unpaired samples and Friedman for several related samples). We estimated the linear slope of LSM for each categorized group of fibrosis (F0-1 versus F2-F4), portal pressure (HVPG < 6 versus HVPG >/=6 mmHg) and control patients using a longitudinal mixed model for repeated measurements (MMRM). Differences between slow and rapid fibrosers (as well as with controls) regarding donor age, liver stiffness, and relevant host-related variables were analyzed by univariate analysis during the first 6 months after LT. Variables showing a P value < 0.05 were included in a multivariate forward stepwise logistic regression analysis to determine the independent predictors of significant fibrosis 1 year after LT. The same procedure was used to identify independent predictors of portal hypertension 1 year after LT. Significant fibrosis or portal hypertension were considered as a positive result and their absence as a negative result. Data from a randomly generated split-sample of 50 (60%) patients were used to estimate the model, and data from the remaining 34 (40%) patients were used to validate the model. A predictive model was constructed by modeling the values of the independent variables and their regression coefficients. Each of the variables included in the model was analyzed to rule out any significant differences between the estimation and the validation groups. Bootstrapping was used to perform an additional internal validation by generating 10,000 resampling sets with replacement. The results of the internal bootstrap validation gave estimates for the area under the receiver operator characteristic (AUROC) curve with the median (5th percentile-95th percentile). The diagnostic accuracy of LSM (at each time point) and of the predictive model (at 6 months) to identify patients at risk to develop significant fibrosis (F >/=2) and portal hypertension (HVPG >/= 6 mmHg) at 1 year after LT were assessed using the AUROC curve. The optimal LSM and score cutoff values were selected on the basis of sensitivity (S), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) to identify significant fibrosis and portal hypertension. We used SAS version 9.1.3 software (SAS Institute Inc., Cary, NC) for the MMRM analysis. All other analyses were done with SPSS 12.0 (SPSS Inc., Chicago, IL).
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