LIVER TRANSPLANTATION 15:876-882, 2009
ORIGINAL ARTICLE
Prognostic Value of Computerized Quantification of Liver Fibrosis in Children with Biliary Atresia Lars Pape,* Karen Olsson,* Claus Petersen,2 Reinhard von Wasilewski,3 and Michael Melter Departments of 1Pediatric Nephrology, Hepatology, and Metabolic Disorders, 2Pediatric Surgery, and 3 Pathology, Hannover Medical School, Hannover, Germany
Biliary atresia (BA) is the leading indication for liver transplantation (LTx) during childhood. Predictive markers for progression are lacking. We investigated the correlation between quantified fibrosis of the liver at the time of Kasai hepatic portoenterostomy (KPE) and the clinical course. Liver tissue was obtained at the time of KPE in 53 children and stained with Picrosirius red. The mean volume of fibrosis per number of periportal fields (Vfib) and the Ishak score were calculated. Vfib was 3.9%. Transplant-free survival was significantly higher in those patients with Vfib ⬍ 2.5% (P ⬍ 0.05, Kaplan-Meier analysis). Vfib ⬍ 2.5% predicted transplant-free survival with a sensitivity of 100% and a specificity of 48% (positive predictive value: 100%, negative predictive value: 17%). Vfib was correlated with bilirubin 6 months after KPE (Bili6m). The negative predictive value could be enhanced to 23% if Vfib ⬍ 2.5% was combined with Bili6m ⬍ 20 mol/L. The Ishak score showed no correlation with transplant-free survival or Vfib. Detection of Vfib at the time of KPE is a valid marker in predicting transplant-free survival in children with BA. This method should be considered an integral part of scoring systems predicting the indication for LTx. Liver Transpl 15:876-882, 2009. © 2009 AASLD. Received July 29, 2008; accepted November 16, 2008.
See Editorial on Page 829 Biliary atresia (BA) is an infantile obstructive cholangiopathy of unknown etiology characterized by progressive fibro-obliteration and obstruction of the extrahepatic and intrahepatic bile ducts. This disease affects approximately 1 of 18,000 live births in Europe. Untreated, this condition leads to biliary cirrhosis and death within the first years of life.1-5 The major problems concerning the management of this rare disease are late referral and diagnosis and unsuccessful surgery.6
The treatment of choice is to restore bile flow into the intestinal tract via Kasai hepatic portoenterostomy (KPE).7 Even with early KPE, progressive inflammation and fibrosis of the intrahepatic bile ducts may develop, leading to biliary cirrhosis and the need for pediatric liver transplantation (LTx) in about 80% of all patients8-10; this makes BA the leading indication for LTx in children. The timing of LTx continues to be one of the main prognostic factors for the overall outcome in patients with BA.11 However, there are no well-defined markers
Abbreviations: BA, biliary atresia; Bili6m, bilirubin 6 months after Kasai hepatic portoenterostomy; FI, fibrosis index; HPE, hepatic portoenterostomy; KPE, Kasai hepatic portoenterostomy; LTx, liver transplantation; PSR, Picrosirius red; ROC, receiver operating characteristic; Vfib, mean volume of fibrosis per number of periportal fields (percentage of fibrosis/periportal field). Lars Pape contributed to the design and conception of the study together with Michael Melter, established the histological procedures, was responsible for the funding, and drafted the first version of the article together with Karen Olsson. Karen Olsson performed the staining procedures, the computerized evaluation of the tissues, and the statistical procedures and drafted the first version of the article together with Lars Pape. Claus Petersen collected clinical data and critically revised the article for important intellectual content. Reinhard von Wasilewski performed the calculation of the Ishak score and critically revised the article for important intellectual content. Michael Melter initiated the study, contributed to the design and conception of the study together with Lars Pape, and critically revised the article for important intellectual content. Karen Olsson is currently affiliated with the Department of Pneumology, Hannover Medical School, Hannover, Germany. Michael Melter is currently affiliated with the Children’s Hospital, University of Regensburg, Regensburg, Germany. *These authors contributed equally to this study. Address reprint requests to Lars Pape, M.D., Department of Pediatric Nephrology, Hepatology, and Metabolic Disorders, Hannover Medical School, Carl-Neuberg-Straße 1, D-30625 Hannover, Germany. Telephone: ⫹49-511-532-5706; FAX: ⫹40-511-530-4830; E-mail:
[email protected] DOI 10.1002/lt.21711 Published online in Wiley InterScience (www.interscience.wiley.com).
© 2009 American Association for the Study of Liver Diseases.
COMPUTERIZED QUANTIFICATION OF LIVER FIBROSIS 877
suggesting the correct therapeutic option at the time of diagnosis of BA. The current prognostic scores are based on clinical and biochemical variables that lack accuracy in detecting the progression of liver fibrosis. Therefore, a major research challenge is to determine markers to reliably predict the overall prognosis and to help in guiding the individual therapeutic strategy in infants with BA. Several studies have demonstrated that the extent of fibrosis or cirrhosis at the time of KPE correlates with the short-term and median-term prognosis,2,12,13 but the amount of fibrosis seems to differ according to different techniques and scoring systems. Unspecific analogue histological classifications such as the Ishak score have been tested but have not proved to be good standards.6 In order to find better monitoring parameters and to improve the value of liver biopsy, computerized quantification of hepatic fibrosis has been tested in this study. Picrosirius red (PSR) is a staining method specifically for collagen I, collagen II, and collagen III, and it was first described in 1964.14 Because of the interaction of the sulfuric groups of PSR and basic groups of collagen, PSR molecules are bound parallel to the collagen molecules and cause an enhancement of birefringence15 under polarized light. PSR has been used as a marker to quantify the extent of fibrosis in kidneys,15,16 and it shows a good correlation between fibrosis and function of native kidneys.17 Recent studies have shown a highly significant correlation between PSR-positive staining and chronic allograft nephropathy. PSR has proved to be a good marker for long-term graft function in kidney transplant patients.18,19 PSR has also proved to be an accurate staining method in animal models.20,21 In 1992, Moragas et al.22 used this method to measure fibrosis in liver biopsies of children undergoing LTx. In this study, we tested whether the computerized quantification of fibrosis by PSR might serve as a predictive marker in BA, adding new supplementary information to established predictors such as serum bilirubin 6 months after KPE (Bili6m).
PATIENTS AND METHODS At the Children’s Hospital of Hannover Medical School, 2 liver specimens from each patient with BA at the time of KPE were stained with PSR and provided for routine histology. PSR staining was performed for all specimens at 1 time point. The diagnosis of BA was confirmed by operative findings and in some cases supported by endoscopic retrograde cholangiogram. All patients were followed up in our children’s hospital by routine laboratory investigation and abdominal ultrasound for at least 6 months after KPE. The mean observation time of the patients until death or the end of the study was 39.28 months (1.78-80.62 months; Table 1). The indication for LTx was set according to the patient’s overall condition with consideration of the following parameters: serum bilirubin ⬎ 340 mol/L, hepatic artery resistance index ⬎ 80, serum albumin ⬍ 33
TABLE 1. Demographic Data for 53 Study Patients with Biliary Atresia Demographic Data
n
Patients Female/male Transplanted patients Deceased patients Transplanted and deceased Retransplantation
Age at KPE (months) Age at LTx (months) Age at death (months) Interval from KPE to LTx (months) Interval from KPE to death (months)) Interval from KPE to LTx or death (months) Observation time until death or 01/02/2005 (months)
53 29/24 26 10 5 1 Median (Range) 1.8 (0.7–4) 10.2 (5.0–54) 9 (3.1–42) 8.4 (2.6–52.1) 6.5 (1.8–38.3) 2.1 (1.8–52.1) 38.3 (1.8–80.6)
Abbreviations: KPE, Kasai hepatic portoenterostomy; LTx, liver transplantation.
mg/dL, and growth arrest.11,23 Transplant-free patient survival (time of KPE to LTx or death in nontransplanted children) was evaluated in association with Bili6M ⬍ 20 mol/L or Bili6M ⬎ 20 mol/L. The correlation between the mean volume of fibrosis per number of periportal fields (Vfib), Ishak score, and Bili6m was analyzed.
Reproducibility/Reliability In order to exclude methodical problems, 8 biopsies of children with cholestatic liver diseases (metabolic disorders, n ⫽ 2; undefined obstructive cholestatic liver disease, n ⫽ 2; glycogenosis, n ⫽ 1; Jeune syndrome, n ⫽ 1; and alpha 1 antitrypsin deficiency and neonatal hepatitis, n ⫽ 1) were stained. Additionally, sections of 1 paraffin block were stained with 4 different sets of reagents and measured by 1 operator on 4 different occasions. The same biopsy was measured by 2 different operators. The coefficients of variance were determined. Vfib in 8 control biopsies was 3.05% (standard deviation: 2.02%). There was no significant difference in the mean amount of fibrosis measured in biopsies stained with 4 different sets of reagents, at 4 different times, or by 2 operators (1-way analysis of variance: P ⫽ 0.93/ 0.97/0.80, respectively).
Histopathology and Ishak Score The use of human tissue for this study was approved by the Ethics Committee of Hannover Medical School, and informed written consent was obtained from parents of all patients. All liver biopsy specimens were wedge bi-
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TABLE 2. Modified Ishak Score Considering Architectural Changes, Fibrosis, and Cirrhosis Liver Changes
Score
No fibrosis Fibrous expansion of some portal areas with or without short fibrous septa Fibrous expansion of most portal areas with or without short fibrous septa Fibrous expansion of most portal areas with occasional portal-to-portal bridging Fibrous expansion of most portal areas with marked bridging (portal-to-portal and portal-to-central bridging) Marked bridging (portal-to-portal and/or portal-to-central bridging) with occasional nodules (incomplete cirrhosis) Cirrhosis, probable or definite
0 1 2 3 4 5 6
NOTE: The Ishak score ranges from 0 (no fibrosis) to the maximum possible score of 6 (cirrhosis).24
opsies and were immediately fixed in formalin/alcohol/ acetic acid and embedded in paraffin wax. Recently, we showed that the level of fibrosis is comparable in wedge and needle biopsies in children with BA (C.P., personal communication, 2008). Routine histology was performed, and liver fibrosis was evaluated semiquantitatively according to the Ishak score (Table 2).24 The fibrosis was assessed retrospectively by a single senior pathologist (R.v.W.). The liver biopsies were classified as follows [median Ishak score: 3 (range: 0-6)]: score of 0, n ⫽ 1; score of 1, n ⫽ 6; score of 2, n ⫽ 5; score of 3, n ⫽ 16; score of 4, n ⫽ 13; score of 5, n ⫽ 7; and score of 6, n ⫽ 5. One biopsy could not be evaluated.
PSR Staining PSR was prepared from a 10-mL 1% Sirius red solution (Aldrich, Munich, Germany) and 90 mL of saturated picric acid (Aldrich).18 For the PSR staining, the whole tissue block was cut, and the slides were stained completely. Two liver tissue specimens from each patient were incubated at 60°C for 1 hour, then deparaffinized in xylene (twice for 10 minutes), hydrated in graded ethanol (100%/90%/70%/50%/20%) for 2 minutes each, washed in distilled water, and stained with PSR overnight. In general, 0.5 mL of PSR per slide was needed. After 24 hours, slides were put into 0.01 N HCl for 2 minutes, dehydrated in ethanol (80%/95%/100%) and xylene for 2 minutes each, and finally cover-slipped in Corbit Balsam (Hecht, Kiel, Germany).
Image Acquisition With a light microscope combined with a video system (Olympus BX 50 and Olympus DP12, Olympus, Hamburg, Germany), a background picture was taken, and periportal fields were counted with the microscope set on overview. Images of the entire biopsy were acquired with 100⫻ magnification under polarized light with crossed polarizers moving in a serpentine progression from one end of the tissue toward the other. Depending on the size of the biopsy, the number of pictures taken varied from 7 to 80 (median: 27). With conventional microscopy, PSR-stained liver tissue shows a light pink/rose color, and connective tissue fibers appear as a deep red and show enhanced
birefringence under polarization microscopy, which cannot be recognized in collagen-negative tissue. In Fig. 1, a section stained with PSR is shown photographed with and without polarized light, along with a specimen with a high level of fibrosis and a specimen with a small amount of fibrosis.
Image Analysis The background picture was subtracted from all pictures with Adobe Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA) in order to eliminate background color. Further image analysis was performed with Scion Image (Scion Corp., Frederick, MD) blinded to the histopathological diagnosis and clinical data of the patient. The same background threshold was used in all biopsy specimens to distinguish black and white parts of the specimen. Subsequently, the amount of white and black was analyzed, and the percentage of white matter, representing PSR-positive tissue (liver fibrosis), was calculated with an automatic macro. The mean fibrosis of 2 specimens from each patient was determined, and the ratio of the calculated mean fibrosis to the number of counted periportal fields (Vfib; percentage of fibrosis/periportal field) was created. Images were stored in sequential fashion on a CD-ROM or a DVD (tiff format). With an experienced operator, the process, including image acquisition, processing, and calculation, took about 5 to 10 minutes for each biopsy.
Statistical Analysis The statistical analysis was realized in collaboration with the Hannover Medical School Institute of Biometry. Data were analyzed with SPSS, version 14 for Windows (SPSS, Inc., Chicago, IL). Normally distributed data are presented as mean ⫾ standard deviation; differences are compared with the t test, and relationships are compared with Pearson’s correlation. Otherwise, medians and ranges are presented and compared with the Mann-Whitney U test. Patient survival was analyzed by Kaplan-Meier analysis, Cox regression, and logistic regression. In other studied parameters, a 1-way analysis of variance or t test (2-tailed distribution) was used as appropriate for comparison between groups. Receiver operating characteristic analysis was used
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COMPUTERIZED QUANTIFICATION OF LIVER FIBROSIS 879
Figure 1. Liver tissue stained with Picrosirius red. (A,B) The same section of a specimen captured under white light and polarized light, respectively. (C) A representative specimen of tissue with a small amount of fibrosis. (D) A representative specimen of tissue with a relatively high grade of fibrosis captured under polarized light. All images were acquired with 100ⴛ magnification.
to determine a cutoff point for Vfib in order to predict early mortality with optimal sensitivity and specificity.
RESULTS Liver tissue was obtained at the time of KPE for 53 children with BA (29 females and 24 males, median age: 54 days, range: 20-122 days, study period: 1998-2003). The median age at KPE was 1.8 months (range: 0.7-4 months). The median time of follow-up was 38 months (range: 54 days to 81 months). Twenty-six of the 53 children underwent LTx. Ten patients died, 5 of them after LTx; 7 of 10 patients died within the first 12 months after KPE. Five patients died before transplantation; 1 of them died because of cardiac failure caused by dilating cardiomyopathy, and 4 died from liver failure on the waiting list for LTx. Twenty-four patients were transplanted within the first 18 months after KPE, whereas only 2 transplants were performed after 40 months. Age at KPE had no significant influence on trans-
plant-free survival (P ⫽ 0.19, t test). The median age at KPE of the children who died before transplantation was 2.2 months versus 1.8 months for those surviving. Vfib of all 53 children was 3.9% (standard deviation: 3.8). In the group of children who died before transplantation, 5 of 5 patients had Vfib ⬎ 2.5 (P ⫽ 0.04, Pearson’s chi-square test). In receiver operating characteristic analysis, Vfib ⬍ 2.5% predicted survival with a sensitivity of 100% and a specificity of 48% (Fig. 2); therefore, we selected a Vfib value of 2.5% as the threshold value. The positive predictive value for transplant-free survival in children with Vfib ⬍ 2.5% was 100%, and the negative predictive value in the group with Vfib ⱕ 2.5% was 17% (P ⫽ 0.04, Pearson’s chisquare test). In Kaplan-Meier analysis, transplant-free survival was significantly higher in children with Vfib ⬍ 2.5% (Fig. 3). Vfib did not correlate with time to transplantation (P ⫽ 0.05); however, there was a tendency of earlier transplantation in those children with higher levels of Vfib. A correlation between Vfib and serum bilirubin at the
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Figure 2. In ROC analysis, Vfib < 2.5% predicted survival with a sensitivity of 100% and a specificity of 48%. Abbreviations: ROC, receiver operating characteristic; Vfib, mean volume of fibrosis per number of periportal fields.
Figure 3. In Kaplan-Meier analysis, transplant-free survival was significantly higher in children with Vfib < 2.5% (P < 0.05). Abbreviations: Cum, cumulative; HPE, hepatic portoenterostomy; LTx, liver transplantation; Vfib, mean volume of fibrosis per number of periportal fields.
time of KPE (r ⫽ 0.43, P ⬍ 0.01) was demonstrated. The median value of Bili6m was 19 mol/L (range: 3.0-534 mol/L). In the group of children who died before transplantation, 2 died before the time point of 6 months after KPE. Therefore, Bili6m could be measured in only 3 of 5 children who did survive until transplantation. All 3 of those children had Bili6m ⬎ 20 mol/L. Ten of 48 children who survived until transplantation had Bili6m ⬎ 20 mol/L. Bili6m ⬍ 20 mol/L could predict transplant-free survival with a specificity of 54% and a sensitivity of 100%. The positive predictive value was 100%, and the negative predictive value was 12.5%. The combined predictive marker of Vfib ⬍ 2.5% and Bili6M ⬍ 20 mol/L could predict transplant-free survival with a specificity of 79%, a sensitivity of 100%, a positive predictive value of 100%, and a negative predictive value of 23% (P ⫽ 0.002, Pearson’s chi-square test).
Figure 4. Vfib values according to the 6 Ishak score groups in patients with BA. No correlation was shown between the 2 parameters (Pearson’s correlation, r ⴝ 0.18, P ⴝ 0.21). Abbreviation: Vfib, mean volume of fibrosis per number of periportal fields.
Groups of patients with different Ishak scores showed no correlation with Vfib (r ⫽ 0.18, P ⫽ 0.21; Fig. 4). There was also no significant correlation of Ishak scoring with time to transplantation (P ⫽ not significant, Cox regression) or incidence of LTx (P ⫽ not significant, Pearson’s chi-square test). Comparing 3 groups with low (1-2), intermediate (3-4), and high (5-6) Ishak scores, we could not determine a correlation with Vfib (r ⫽ 0.172, P ⫽ not significant), transplant-free patient survival (P ⫽ not significant, Pearson’s chi-square test), time to transplantation (P ⫽ not significant, Cox regression), or incidence of LTx (P ⫽ 0.184, Pearson’s chisquare test). No significant correlation was demonstrated with transplant-free patient survival (P ⫽ not significant, log rank) or time from transplantation to death for the different Ishak groups (P ⫽ not significant, log rank)
DISCUSSION Computerized quantification of fibrosis by PSR staining is fast, simple, reliable, reproducible, and cheap. A correlation between Vfib and Bili6m, the most important parameter currently used for the prediction of outcome of KPE and timing for LTx,6,25 could be determined. Furthermore, a combination of both parameters increased the negative predictive value for survival with a positive predictive value of 100%. In larger studies, it should be confirmed whether the combined surrogate marker can also predict time to LTx in a larger group of children. Bili6m has proven to be a prognostic marker with high sensitivity and specificity.6 Vfib provides the clinician with the advantage of having a predictor 6 months before, at the time of KPE. In our patient collective, positive and negative predictive values of both markers were comparable. It might be speculated that by the
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COMPUTERIZED QUANTIFICATION OF LIVER FIBROSIS 881
measurement of Vfib at the time of KPE, patients who may benefit from anti-inflammatory therapy or trials of antifibrotic agents, which have been recently introduced in the treatment of BA,26-28 could be detected earlier. We could show that a combination of both markers enhances the predictive value. Therefore, it may be of special interest to validate a combined score of Bili6M and Vfib in further prospective studies that include liver biopsies post-Kasai and explanted livers also to evaluate Vfib as a useful follow-up parameter. Various attempts have been made to develop29 and improve unique scoring systems such as the Pediatric End-Stage Liver Disease model30,31 to set the indication for LTx and to determine the multiple factors influencing the outcome of children with BA.12,32,33 The integration of Vfib may be another element to improve the predictive value of those scoring systems. The Ishak score24 showed only a weak association with the clinical course of children with BA. We conclude that this result is caused by the semiquantitative evaluation of fibrosis and by interobserver variability. Furthermore, a sampling error was reported by Ishak et al.24 when the observed area of biopsy was not representative or when areas differed in the extent of fibrosis. It has to be taken into account that the Ishak score was originally developed and validated only for the grading and staging of chronic hepatitis B and not for other conditions with possibly different distribution patterns of fibrosis such as BA. Although there are studies showing no correlation between the degree of liver fibrosis and poor outcome,25,27,34-37 hepatic fibrosis as a predictor for a poor outcome of KPE or early mortality has been previously determined by several authors.36,38-40 However, apart from the study of Tanano et al.,34 in all of those studies, fibrosis was determined semiquantitatively with histological criteria or scoring systems. Therefore, we conclude that the discrepancy in the results may be due to the interobserver variability in conventional histology. Overcoming this confounding factor is of particular interest and can be achieved by PSR staining computerized quantification. Tanano et al. used computerized quantification to define a fibrosis index (FI) in Azan Mallory–stained liver tissue of 46 children with BA at the time of KPE and stoma closure to detect if the FI could reflect liver function. They were able to show a positive correlation between the chronological difference rate [(FI at stoma closure FI at KPE)/interval from KPE to stoma closure] and clinical outcome but did not find a correlation between the FI at the time of KPE and clinical outcome. Therefore, the predictive value of this method is exclusively based on 2 liver biopsies, one during the KPE procedure and a second during the closure of the primary established stoma. Because usually no primary stoma is constructed at the time of KPE anymore, in contrast to our method, this is unsuitable for routine clinical use. We therefore conclude that PSR staining of liver biopsies is a new tool for predicting the course of liver function as early as the time of KPE and for improving the known predictive value of Bili6m and classical pa-
thology. Vfib should therefore be discussed as an integral part of scoring systems calculating the indication and time to LTx in children with BA and be integrated into prospective interventional multicenter studies.
ACKNOWLEDGMENT We thank Dr. Michael Mengel on behalf of the Hannover Medical School Department of Pathology for providing the facilities for staining and image acquisition and Dr. Ludwig Hoy of the Hannover Medical School Institute of Biometry for his excellent cooperation.
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LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases
