Abstract
The frequency with which florid duct lesions are seen in needle-biopsy specimens of the liver was assessed in patients with primary biliary cirrhosis (PBC) enrolled in a 2-year randomized, double-blind, placebo-controlled trial of ursodeoxycholic acid (UDCA) versus placebo. Paired biopsy specimens obtained at entry and after 2 years on medication were reviewed blindly and mostly simultaneously by a panel of 5 hepatopathologists who, earlier, had characterized the florid duct lesion, which has been well described in the pathology literature. Florid duct lesions at entry were identified in approximately 36%. Patients with earlier disease showed florid duct lesions much more frequently than those with more advanced disease. The prevalence of florid duct lesions in 60 patients receiving placebo medication fell from 38.3% to 21.7%, P = .025, over the period of 2 years. The prevalence of florid duct lesions also decreased in the 55 patients receiving UDCA, from 32.7% to 18.2%, P = .046. The prevalences of these lesions in the placebo and UDCA patients at entry and at 2 years were not significantly different from each other. The findings suggest that UDCA does not prevent ongoing bile duct destruction in patients with PBC. Instead, they support the impression that UDCA exerts its beneficial effects by protecting against the consequences of bile duct destruction.
Destruction of interlobular and septal bile ducts is the hallmark histological lesion of primary biliary cirrhosis (PBC).1 The damaged ducts are almost invariably inflamed and engulfed by a mononuclear cell infiltrate that includes lymphocytes and plasma cells and may have granulomatous characteristics. The lesion is commonly referred to as nonsuppurative destructive cholangitis1 or more simply as the florid duct lesion.2 With time, the damaged ducts disappear and ductopenia ensues as the pattern of cholestasis progresses to cirrhosis with its attendant complications. Intuitively, the rate of progression of PBC must be dependent in part on the rate of development of florid duct lesions and it follows that the most effective primary therapy of PBC should be one that prevents development of additional duct destruction.
In recent years, ursodeoxycholic acid (UDCA) has become the treatment of choice in PBC because it rapidly results in improvement of laboratory markers of cholestasis and hepatic inflammation as well as in some histological features and symptoms. It may delay the time to development of serious complications, and it is well tolerated and safe.3–14 Although there is evidence that piecemeal necrosis and portal inflammation are improved by UDCA, the effect of this agent on the frequency of the florid duct lesion is unknown. This report addresses this issue.
PATIENTS AND METHODS
Patients with PBC defined by the criteria of (1) a cholestatic liver disease of at least 6 months’ duration; (2) a serum alkaline phosphatase value at least one and a half times the upper limit of normal; (3) a positive antimitochondrial antibody test; (4) exclusion of biliary obstruction by ultrasonography, computed tomography, or endoscopic cholangiography; and (5) a liver biopsy specimen compatible with the diagnosis of PBC, were enrolled in a 2-year randomized, double-blind, controlled treatment trial of UDCA versus placebo. Seventy-seven patients received UDCA in a single bedtime dose of 10 to 12 mg/kg/d and 74 received placebo medication. Patients were stratified into 4 groups on the basis of (1) a serum bilirubin of less than 2 mg/dL or 2 mg/dL and greater and (2) liver histology either stages I and II or stages III and IV, as defined by Ludwig et al.15 Patients in stratum 1 had a serum bilirubin less than 2, and stage I or II histology; in stratum 2, patients had a bilirubin of less than 2 and stage III or IV histology; in stratum 3, patients had a bilirubin of 2 or greater and stage I or II histology; and in stratum 4, a bilirubin of 2 or greater and stage III or IV histology. The protocol was approved by the institutional review boards at each of our 6 treatment centers.
A needle liver biopsy specimen was obtained within 6 months before starting the trial (entry biopsy) and again after 2 years on trial medication. The biopsy specimens were reviewed by a panel of 5 hepatopathologists. At a pretrial meeting, the pathologists simultaneously reviewed 35 biopsy specimens obtained from patients with documented PBC, using a multiheaded microscope. The grading system for stage was standardized because this was a criterion used for stratification of patients at entry into the trial. Florid duct lesions were characterized. The ducts in such lesions are largely interlobular and septal ducts with a lumen size estimated to being between 30 to 80 μm.16 The walls of the ducts show varying degrees of inflammation, vacuolar change, and necrosis. The ducts are usually surrounded by an intense lymphocytic infiltrate, which may contain a few neutrophils, eosinophils, and plasma cells. The inflammatory infiltrate may have granulomatous characteristics or contain well-formed granulomas. However, granulomas were absent in the majority of the lesions, and their presence was not required for the designation of florid duct lesions. In addition, a grading system for piecemeal necrosis, portal inflammation, fibrosis, and cholate injury was established. Liver biopsy specimens were staged at each center. Unstained slides were sent to our pathology core center at Nebraska where they were stained and coded. The biopsy specimens were then assessed blindly and mostly simultaneously by the 5 pathologists. Highlights of the analysis of piecemeal necrosis, portal inflammation, fibrosis, and cholate injury were presented in an earlier report.9 The current report focuses on the effect of UDCA on florid duct lesions.
The statistical significance of differences in the incidence of these lesions in the placebo and UDCA groups at entry and at 2 years was assessed by χ2 tests. The statistical significance of differences in incidence of florid duct lesions in the paired biopsy specimens 1 and 2 for placebo and UDCA was assessed by McNemar’s test.17
RESULTS
Patient Characteristics at Entry
Demographic, laboratory, and histological characteristics of the patients at entry were described in detail in an earlier publication.9 The results of initial laboratory tests and histological staging were comparable in patients randomized to receive either placebo or UDCA. Approximately 70% of patients had an entry serum bilirubin less than 2 mg/dL. One third showed stage I and II histology; two thirds showed stage III and IV histology.
Number of Biopsy Specimens Analyzed
The presence or absence of florid duct lesions was recorded for 119 paired liver biopsy specimens that were in the vast majority at least 2 cm in length. Thirty-eight pairs were from patients in stratum 1 (17 in the placebo group and 21 in the UDCA group); 48 were in stratum 2 (28 in the placebo group and 20 in the UDCA group); 4 were in stratum 3 (2 in the placebo group and 2 in the UDCA group); and 29 were in stratum 4 (15 in the placebo group and 14 in the UDCA group). Patients in stratum 3 were excluded from subsequent analyses because there were so few of them.
Florid Duct Lesions at Entry
Florid duct lesions were identified in 41 of the 115 patients (35.6%) with paired liver biopsy specimens. The frequency with which these lesions were described differed significantly in stratum 1 (52.6%); for stratum 2 (31.2%) and stratum 4 (20.6%), χ2 = 7.6, degrees of freedom = 2, P = .02 (Table 1).
Table 1.
Prevalence of Florid Duct Lesions in Primary Biliary Cirrhosis at Entry
| N | Florid Duct Lesions
|
||
|---|---|---|---|
| N | Percent | ||
| All Patients | 115 | 41 | 35.6 |
| Stratum 1 | 38 | 20 | 52.6 |
| Stratum 2 | 48 | 15 | 31.2 |
| Stratum 4 | 29 | 6 | 20.6 |
NOTE. χ2 = 7.6; degrees of freedom = 2; P = .02.
Florid Duct Lesions at Two Years
Comparison With Entry Values
The numbers of patients with florid duct lesions described in biopsy 1 and biopsy 2 are listed in Table 2. Moreover, the number of patients who exhibited florid duct lesions in both biopsy specimens, in either the first or second biopsy specimen, or in neither biopsy specimen, is also summarized in Table 2.
Table 2.
Prevalence of Florid Duct Lesions at Entry and After Two Years on Placebo or UDCA
| Patient Group |
Florid Duct Lesions
|
||||||
|---|---|---|---|---|---|---|---|
| B1 (+) |
B2 (+) |
B1 B2 (+) (+) |
B1 B2 (+) (−) |
B1 B2 (−) (+) |
B1 B2 (−) (−) |
||
| Placebo | |||||||
| All patients | 60 | 23 | 13 | 8 | 15 | 5 | 32 |
| Stratum 1 | 17 | 11 | 6 | 4 | 7 | 2 | 4 |
| Stratum 2 | 28 | 8 | 5 | 2 | 6 | 3 | 17 |
| Stratum 4 | 15 | 4 | 2 | 2 | 2 | 0 | 11 |
| UDCA | |||||||
| All patients | 55 | 18 | 10 | 6 | 12 | 4 | 33 |
| Stratum 1 | 21 | 9 | 5 | 5 | 4 | 0 | 12 |
| Stratum 2 | 20 | 7 | 5 | 1 | 6 | 4 | 9 |
| Stratum 4 | 14 | 2 | 0 | 0 | 2 | 0 | 12 |
Abbreviations: B1, biopsy 1; B2, biopsy 2.
Placebo
The prevalence of florid duct lesions decreased significantly in all untreated patients, from 38.3% to 21.7%, P = .025 (Table 3). The prevalence also decreased in patients in strata 1, 2, and 4. However, none of these stratal changes were statistically significant.
Table 3.
Statistical Significance of Differences in Prevalence of Florid Duct Lesions at Entry and After Two Years on Placebo or UDCA
| Placebo | UDCA | Placebo vs. UDCA | |
|---|---|---|---|
| All patients | |||
| Total n | 60 | 55 | χ2 |
| Florid duct n at entry | 23 | 18 | P = .4* |
| 2 yr | 13 | 10 | P = .6* |
| 2 yr vs. entry† | P = .025 | P = .046 | |
| Stratum 1 | |||
| Total n | 17 | 21 | |
| Florid duct n at entry | 11 | 9 | P = .2 |
| 2 yr | 6 | 5 | P = .4 |
| 2 yr vs. entry† | P = .096 | P = .046 | |
| Stratum 2 | |||
| Total n | 28 | 20 | |
| Florid duct n at entry | 8 | 7 | P = .6 |
| 2 yr | 5 | 5 | P = .5 |
| 2 yr vs. entry† | P = .3 | P = .5 | |
| Stratum 4 | |||
| Total n | 15 | 14 | |
| Florid duct n at entry | 4 | 2 | P = .7‡ |
| 2 yr | 2 | 0 | P = .5‡ |
| 2 yr vs. entry† | P = .2 | P = .2 | |
Mantel-Haenszel χ2, adjusting for stratum.
McNemar’s test.
Yates correction used for data in Stratum 4.
UDCA
The prevalence of florid duct lesions also decreased in UDCA-treated patients from 32.7% to 18.2%, P = .046, and in patients in strata 1, 2, and 4. The decrease was statistically significant in stratum 1, but not in strata 2 or 4.
Placebo Versus UDCA
The prevalences of florid duct lesions in the various placebo and UDCA groups at entry and at 2 years were not significantly different from each other.
DISCUSSION
The florid duct lesion is the histological hallmark of PBC and is virtually diagnostic of this disease.1,2,18 It is usually a marker of duct necrosis and destruction, although it is uncertain whether necrosis is caused by components of the intense portal inflammatory infiltrate that surrounds the duct, or whether the infiltrate is a response to contents of a necrotic duct that have leaked into the surrounding portal tract. Because it is virtually impossible to follow the evolution of the florid duct lesion, it cannot be concluded that it invariably is followed by permanent duct destruction or that it is the only marker of duct destruction. Nevertheless, the frequent association of bile duct destruction with a prominent cellular reaction in serial section observations16 and three-dimensional reconstructions19 of liver, particularly early in the course of PBC, strongly supports the judgment that the florid duct lesion is a major, if not the only, marker of the underlying primary bile duct destructive lesion.
The current assessment of florid duct lesions was performed on needle biopsy specimens of the liver that were largely at least 2 cm in length. Because such lesions are focal and segmental, it is not surprising that they are detected in only some of the biopsy specimens. Moreover, it is impossible to quantitate the number of florid duct lesions present in a given liver. Thus, in the present communication, we are only providing information on whether such lesions were detected or not. The assumptions made are that problems with sampling are comparable in patients who were first stratified on the basis of severity of disease and were then randomized within strata to receive either placebo medication or UDCA. Changes in the prevalence of florid duct lesions with time were felt to reflect the combination of disappearance of old lesions and appearance of new lesions. In placebo-treated patients, the change in prevalence was felt to reflect these alterations occurring during the natural history of the disease.
Overall, florid duct lesions at baseline were described in 35.6% of the patients, but there was a significant difference in prevalence in that patients with earlier disease showed florid duct lesions much more frequently than those with more advanced PBC (see Table 1). A similar finding of increased frequency of bile duct destructive lesions in earlier histological stages of PBC was reported previously by Portmann et al.20 These findings may simply reflect the development of ductopenia as the disease progresses and, thus, the presence of fewer ducts in which destructive lesions can occur.
Of considerable interest is the observed decline in prevalence of florid duct lesions over 2 years in patients who were untreated (placebo group). This trend was present in each of the strata, but was statistically significant only when data for all the placebo-treated patients were combined (Table 3).
The prevalence of florid duct lesions also declined over 2 years in patients randomized to receive UDCA. The extent of the decline, however, was comparable in the UDCA- and placebo-treated patients. In the analyses of the subgroups, the prevalence of florid duct lesions in stratum 1 fell from 11 at entry to 6 at 2 years, P = .096 in the placebo group, and from 9 to 5, P = .046 in the UDCA-treated group. These decreases in prevalence are similar, and the statistical differences found in the stratum 1, placebo, and UDCA subgroups are marginal, with the placebo group P value slightly above .05 and the UDCA group P value slightly below .05. Fundamentally, we do not believe that these small statistical differences indicate an advantage of UDCA over placebo in stratum 1 patients. It is not unusual to find such minor statistical differences in analyses that involve small sample sizes and/or multiple testing of subgroups.
As indicated earlier, the change in prevalence of florid duct lesions over a period of 2 years must reflect some combination of disappearance of existing lesions plus the appearance of new lesions. There can exist three explanations for the findings of a comparable prevalence of florid duct lesions at entry in patients eventually randomized to receive either UDCA or placebo medication, and of a comparable prevalence and decline in florid duct lesions after 2 years on UDCA or placebo (Table 3). Thus, in UDCA-treated patients, florid duct lesions could both disappear and appear more slowly than during the natural history of the disease (i.e., placebo-treated patients); or these lesions could both disappear and appear more rapidly; or florid duct lesions could appear and disappear at comparable rates in UDCA- and placebo-treated patients. It seems unlikely that UDCA would increase the rate at which new bile duct lesions occur. We cannot exclude the possibility that UDCA did decrease the rate of appearance of new duct lesions. For this to be the case, it would have been necessary for UDCA to simultaneously decrease the rate at which existing duct lesions disappear. That such a combination of events would yield a comparable decline in prevalence of florid duct lesions at 2 years in the 2 treatment groups also seems unlikely. Rather, we feel it is reasonable to speculate that UDCA did not affect the primary destructive duct lesion of PBC.
Duct destruction leads to impaired transport of compounds into bile (cholestasis) with its consequences, pruritus and hyperbilirubinemia. Cytotoxic aspects of bile components likely to be present in increased concentrations in obstructed areas of liver presumably cause hepatocyte injury and death. The dihydroxy bile acids, deoxycholic acid and chenodeoxycholic acid, and the monohydroxy bile acid, lithocholic acid, undoubtedly contribute to this and have the additional potential to impair biliary transport in nondamaged hepatocytes.21 Portal triads usually contain a prominent mononuclear infiltrate with a variable small number of polymorphonuclear cells. Such cells are able to generate cytokines that may themselves have cytotoxic potential and stimulate fibrogenesis. Thus, interactions between components of bile and products of inflammatory cells appear likely to contribute to scarring and hepatocyte injury, resulting in progressive fibrosis and cirrhosis with its attendant complications.
The mechanisms by which UDCA exerts its favorable effects in PBC are still uncertain. The current data strongly suggest that UDCA does not prevent ongoing bile duct destruction. Rather, they support the impression that UDCA protects against the consequences of bile duct destruction. Protection against the cytotoxic effects of increased concentrations of bile acids that accumulate in PBC is one important effect.21 A decrease in immunologically-mediated injury via modulation of expression of human leukocyte antigens on cell surfaces is postulated to be another.22
It seems likely that if bile duct injury continues unabated during UDCA therapy, eventually the desirable hepatoprotective effects of UDCA will no longer delay progression of the disease. Renewed attention should be directed, therefore, at developing therapies that prevent bile duct destruction. Assessment of the influence of such therapies on the prevalence of florid duct lesions in appropriately controlled trials may provide an early surrogate marker of progression of the disease. The group of PBC patients likely to best provide this assessment is the stratum 1 group. This group (bilirubin less than 2 mg/dL with stage I and II histology) had the highest prevalence of florid duct lesions at entry and showed a significant, spontaneous decrease in this prevalence in 2 years. An effective therapy should show an even greater decrease than a corresponding placebo group and could do so in a relatively short period of time.
Acknowledgments
Supported in part by a research grant from Ciba-Geigy; NIH General Clinical Research Grants to UT Southwestern (MO1-RR00633), Yale (MO1-RR00125), Medical College of Virginia (MO1-RR00065), Washington University, St. Louis (MO1-RR00036); NIH grant to Yale (P30-DK34989); Institutional Funds: Thomas Jefferson (Louis A. Rosen Fund for Liver Research); Nebraska (Clinical Research funds of University of Nebraska Medical Center).
The authors are indebted to Renate Davis for preparation of the manuscript and to Beverly Adams Huet, UT Southwestern GCRC biostatistician, for statistical advice.
Abbreviations
- PBC
primary biliary cirrhosis
- UDCA
ursodeoxycholic acid
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