Here we describe the morphologic, immunohistochemical, and ultrastructural features in 12 patients with genetically confirmed PFIC2. All liver samples showed lobular cholestasis, and most had bile plugs in canaliculi. Other common features included lobular and/or portal inflammation, giant cell transformation, hepatocellular necrosis, and hepatocellular swelling. This neonatal hepatitis-like pattern has been described in PFIC2, particularly in early biopsy specimens.1,4,6,12,14,15,21
A comprehensive histopathologic examination of 84 liver biopsy specimens in the setting of PFIC identified similar features in some specimens, including hepatocellular and canalicular cholestasis, ballooned hepatocytes, and giant cell transformation, and prominent bile duct loss and injury,2
but this study was carried out before the genetic basis of PFIC2 was known.19
Thus, it may have included cases of PFIC1, a related form of cholestatic liver disease caused by mutations in familial intrahepatic cholestasis, type 1 (FIC1
that clinically can resemble PFIC2.9
The bile duct loss and injury reported2
might have been observed in patients with PFIC1, as these changes were not characteristic in our patients with PFIC2.
A comparison of histopathologic differences between PFIC1 and PFIC2 reported portal and lobular fibrosis, many giant hepatocytes, and occasional hepatocyte necrosis in PFIC2, just as we have observed.5
Chart review of patients with PFIC1 and patients with PFIC2 also identified giant or multinucleate hepatocytes in most patients with PFIC2,16
whereas giant hepatocytes and necrosis were rare in PFIC1.5,16
In our study, giant cells were present at least rarely in all samples, and were prominent in most samples obtained during infancy (<12 mo of age). Thus, our observations are in keeping with the major histologic findings of these 2 recent studies, and support the proposition that giant cells (even in patients older than 1 y), portal and lobular fibrosis, and occasional hepatocyte necrosis characterize PFIC2.
Our study included 5 patients with multiple liver samples taken at least 6 months apart, which let us examine changes in histopathologic features of PFIC2 over time (). Interestingly, the prominence of neonatal hepatitis-like findings varied greatly among patients and within samples from individual patients, and we observed no clear trends. For example, in 3 patients, the frequency of giant cells remained approximately the same over time, whereas in 2 patients the frequency decreased. Lobular inflammation fluctuated in 2 patients and remained similar in 3 patients. Hepatocellular swelling fluctuated in 1 patient, remained similar in 3 patients, and decreased in 1 patient. However, our finding that giant cells and lobular inflammation were either rare or absent in some biopsy specimens indicates that the absence of classic neonatal hepatitis-like features on light microscopy does not exclude the diagnosis of PFIC2. Importantly, biopsy specimens from 3 of our oldest patients (patients 7, 9, and 11; ages 9, 7, and 5 y, respectively) continued to show at least some neonatal hepatitis-like features, including giant cells, hepatocyte swelling, and inflammation. This finding indicates that regardless of age, patients with PFIC2 are likely to show at least some neonatal hepatitis-like changes on biopsy.
All patients had some portal-based fibrosis in at least 1 sample. Variation in portal fibrosis, including progression to micronodular cirrhosis, has been reported in PFIC2.4,5,10,12,14–17,20,21
All patients in this study had sinusoidal/centrizonal fibrosis in at least 1 sample. Sinusoidal fibrosis was usually prominent, and often extended beyond zone 3. The pronounced lobular fibrosis in PFIC2 is distinct from portal-based or post-necrotic fibrosis. Lobular fibrosis, together with lobular inflammation, giant cells, and cell swelling, may be helpful clues to the possibility of PFIC2 on initial light microscopic examination in the appropriate clinical setting, and should perhaps prompt immunohistochemical and genetic studies.
Mutations in ABCB11
are typically associated with loss of immunohistochemically demonstrable BSEP expression, indicating that immunohistochemical staining is a useful diagnostic tool in PFIC2.5,7,10,14,15,17,20,21
In our study, 10 patients lacked BSEP expression on immunohistochemical analysis. However, patients with clinical PFIC2 and mutations in ABCB11
may still have immunohistochemically detectable BSEP20
; in such patients the protein expressed may be quantitatively insufficient or not fully functional. Patients 3 and 7 expressed BSEP on immunohistochemical analysis. Both had missense mutations (p.G116E and p.V1212F) predicted to affect the highly conserved nucleotide-binding folds of BSEP. These results are in keeping with the findings of Strautnieks et al,20
who detected BSEP expression in 28% of patients with PFIC2, most of whom had missense mutations within the nucleotide-binding folds.
An interesting question is whether the presence of intact BSEP expression correlates with clinical and/or histologic findings. The 2 BSEP-expressing patients in this study have followed a relatively slow progressive clinical course. Patient 7 was treated with ileal exclusion at 9 years of age and has not required additional surgical intervention. Patient 3 has not yet required surgical intervention till age 30 months. All biopsy specimens from these 2 patients showed only minimal or mild portal fibrosis (stages 1 or 2). These findings suggest that in PFIC2, patients with intact BSEP expression tend to develop less portal fibrosis than patients without BSEP expression, and follow a correspondingly more indolent clinical course. However, sinusoidal fibrosis, inflammatory features, and cholestatic changes in these BSEP-expressing patients varied, and did not substantially differ from those in patients without BSEP expression.
A related issue is whether specific genetic mutations are correlated with clinical and/or histologic findings. In this study, we found 6 ABCB11
mutations associated with clinical PFIC2 that have not been previously published, and identified 10 mutations that were previously published ().5,7,10,13,16,19,20
The diversity of ABCB11
mutations and the wide spectrum of findings on light microscopy, particularly the variations in samples from individual patients over time, indicate that severity of inflammatory and/or cholestatic features cannot be correlated with specific genetic mutations. Perhaps some of the variation reflects the realities of tissue sampling in a heterogeneous setting or reflects intercurrent, non-PFIC2-related hepatic injury. In a large study of patients with PFIC2, Strautnieks et al20
found that patients with 2 predicted protein-truncating mutations were at increased risk of developing hepatobiliary malignancy compared with other patients with PFIC2. That study raised the possibility that certain types of genetic mutations in PFIC2 may be correlated with clinical outcomes. A study of 180 families with PFIC1 or benign recurrent intra-hepatic cholestasis, type 1 (BRIC1), both caused by mutation in ATP8B1
, correlated mutation type/location with clinical severity.8
An analogous study for ABCB11
, correlating clinical presentations of PFIC2 and BRIC2 with mutation class, has not yet been conducted. Descriptive analysis in our small sample suggests that mutations predicted to abolish ABCB11
transcription are linked through lost BSEP expression, by more necrotic hepatocytes and more fibrosis, to worse outcome. Assessment in larger cohorts may validate this impression.
In summary, key light microscopy findings in PFIC2 include cholestasis, lobular fibrosis, lobular inflammation, portal inflammation, portal fibrosis, giant cell transformation, hepatocellular necrosis, and hepatocellular swelling. EM shows canalicular dilatation, partial-to-complete loss of microvilli, abnormal mitochondrial internal structure, and varying accumulation of finely granular bile. Our study underscores the variation in histologic findings that may be seen in PFIC2, both among patients and within the same patient over time. Light microscopic features are not clearly associated with clinical course in patients with PFIC2. Thus, although morphologic features can provide some support for the diagnosis of PFIC2, immunohistochemical or genetic studies, sometimes in combination, are required to establish the diagnosis.