BACKGROUND
Within the spectrum of autoimmune liver diseases, there are patients who manifest features of more than one disease, previously identified as having an “overlap syndrome” (1, 2), now referred to as variant syndromes. The most common variant syndrome is between primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH). Typically, AIH presents with elevated serum immunoglobulin G (IgG) while PBC is associated with elevated serum immunoglobulin M (IgM) (3, 4). Previous studies have suggested that plasma cells in liver biopsies of AIH patients are predominantly IgG+ while in PBC, there is an abundance of IgM+ cells (5, 6). We wanted to determine the immunostaining pattern for IgG and IgM of liver plasma cells among Hispanic patients in Los Angeles with features of both PBC-AIH compared to those with PBC or AIH alone.
METHODS
A retrospective review of the LAC+USC Hospital pathology database from 2007 to 2016 was performed. Adult patients with PBC, AIH, or PBC-AIH and a pretreatment liver biopsy were selected. Patients with AIH met the simplified IAIHG criteria for probable/definitive diagnosis of AIH. PBC patients met two of the following: biochemical evidence of cholestasis, presence of antimitochondrial antibody, or florid duct lesions. PBC-AIH patients met the Paris Criteria (7).
Immunostaining of plasma cells for IgG and IgM was performed on liver tissue specimens in a subgroup of patients. Specimens with cirrhosis, lacking portal tracts, fragmentation, or scant amount of tissue were excluded. IgG and IgM antibodies from Dako North America (Carpinteria, CA) were used at a concentration of 1:1000. The Leica IHC automation stainer (Bond III) was used with BOND Polymer Refine Detection. Plasma cells were counted by an expert liver pathologist (GK) who was blinded to clinical data and diagnosis. Plasma cell IgG/IgM ratios were calculated by dividing the total IgG-plasma cells by the total IgM-plasma cells per portal tract for each patient. Values were expressed as medians (interquartile range (IQR)). Mann-Whitney U or Kruskal-Wallis test was used to compare differences. All statistical analyses were performed using R 3.5.1 (R Foundation for Statistical Computing, Vienna, Austria).
RESULTS
A total of 114 liver biopsies from patients with a diagnosis of AIH, PBC or PBC-AIH (55, 31, and 25 patients, respectively) were identified. Ninety-one percent were female and 87% were Hispanic with a mean age of 49 years. A total of 54 non-cirrhotic, non-fragmented liver biopsy specimens were randomly chosen for immunostaining for IgG and IgM (19 AIH, 22 PBC, and 13 PBC-AIH) (Figure 1A). The number of portal tracts did not differ across subgroups. The number of IgG-plasma cells per portal tract was similar amongst all groups. However, the number of IgM-plasma cells per portal tract was higher in PBC and PBC-AIH compared to AIH. The calculated IgG/IgM plasma cell ratio was lower in PBC and PBC-AIH compared to AIH (1.2 (IQR: 0.6-1.7) and 1.1 (IQR: 0.7-1.5) versus 5.2 (IQR: 2.6-13.3), respectively, p<0.01) (Figure 1B).
Figure 1.
A) Representative immunostaining of plasma cells on liver biopsy. Top row represents IgG, bottom row IgM. Left column represents autoimmune hepatitis (AIH), middle column primary biliary cholangitis (PBC), right column, PBC-AIH.
B) Boxplot of histologic IgG/IgM ratios of plasma cells (N=54, p<0.01).
DISCUSSION
We studied the immune-phenotype of a large cohort of Hispanic patients of North and Central American Ancestry with autoimmune liver disease. Immunostaining of liver tissue for IgG and IgM plasma cells suggests that the phenotype of patients with PBC-AIH in our cohort predominantly resembles that of PBC. In addition, a positive correlation was seen between serum and histologic IgG/IgM ratios (data not shown).
The classification of patients with variant syndromes has been controversial and not well-defined. The IAIHG does not consider variant syndromes a distinct entity, instead suggesting that patients with autoimmune liver disease be categorized based on the predominant disease (8). However, it is not clear what should be considered the predominant pathologic feature in PBC-AIH patients when there is simultaneous nonsuppurative duct damage, severe interface hepatitis, and lobular inflammation. Therefore, we decided to use immunostaining of plasma cells for IgG/IgM to determine the predominant immune-phenotype histologically. Interestingly, IgG/IgM-plasma cell ratios in PBC-AIH patients were similar to those with PBC alone and significantly lower than ratios in AIH. This suggests that our patients with PBC-AIH exemplify an immune-phenotype similar to that in PBC.
Our study is limited by a cohort of primarily Hispanic descent and a retrospective design. Histology was reviewed by a single pathologist. Due to tissue quality, immunostaining was limited to a subset of patients encompassing a small, but representative, proportion of patients.
In conclusion, by using immunostaining of plasma cells for IgG and IgM, we determined that our Hispanic patients with PBC-AIH resemble PBC. Larger prospective studies evaluating the IgG/IgM ratio as a biomarker for distinguishing autoimmune liver diseases are warranted.
Acknowledgement:
This work was supported by the Southern California Clinical and Translational Science Institute UL1TR001855. Microscopy was performed at the Cell and Tissue Imaging Core of the USC Research Center for Liver Diseases, NIH grant No. P30 DK048522 and S10 RR022508.
ABBREVIATIONS:
- ALT
alanine transaminase
- ALP
alkaline phosphatase
- AMA
antimitochondrial antibody
- ANA
antinuclear antibody
- AST
aspartate transaminase
- AIH
autoimmune hepatitis
- CI
confidence intervals
- HR
hazard ratio
- IgG
immunoglobulin G
- IgM
immunoglobulin M
- IQR
interquartile range
- PBC
primary biliary cholangitis
- ULN
upper limit of normal
- UDCA
ursodeoxycholic acid
Footnotes
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Disclosures: There are no personal or financial disclosures to declare.
Conflict of interest statement:
There are no conflicts of interest (including personal or financial) to declare from all of the authors.
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