Optimizing the detection of biliary dysplasia in primary sclerosing cholangitis before liver transplantation

<p><b>Background:</b> Patients with primary sclerosing cholangitis (PSC) have increased risk of cholangiocarcinoma (CCA). We evaluated pre-transplant work-up in PSC patients, to search for the most effective strategy for the detection of biliary dysplasia or early CCA.</p> <p><b>Methods:</b> Two hundred and twenty five consecutive PSC patients undergoing liver transplantation (LTx) in Sweden between 1999 and 2013 were studied. Patients with CCA or dysplasia in the explanted liver were compared with those with benign histopathology. Measures of test performance were calculated for patients having brush cytology on one endoscopic retrograde cholangiopancreaticography (ERCP) occasion, for those having repeated examinations with or without cholangioscopy, and for fluorescence <i>in situ</i> hybridization (FISH). Survival after LTx was analyzed.</p> <p><b>Results:</b> Brush cytology on a single ERCP occasion had moderate sensitivity (57%) and high specificity (94%) for the detection of CCA/high grade dysplasia (HGD) in the explanted liver. The corresponding sensitivity and specificity for FISH were 84% and 90%, respectively. Utilizing repeated ERCP and brush cytology to confirm the initial finding improved sensitivity to 82%. Using single operator cholangioscopy (SOC) for targeted examination at the second ERCP improved sensitivity (100%) and specificity (97%) significantly. Mortality rate in patients with incidentally discovered CCA (<i>n</i> = 16) in the explanted liver was significantly higher than in patients with HGD or benign histopathology (HR 16.0; 95% CI, 5.6–45.4; <i>p</i> < .001).</p> <p><b>Conclusions:</b> Repeated brush cytology especially when combined with targeted examination under SOC guidance is superior to single brush examinations. This strategy improves the detection of malignancy in PSC and is of importance for selection of patients for LTx.</p

<i>In Situ</i> Characterization of Intrahepatic Non-Parenchymal Cells in PSC Reveals Phenotypic Patterns Associated with Disease Severity

<div><p>Liver-infiltrating T cells have been implicated in the pathogenesis of primary sclerosing cholangitis (PSC), however little information is available about changes in other cellular compartments in the liver during PSC. This study aimed to characterize non-parenchymal intrahepatic cells in PSC livers and to find associations between phenotypes and disease severity. Using immunohistochemistry, followed by automated image analysis and quantification and a principal component analysis, we have studied non-parenchymal intrahepatic cells in PSC-patient livers (n = 17) and controls (n = 17). We observed a significant increase of T cells in the PSC patients, localized to the fibrotic areas. MAIT cells, normally present at high numbers in the liver, were not increased to the same extent. PSC patients had lower expression of MHC class I than controls. However, the levels of NKp46+ NK cells were similar between patients and controls, nevertheless, NKp46 was identified as a phenotypic marker that distinguished PSC patients with mild from those with severe fibrosis. Beyond that, a group of PSC patients had lost expression of Caldesmon and this was associated with more extensive bile duct proliferation and higher numbers of T cells. Our data reveals phenotypic patterns in PSC patients associated with disease severity.</p></div

Control and PSC-patient characteristics.

<p>Abbreviations: IBD = inflammatory bowel disease; UC = ulcerative colitis; CD = Crohńs disease; NA = not applicable, Ltx = liver transplantation, MELD = Model for End-Stage Liver Disease, S.D = standard deviation, ALP = alkaline phosphatase, ALT = alanine aminotransferase, CRP = C-reactive protein, Urso = ursodeoxycholic acid.</p>1<p>Information not available for four controls.</p

IHC characterization and quantitative image analysis of non-parenchymal non-immune cells of PSC patient livers.

<p>Representative immunostaining and quantification analysis of (A) CK19, (B) CK7, (C) EpCAM, (D) CD31 expression in controls (n = 14–17) and PSC patients (n = 14–17). Samples were counterstained with hematoxylin and dark brown indicate immunoreaction product. Quantification was performed to calculate mean intensity of the stainings (ACIA) and frequency of expression (percentage of stained area out of total area).</p

Principal component analysis of non-parenchymal liver cells in PSC.

<p>(A) PCA plot showing the parameters that most strongly separate PSC-patients (n = 17) from controls (n = 17) out of totally 20 measured parameters. (B) Comparison of parameters separating PSC-patients with high stage fibrosis (n = 13) from patients with low stage fibrosis (n = 4) from a total of 23 measured parameters. (C) Comparison of parameters that discriminate PSC-patients with severe bile duct proliferation (n = 12) from patients with low bile duct proliferation (n = 5) from a total of 23 measured parameters.</p

Low expression of Caldesmon in a distinct group of PSC-patients correlates with higher numbers of T cells and more bile duct proliferation.

<p>(A) Representative Caldesmon stainings of one control and two PSC-patients (left) and image analysis of Caldesmon expression dividing PSC-patients into groups of high (pos) and low expression (neg) (right). (B and C) Quantification with image analysis of CK19, EpCAM, CD3, NKp46, CD163, and MHC class I expression in controls and PSC patients with pos or neg expression of Caldesmon. (D) Linear regression analysis for Caldesmon and histological parameters of liver disease for the PSC-patients.</p

T cells localize to fibrosis-affected areas in PSC patient-livers.

<p>(A) Representative photomicrograph for identification of affected and unaffected (striped) areas from one PSC-patient (left) and quantifications of fibrosis affected or non-affected areas in the PSC-patients (right). Immunostainings and quantitative determination of intensity of staining (ACIA) of (B) CD3, (C) TCR-Vα7.2, (D) NKp46, and (E) CD163, in affected and non-affected areas in the patients (CD3, n = 14; TCR-Vα7.2, n = 10; NKp46, n = 13; and CD163, n = 14), and in comparison with total area of controls (n = 14–17).</p

T cells infiltrate PSC-patient livers.

<p>Representative immunostaining and quantification analysis of (A) CD3, (B) TCR-Vα7.2, (C) NKp46, (D) CD163, and (E) MHC class I expression in controls (n = 14–17) and PSC patients (n = 14–17). Samples were counterstained with hematoxylin and dark brown indicate immunoreaction product. Quantification was performed to calculate mean intensity of the stainings (ACIA) and frequency of expression (percentage of stained area out of total area).</p

Conditional logistic regression analyses within the major histocompatibility complex (MHC) in primary sclerosing cholangitis (PSC).

<p>The three various conditional strategies employed and their conditional steps are indicated by Ia-Id, IIa-IIc and IIIa-IIIc. For all panels the conditional <i>P</i>-values (y-axis) are plotted against the position on chromosome 6 shown in million base pairs (Mb, x-axis). HLA alleles, <i>DRB1-DQB1</i> haplotypes, SNPs, the MICA5.1 allele and KIR ligands are distinctly designated (see legend in panel Ia). The significance threshold (<i>P</i>≤2.66×10⁻⁶) is indicated by the black, horizontal dotted line.</p

The extension of nine significantly primary sclerosing cholangitis (PSC) associated major histocompatibility complex (MHC) haplotypes, observed in the unconditional and conditional regression analyses combined.

<p>The extension of the haplotypes is determined by three categories of linkage disequilibrium (<i>r</i>²>0.2 (green), <i>r</i>²>0.5 (purple) and <i>r</i>²>0.8 (black), respectively) between a single haplotype marker (listed in the figure box) and any single nucleotide polymorphism (SNP) in the dataset. Haplotype 1; AH8.1 (A*01-C*07-B*08-DRB3*01:01-DRB1*03:01-DQA1*05:01-DQB1*02:01). Haplotype 2; HLA-DRB1*13:01-DQB1*06:03. Haplotype 3; HLA-DRB1*04-DQB1*03:01. Haplotype 4; HLA-DRB1*04-DQB1*03:02. Haplotype 5; HLA-DRB1*15:01-DQB1*06:02. Haplotype 6; HLA-B*07. Haplotype 7; HLA-DRB3*03:01-DRB1*13:02-DQB1*06:04. Haplotype 8, HLA-DRB1*07:01-DQB1*03:03. Haplotype 9; an independent LD-block centromeric of <i>NOTCH4</i> in class III, tagged by rs116212904. DRB1*15:01 is part of the DR2 serotype group.</p