Journal Watch November-December 2016

American Journal of Gastroenterology

Vitamin D is not associated with severity in NAFLD: Results of a paired clinical and gene expression profile analysis
Patel YA, Henao R, Moylan CA, Guy CD, Piercy DL, Diehl AM, Abdelmalek MF. Am J Gastroenterol 2016;111:1591-1598.

Vitamin D deficiency is associated with obesity, sedentary lifestyle, and insulin resistance.  Recent studies have implicated vitamin D in the pathogenesis of NAFLD.  The authors of this study correlated vitamin D levels with clinical, histologic, and gene expression differences in patients with NAFLD.  Vitamin D levels decreased with increased BMI but no differences were seen in vitamin D levels between patients with NAFLD and control patients.  There was no association between vitamin D levels and steatosis, lobular inflammation, ballooning, or overall NAS score.  Interestingly, vitamin D levels increased with increasing fibrosis up to stage 3 but were decreased in cirrhotic patients.  When comparing stage 0-1 to stage 3-4 patients, there only minor differences in expression of genes associated with vitamin D metabolism.


Zhou S, Venkatramani R, Gomulia E, Shillingford N, Wang L. Histopathol 2016; Nov;69(5):822-830
The authors studied the diagnostic and prognostic utility of SALL4 in hepatoblastomas (HB). This marker was present in 100% and 41% respectively of embryonal and fetal components of HB, but not in the small cell or mesenchymal components. Also SALL4 expression correlated with poor overall survival and other prognostic indices.
AIMS: To investigate the expression of spalt-like transcription factor 4 (SALL4), a regulator of embryonal development, in three epithelial components of hepatoblastoma (HB) and the relationship between SALL4 expression levels and patients’ clinicopathological features.
METHODS AND RESULTS:  A total of 115 specimens from 79 patients with HB were selected for immunostaining of SALL4. Nuclear staining was semi-quantified using the immunoreactive score (IS; range: 0-12). SALL4 expression was seen in all embryonal components (mean IS = 8.58) and in 41% of fetal components (mean IS = 0.78). No SALL4 expression was seen in either small cell undifferentiated or mesenchymal components of HB. Neither chemotherapy nor metastasis altered SALL4 expression significantly. High SALL4 expression levels were associated significantly with decreased overall survival (OS) (P = 0.004), event-free survival (EFS) (P = 0.003) and the presence of metastasis (P = 0.049) on univariate analysis. Multivariate analysis identified SALL4 as an independent prognostic predictor for OS (P = 0.029).
CONCLUSIONS:  SALL4 is useful for subtyping HB, and high SALL4 expression is associated with decreased survival in HB.
Piotti KC, Yantiss RK, Chen Z, Jessurun J. Histopathol 2016; Dec;69(6):937-942
AIMS:  Serum amyloid A is an acute phase reactant that is produced by hepatocytes in response to either inflammatory or neoplastic conditions. Because inflammatory adenomas produce this protein, serum amyloid A immunohistochemistry has been used in the evaluation of hepatocellular neoplasms. However, studies evaluating the expression of this protein in hepatitis are lacking. The aim of this study was to perform serum amyloid A immunostains on medical liver biopsy specimens of patients with common chronic liver diseases and correlate them with disease activity and stage.
METHODS AND RESULTS:  We performed serum amyloid A immunostains on 160 medical liver biopsies, including 100 cases of hepatitis C virus infection at different stages (20 cases of each) and 20 cases each of hepatitis B viral infection, steatohepatitis and autoimmune hepatitis. The extent and location of staining was recorded and correlated with grade, stage and laboratory values (transaminases, bilirubin and viral load). Data were analyzed using the Cochran-Mantel-Haenszel χ2 test for trend. Serum amyloid A staining was present in 130 (81%) cases and was limited to zone 3 perivenular hepatocytes in 66 (41%). Biopsy specimens with less fibrosis and/or mild portal inflammation showed significantly more staining than those with cirrhosis (P < 0.001), or at least moderate inflammatory activity (P < 0.001). There was no significant association between lobular inflammation (P = 0.06), bilirubin levels or viral load and immunohistochemical staining for serum amyloid A.
CONCLUSIONS:  Our results show that liver biopsy specimens with mildly active chronic hepatitis, early fibrosis and normal serum transaminases show more serum amyloid A immunopositivity compared with cases with more inflammatory activity, fibrosis or transaminitis. These findings indicate that serum amyloid A is expressed primarily in the early phases of disease and might influence progression and/or response to treatment.
Shalaby A, Hajhosseiny R, Zen Y, Davenport M, Quaglia A. Histopathol 2016; Dec;69(6):943-949
Aims: Orientation and digital analysis of the biliary remnants in the resected porta hepatis in infants with biliary atresia.
Methods and results:  Samples were orientated intra-operatively then stained with haematoxylin and eosin and immunostained for cytokeratin 7 (CK7). Sections were then digitized and analysed. Most proximal transected surface area was defined as the porta hepatis area (PHA) and the biliary epithelial area was defined as ‘BEA’. Data are quoted as median (range). Non-parametric statistical comparisons were made as appropriate. P < 0.05 was regarded as significant. Thirty-eight infants underwent surgery [median age 53 (16–120) days]. Eight specimens were excluded from the study due to technical reasons, leaving 30 specimens as the study cohort. Median PHA was 70 (30–133) mm2, median BEA 0.57 (0.07–5.5) mm2 (r = 0.51; P < 0.002). The median BEA/PHA ratio was 9.6 × 10−3 (1.9–104 × 10−3). There was a marked correlation of PHA with plasma γ-glutamyl transpeptidase (r = −0.51; P = 0.001). Both total BEA and the BEA/PHA ratio correlated with alkaline phosphatase (r = −0.35; P = 0.03 and r = −0.47; P = 0.005, respectively). Age at surgery correlated inversely with BEA (r = −0.44; P = 0.01) but not PHA (P = 0.1).
Conclusions: Precise quantification of biliary remnants is possible and correlates with biochemical variables. Values for BEA were associated with and declined demonstrably with increasing age at surgery.
Fujikura K, Fukumoto T, Ajiki T, Otani K, Kanzawa M, Akita M, Kido M, Ku Y, Itoh T, Zen Y. Histopathol 2016; Dec;69(6):950-961.
Aims: The aim of this study was to achieve a better definition of intraductal papillary neoplasms of the bile duct (IPNBs).
Methods and results: Biliary tumours that showed predominantly intraductal papillary growth were provisionally classified as IPNBs (n = 25) and papillary cholangiocarcinomas (n = 27). IPNB was defined as a neoplasm that is confined to the epithelium or is regularly arranged in a high-papillary architecture along thin fibrovascular stalks, whereas the term ‘papillary cholangiocarcinoma’ was used for tumours with more complex papillary structures (e.g. irregular papillary branching or mixed with solid–tubular growth). In our consecutive cohort of biliary neoplasms, 5% were classified as IPNBs, and 10% as papillary cholangiocarcinomas. IPNBs differed from papillary cholangiocarcinomas by less advanced invasion, gross mucin overproduction (72% versus 7%), and their prevalent location (84% of IPNBs in intrahepatic/hilar ducts; 70% of papillary cholangiocarcinomas in extrahepatic ducts). Gastric-type and oncocytic-type tumours were only detected in IPNBs. Expression of mucin core proteins and cytokeratin 20 significantly differed between the two groups. KRAS and GNAS were wild-type genotypes in all but one case of KRAS-mutated IPNB. Patients with IPNB had better recurrence-free survival than those with papillary cholangiocarcinoma (P = 0.007). In multivariate analysis, in which several other prognostic factors (e.g. stromal invasion and lymph node metastasis) were applied, the classification of the two papillary tumours was an independent prognostic factor (P = 0.040).
Conclusions: Given the significant contrast in clinicopathological features between IPNBs and papillary cholangiocarcinomas, it may be more appropriate to use the diagnostic term ‘IPNB’ for selected tumours that show regular papillary growth, separately from papillary cholangiocarcinomas.
Chan AW, Yu S, Yu YH, Tong JH, Wang L, Tin EK, Chong CC, Chan SL, Wong GL, Wong VW, Chan HL, Lai PB, To KF. Histopathol 2016; Dec;69(6):971-984.
Aims: Steatosis in hepatocellular carcinoma (HCC) has been recognized for decades and found most commonly in small, well-differentiated HCC. However, the clinicopathological features and pathogenesis of HCC with steatosis is not well characterized. There are few data concerning whether HCC with steatosis should be regarded a distinct histological variant, steatotic HCC.
Methods and results: A retrospective cohort of 516 patients undergoing curative surgery for primary HCC was recruited. The median follow-up was 45.5 (range: 0.2–166.0) months. Steatotic HCC was defined as HCC with significant steatosis (≥5% of tumour cells). Associations with immunohistochemical expression of fatty acid binding protein-1 (FABP1), sonic hedgehog (SHH) and gene polymorphism of patatin-like phospholipase 3 (PNPLA3) were analysed. Steatotic HCC was found in 21.1% of patients and was associated with higher metabolic risks [diabetes mellitus (36.7% versus 18.2%) and hypertension (44.0% versus 28.7%)], underlying fatty liver (60.6% versus 37.8%), steatohepatitis (30.3% versus 13.0%), smaller tumour size, lower frequency of major vessel (1.8% versus 11.3%) and microvascular invasion (20.2% versus 32.4%), earlier tumour stages and lower serum alpha-fetoprotein. It was associated with developing late tumour relapse (hazard ratio 2.15, P = 0.002) independently of underlying cirrhosis and non-anatomical excision. Steatotic HCC did not differ from HCC without significant steatosis in immunohistochemical expression of FABP1 and SHH and PNPLA3 gene polymorphism.
Conclusion: Steatotic HCC is a common histological variant of HCC with distinct association with underlying fatty liver, steatohepatitis and metabolic risks. Despite more favourable baseline tumour features, it was associated with late tumour relapse.
Walter D, Herrmann E, Winkelmann R, Albert JG, Liese J, Schnitzbauer A, Zeuzem S, Hansmann ML, Peveling-Oberhag J, Hartmann S. Histopathol 2016; Dec;69(6):962-970.
Background and aims: CD15 is expressed by various cancer types; among these are intrahepatic and perihilar cholangiocarcinoma (CCA). The aim of this study was to elucidate CD15 expression in distal CCA as well as in dysplastic biliary tissue and to determine its prognostic significance.
Methods and results: Tissue samples from patients with intrahepatic (iCCA, n = 22), perihilar (pCCA, n = 7) and distal CCA (dCCA, n = 15), who underwent surgical resection in the period from 2010 to 2015 were evaluated for CD15 expression. Tissue of synchronous lymph node metastasis (n = 13), CCA-associated dysplasia (n = 20), dysplasia in intraductal biopsies (n = 10) and benign proliferations (n = 12), as well as inflammatory biliary lesions (n = 28) and non-inflammatory bile ducts (n = 23), were evaluated equally for CD15 expression. CD15 was found to be expressed highly in iCCA (81.8%), pCCA (85.7%), dCCA (73.3%), CCA-associated dysplasia (70.0%), dysplasia in intraductal biopsies (100%) and metastatic tissue (84.6%). CD15 expression was negative in 58 of 64 benign bile duct alterations resulting in an overall sensitivity and specificity of CD15 in CCA of 80.7 and 90.6% patients, respectively. CD15 expression was correlated significantly with a decreased overall survival in patients with CD15-positive CCA associated dysplasia (P = 0.003). However, CD15 expression in the invasive tumour component was not correlated with clinical outcome.
Conclusion: CD15 is a sensitive and specific marker for intraepithelial and invasive neoplasias of the bile duct. Therefore, it can be helpful in the delineation of dysplastic and neoplastic biliary cells from non-neoplastic tissue, which frequently causes a diagnostic problem in indeterminate biliary stricture.

Modern Pathology

Hale G, Liu X, Hu J, Xu Z, Che L, Solomon D, Tsokos C, Shafizadeh N, Chen X, Gill R, Kakar S. Mod Pathol 2016; Nov; 29(11):1370-1380.

The study examines the correlation between glutamine synthetase staining patterns and β-catenin mutations in 15 typical hepatocellular adenomas, 5 atypical hepatocellular neoplasms and 60 hepatocellular carcinomas. Glutamine synthetase staining was classified as: (1) diffuse homogeneous: moderate-to-strong cytoplasmic staining in >90% of lesional cells, without a map-like pattern, (2) diffuse heterogeneous: moderate-to-strong staining in 50-90% of lesional cells, without a map-like pattern, and (3) patchy: moderate-to-strong staining in <50% of lesional cells (often perivascular), or weak staining irrespective of the extent, and all other staining patterns (including negative cases). Sanger sequencing of CTNNB1 exon 3 was performed in all cases. Of hepatocellular tumors with diffuse glutamine synthetase staining (homogeneous or heterogeneous), an exon 3 β-catenin mutation was detected in 33% (2/6) of typical hepatocellular adenoma, 75% (3/4) of atypical hepatocellular neoplasm and 17% (8/47) of hepatocellular carcinomas. An exon 3 mutation was also observed in 15% (2/13) of hepatocellular carcinomas with patchy glutamine synthetase staining. The results show a modest correlation between diffuse glutamine synthetase immunostaining and exon 3 β-catenin mutations in hepatocellular adenoma and hepatocellular carcinoma with discrepancy rates >50% in both hepatocellular adenoma and hepatocellular carcinoma. The interpretation of β-catenin activation based on glutamine synthetase staining should be performed with caution, and the undetermined significance of various glutamine synthetase patterns should be highlighted in pathology reports.

Journal of Hepatology

Solà E, Kerbert A, Verspaget HW, et al. J Hepatol 2016; 65:914-920.
Clinical decompensation of cirrhosis is mainly related to a progressive increase in portal hypertension and circulatory dysfunction. Copeptin is the C-terminal fragment of the vasopressin (AVP) precursor and is secreted from neurohypophysis together with AVP. This prospective study included 321 patients and investigated the level of copeptin in disease progression and prognosis of cirrhosis.  The plasma copeptin level showed a significant correlation with plasma AVP level and endogenous vasoactive systems in cirrhotic patients. Compared to the patients with compensated cirrhosis, the patients with decompensation showed significantly higher plasma copeptin levels. Multivariate analysis revealed plasma copeptin was an independent predictor of development of complications of cirrhosis and 3-months mortality. The data suggests that copeptin is reliable marker for disease progression, decompensation and poor prognosis in patients with cirrhosis.
Pais R, Barritt AS 4th, Calmus Y, Scatton O, Runge T, Lebray P, Poynard T, Ratziu V, Conti F. J Hepatol 2016; 65:1245-1257.
NAFLD has become one of the most frequent cause of chronic liver disease, cryptogenic cirrhosis, and hepatocellular carcinoma, and is the second leading cause of liver transplantation (LT) in the United States. In this review article, the authors discuss the multifaceted impact of NAFLD on liver transplantation and how to optimize the outcome of NAFLD patients undergoing LT. Several key points are raised: patients with NAFLD listed for LT are more often removed from the waiting list because of associated comorbidities and older age; the shortage of liver grafts will increase in the future and the use of fatty liver grafts will require optimization of surgical and preservation techniques; patients with NAFLD are at higher risk of cardiovascular morbidity and mortality following LT; NAFLD recurrence is frequent after LT.

American Journal of Surgical Pathology

Russo P1, Magee JC, Anders RA, Bove KE, Chung C, Cummings OW, Finegold MJ, Finn LS, Kim GE, Lovell MA, Magid MS, Melin-Aldana H, Ranganathan S, Shehata BM, Wang LL, White FV, Chen Z, Spino C; Childhood Liver Disease Research Network (ChiLDReN). Am J Surg Pathol 2016; Dec;40(12):1601-1615.
Given variability in data and limitation to single institution cohorts in previous studies, the goal of this project was to better define which histologic features are the strongest predictors of biliary atresia (BA) and identify parameters that may be of prognostic significance.  This study utilized data and slide review from cholestatic infants that were prospectively enrolled in the multicenter ChiLDReN network to determine which histologic features: (1) could distinguish BA from non-BA causes of cholestasis [N=227]; (2) varied with respect to clinical parameters (including age); and (3) correlated with clinical outcome in BA patients after hepatoportoenterostomy (HPE) [N=316].  Except for patient age, central review pathologists were blinded to all clinical information and scored 26 histologic features based on consensus.  Bile plugs in portal tracts and portal tract edema, when seen without bile duct paucity or features of idiopathic neonatal hepatitis (giant cell transformation and extramedullary hematopoiesis), were most associated with BA (diagnostic accuracy 90%).  With regard to clinical outcomes, elevated bilirubin 6 months after HPE was associated with older age, but showed no strong correlation with histologic changes.  Advanced fibrosis (Batts-Ludwig stage ≥3 fibrosis), ductal plate malformation, bile duct injury, older age at HPE, and elevated INR were associated with an increased risk for transplantation.  In contrast to previous literature, this study found that age was not a significant factor in assigning the biopsy to the correct category of disease (BA vs. non-BA), and that there was substantial variability in the range of histologic changes in cases of BA that were clinically well-characterized.  Common causes of misinterpretation of findings in BA cases as non-BA included an overestimation of bile duct paucity and underestimation of portal tract edema as well as known mimickers of BA (cystic fibrosis and α-1 antitrypsin deficiency) in needle biopsies.

Archives of Pathology and Laboratory Medicine

Ainechi S, Lee H. Arch Pathol Lab Med 2016; Nov;140(11):1285-1289.
This resident short review summarizes current terminology and disease concepts for precancerous lesions of the biliary tract.  Normal biliary tract histology, categories and features of biliary neoplasia (biliary intraepithelial neoplasia, intraductal papillary neoplasm of the bile duct, intraductal tubular neoplasm, mucinous cystic neoplasm, differences in immunophenotype), geographic differences in disease incidence and risk factors for biliary neoplasia are discussed in this manuscript.


Happaerts S, Foucault A, Billiard JS, Nguyen B, Vandenbroucke-Menu F. Hepatol 2016; 64(5): 1800-1802.
The authors report a case of a 27 year-old woman with a past medical history of tetralogy of Fallot and Abernathy malformation (congenital extrahepatic portosystemic shunt), who had a 22 cm combined hepatocellular-cholangiocarcinoma (HCC-CC) resected.  Non-neoplastic tissue adjacent to the carcinoma showed FNH-like reaction, consistent with the pre-operative imaging studies which suggested nodular regenerative hyperplasia, FNH, and FNH-like lesions in addition to the carcinoma. Such lesions, including hepatocellular carcinoma (HCC), have been reported in the past, but this is the first report of HCC-CC in the setting of Abernathy malformation. Of particular interest is a 2011 paper referenced by the authors, which describes in detail the various histologic findings seen in Abernathy malformation. These include absence of portal veins in small portal tracts, hypoplastic portal veins in larger portal tracts, lobular unpaired arterioles, hepatic artery hypertrophy, and nodular regenerative hyperplasia, all changes which likely reflect the absence of a portal venous system with compensatory hepatic artery buffer response.
Calderaro J, Rousseau B, Amaddeo G, Mercey M, Charpy C, Costentin C, Luciani A, Zafrani ES, Laurent A, Azoulay D, Lafdil F, Pawlotsky JM. Hepatol 2016; 64(6): 2038-2046.
The authors investigated the PD-L1 status of 217 hepatocellular carcinomas. PD-L1 expression by neoplastic cells was associated with elevated serum AFP, satellite nodules, lymphovascular space invasion, poor differentiation, and CK19 expression. PD-L1 expression by inflammatory cells associated with the tumor was associated with elevated serum AFP, lymphovascular space invasion, poor differentiation, and lymphoepithelial histologic subtype of HCC. No association was seen with B-catenin nuclear staining or with diffuse glutamine synthetase expression.

Rebouissou S, Franconi A, Calderaro J, Letouzé E, Imbeaud S, Pilati C, Nault JC, Couchy G, Laurent A, Balabaud C, Bioulac-Sage P, Zucman-Rossi J. Hepatol 2016; 64(6):2047-2061.
This paper is by the French group which has reported much of the data on hepatocellular adenoma (HA) subtypes. In this study, the authors investigated B-catenin mutation status and B-catenin activity levels on fresh frozen tissue from 220 HAs, 373 HCCs, and 17 borderline HA/HCC lesions. Overall B-catenin activity was higher in HCCs than HAs, and this was related to mutation type. In their HAs, they divided the B-catenin activation into three categories, which was related to specific mutations:
1) Weak activation: S45, K335, and N387 mutations.
2) Moderate activation: T41 mutations.
3) High activation: Exon 3 deletions (most commonly seen in borderline lesions) and D32-S37 mutations.
Tumors with highly active mutations demonstrated strong and homogenous GS staining. Weak mutants demonstrated heterogenous and patchy GS staining. Also, highly active mutants typically showed nuclear B-catenin IHC staining, whereas weakly active mutants did not. Thus, weak mutants would not show typical high risk IHC staining, whereas highly active mutants would. S45, however, is a notable exception which was a mutation seen in both HAs and HCCs. Although this mutation could be seen in malignancy, particularly if duplicated, GS IHC staining would not pick it up. In HCCs with weakly active mutations, the authors demonstrated B-catenin to be activated through additional events, such as loss of heterozygosity (LOH) at 3p or a second B-catenin mutation. Overall, the study is the largest to date correlating B-catenin mutation status with GS IHC staining pattern.

Prepared by:
Editor, Daniela Allende, MD, Cleveland Clinic
Oyedele Adeyi, MD; University of Toronto
Wenqing Cao, MD; New York University
Cindy Guy, MD; Duke University
Sanjay Kakar, MD; University of California, San Francisco
Jingmei Lin, MD, PhD; Indiana University
Rish Pai, MD, PhD; Mayo Clinic Arizona
Eric Yee, MD; University of Oklahoma
Nafis Shafizadeh, MD; Southern California Permanente Medical Group

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