Validation of Metallothionein Immunohistochemistry as a Highly Sensitive Screening Test for Wilson Disease.

Wilson's disease (WD) is a rare autosomal recessive condition with protean clinical manifestations that results from biallelic ATP7B mutations. However, non-destructive tissue tests to be applied clinically to tissue specimens are not widely available to effectively assess patients for possible WD. Previously, we showed that metallothionein (MTH) immunohistochemistry has a high sensitivity and specificity for WD diagnosis and, thus, represents a potentially powerful diagnostic tool that can be used in routine histologic sections. We sought to validate this finding in a large cohort of bona fide WD patients and to correlate metallothionein expression with other histologic features. We identified 91 cases of WD, which included 28 needle biopsies and 64 explants from 14 centers worldwide. Histologic features were evaluated, and a histopathological pattern was assigned to each case. All cases were evaluated with Masson trichrome and MTH immunohistochemistry (clone UC1MT, Abcam) using previously published technique. Liver tissues from chronic cholestatic diseases (n= 42) were used as controls. The median age of the cohort was 28.5 years. Out of 91 total cases, 83 were positive for MTH immunostain. In the controls, all 42 cases were negative for MTH immunostain. The sensitivity and specificity of MTH immunostain for WD were 91.20% and 100%, respectively. MTH immunohistochemistry is a highly sensitive and specific cost-effective screening tool for WD. It can be used for patients across age groups, varied histologic patterns, and fibrosis stages. This marker could prove to be a valuable tool in the evaluation of patients with possible WD.

Simultaneous total internal biliary diversion during liver transplantation for progressive familial intrahepatic cholestasis type 1: Standard of care?

Patients post liver transplant (LT) with progressive familial intrahepatic cholestasis type 1 (PFIC-1) often develop progressive graft steatohepatitis, intractable diarrhea, and growth failure. A total internal biliary diversion (TIBD) during an LT may prevent or reverse these adverse events. Children with PFIC-1 who underwent an LT at our institute were divided into 2 groups, A and B based on the timeline where we started offering a TIBD in association with LT. Pre-LT parameters, intraoperative details, and posttransplant complications like graft steatosis and diarrhea were also analyzed between the 2 groups, and their growth velocity was measured in the follow-up period. Of 550 pediatric LT performed between 2011 and 2022, 13 children underwent LT for PFIC-1. Group A had 7 patients (A1-A7) and group B had 6 (B1-B6). Patients A1, A4, B4, and B5 had a failed partial internal biliary diversion before offering them an LT. Patients A1, A2, and A6 in group A died in the post-LT period (2 early allograft dysfunction and 1 posttransplant lymphoproliferative disorder) whereas A3, A4, and A5 had graft steatosis in the follow-up period. A4 was offered a TIBD 4 years after LT following which the graft steatosis fully resolved. In group B, B1, B2, B5, and B6 underwent TIBD during LT, and B3 and B4 had it 24 and 5 months subsequently for intractable diarrhea and graft steatosis. None of the patients in group B demonstrated graft steatosis or diarrhea and had good growth catch-up during follow-up. We demonstrate that simultaneous TIBD in patients undergoing LT should be a standard practice as it helps dramatically improve outcomes in PFIC-1 as it prevents graft steatosis and/or fibrosis, diarrhea, and improves growth catch-up.

Artificial intelligence-based pathology as a biomarker of sensitivity to atezolizumab-bevacizumab in patients with hepatocellular carcinoma: a multicentre retrospective study.

BACKGROUND: Clinical benefits of atezolizumab plus bevacizumab (atezolizumab-bevacizumab) are observed only in a subset of patients with hepatocellular carcinoma and the development of biomarkers is needed to improve therapeutic strategies. The atezolizumab-bevacizumab response signature (ABRS), assessed by molecular biology profiling techniques, has been shown to be associated with progression-free survival after treatment initiation. The primary objective of our study was to develop an artificial intelligence (AI) model able to estimate ABRS expression directly from histological slides, and to evaluate if model predictions were associated with progression-free survival. METHODS: In this multicentre retrospective study, we developed a model (ABRS-prediction; ABRS-P), which was derived from the previously published clustering-constrained attention multiple instance learning (or CLAM) pipeline. We trained the model fit for regression analysis using a multicentre dataset from The Cancer Genome Atlas (patients treated by surgical resection, n=336). The ABRS-P model was externally validated on two independent series of samples from patients with hepatocellular carcinoma (a surgical resection series, n=225; and a biopsy series, n=157). The predictive value of the model was further tested in a series of biopsy samples from a multicentre cohort of patients with hepatocellular carcinoma treated with atezolizumab-bevacizumab (n=122). All samples in the study were from adults (aged ≥18 years). The validation sets were sampled between Jan 1, 2008, to Jan 1, 2023. For the multicentre validation set, the primary objective was to assess the association of high versus low ABRS-P values, defined relative to cross-validation median split thresholds in the first biopsy series, with progression-free survival after treatment initiation. Finally, we performed spatial transcriptomics and matched prediction heatmaps with in situ expression profiles. FINDINGS: Of the 840 patients sampled, 641 (76%) were male and 199 (24%) were female. Across the development and validation datasets, hepatocellular carcinoma risk factors included alcohol intake, hepatitis B and C virus infections, and non-alcoholic steatohepatitis. Using cross-validation in the development series, the mean Pearson's correlation between ABRS-P values and ABRS score (mean expression of ABRS genes) was r=0·62 (SD 0·09; mean p<0·0001, SD<0·0001). The ABRS-P generalised well on the external validation series (surgical resection series, r=0·60 [95% CI 0·51-0·68], p<0·0001; biopsy series, r=0·53 [0·40-0·63], p<0·0001). In the 122 patients treated with atezolizumab-bevacizumab, those with ABRS-P-high tumours (n=74) showed significantly longer median progression-free survival than those with ABRS-P-low tumours (n=48) after treatment initiation (12 months [95% CI 7-not reached] vs 7 months [4-9]; p=0·014). Spatial transcriptomics showed significantly higher ABRS score, along with upregulation of various other immune effectors, in tumour areas with high ABRS-P values versus areas with low ABRS-P values. INTERPRETATION: Our study indicates that AI applied on hepatocellular carcinoma digital slides is able to serve as a biomarker for progression-free survival in patients treated with atezolizumab-bevacizumab. This approach could be used in the development of inexpensive and fast biomarkers for targeted therapies. The combination of AI heatmaps with spatial transcriptomics provides insight on the molecular features associated with predictions. This methodology could be applied to other cancers or diseases and improve understanding of the biological mechanisms that drive responses to treatments. FUNDING: Institut National du Cancer, Fondation ARC, China Scholarship Council, Ligue Contre le Cancer du Val de Marne, Fondation de l'Avenir, Ipsen, and Fondation Bristol Myers Squibb Pour la Recherche en Immuno-Oncologie.

Outcomes of liver transplantation in children with Langerhans cell histiocytosis: Experience from a quaternary care center.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare but important cause of end-stage liver disease in children. Conventional chemotherapeutic agents that are otherwise the standard-of-care in LCH may be counterproductive in patients with hepatic decompensation. Furthermore, the precise role of liver transplantation (LT) in the management of LCH remains unclear. METHODS: Review of a prospectively collected database (January 2014 to December 2020) of children with liver disease was performed. All clinical details of patients with LCH managed at our center were collected and data analyzed. Based on the outcomes, a management algorithm was proposed. RESULTS: Of the eight (five male) patients referred to our unit, six (75%) underwent LT (four and two for compensated and decompensated cirrhosis, respectively). Median age at diagnosis of LCH was 25 (range: 9-48) months. Two patients, who had previously completed LCH-specific chemotherapy, underwent upfront LT for compensated cirrhosis. Other two patients with compensated cirrhosis showed evidence of active disease. They underwent LT following completion of chemotherapy. Two children with decompensated cirrhosis also had evidence of active disease and were started on modified chemotherapy Both of them had progression of liver disease while on chemotherapy. Hence, an urgent LT was performed which was followed by completion of chemotherapy in these patients. On a median follow-up of 30.5 (10.5-50) months, all post-LT patients were alive with stable graft function and showed no disease recurrence. CONCLUSION: We demonstrate that an algorithmic approach, along with newer chemotherapeutic agents, results in excellent outcomes in LCH patients with liver involvement. Larger multicentric studies on this rare disease are, however, needed to validate our findings.

Frequent expression of PD-L1 in lymphocyte-rich hepatocellular carcinoma: A report of 4 cases.

BACKGROUND: Programmed death ligand 1 (PD-L1) is an immune checkpoint inhibitor. PD-L1 binds to its receptor programmed death receptor (PD-1) expressed by immune cells and plays a key role in regulating immune responses. Engagement of PD-L1 on cancer cells and PD-1 on immune cells avoid destruction of tumour cells by immune cells. Immunostaining with PD-L1 has been suggested as a biomarker predictive of antiPD-L1 immunotherapy. Lymphocyte-rich hepatocellular carcinoma (LrHCC) is a rare histological HCC subtype which is characterised by neoplastic epithelial cells intermixed with numerous immune cells. METHODS: Here in we investigated immunohistochemical PD-L1 expression in 4 cases of LrHCC. Tumour proportion score (TPS) and immune cell score was recorded. Immunophenotypic characterization of the tumour and inflammatory cells was also done. Epstein-Barr encoding region (EBER) in situ hybridization (ISH) assay as performed in all four tumours. RESULTS: Expression of PD-L1 was demonstrated in tumour epithelial cells and immune cells in all four cases. Incomplete to membranous staining was demonstrated in the tumour cells. Tumour proportion score (TPS) was 1.2-20 %. Immune cells demonstrated membranous and cytoplasmic immunostaining. Immune cell score was ≥1 % to >10 %. CONCLUSION: PD-L1 expression in both tumour and immune cells suggests distinct immunogenic feature and potential role of antiPD-L1 therapies in cases with inoperable disease.

Pathologic and Immunophenotypic Characterization of Syncytial Giant Cell Variant of Pediatric Hepatocellular Carcinoma. A Distinct Subtype.

INTRODUCTION: Hepatocellular carcinoma (HCC) in pediatrics has a uniformly poor prognosis. Complete surgical resection or liver transplantation remain the only curative options. In contrast to adult HCC, literature on pediatric HCC is sparse and a majority of the distinct subtypes are undefined with regards to their histology, immunohistochemistry and prognosis. CASE REPORT: Two infants, one with biliary atresia and another with transaldolase deficiency, underwent living donor liver transplants. Explant-liver histopathology revealed tumor with diffuse neoplastic syncytial giant cell pattern. Immunophenotypic characterization highlighted expression of epithelial cell adhesion molecule, alpha fetoprotein and metallothionein. CONCLUSION: HCC with syncytial giant cells variant can occur in infants with underlying liver disease, specifically in our experience, with biliary atresia and another with transaldolase deficiency.

First report of auxiliary liver transplantation for severe cholangiopathy after SARS-CoV-2 respiratory infection.

Post-coronavirus disease 2019 (COVID-19) cholangiopathy (PCC) is a new entity observed in patients recovering from severe COVID-19 pneumonia. Most patients recover with cholestasis improving over a period of time. In some patients, cholestasis is severe and persists or progresses to liver failure necessitating liver transplant. We present a previously healthy 50-year-old man who developed PCC with peak total bilirubin of 42.4 mg/dl and did not improve with medical management. He underwent living donor auxiliary right lobe liver transplantation. He recovered well after transplant and remains asymptomatic at 6 months follow-up with good graft function and recovering function in native liver remnant.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

BACKGROUND & AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. METHODS: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. RESULTS: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. CONCLUSION: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. LAY SUMMARY: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Biallelic Mutations in Ubiquitin-Specific Peptidase 53 (USP53) Causing Progressive Intrahepatic Cholestasis. Report of a Case With Review of Literature.

Whole-exome sequencing studies have recently identified novel genes implicated in normal- or low-GGT pediatric cholestasis including ubiquitin-specific peptidase 53 (USP53). We identified novel biallelic mutations in the USP53 gene in a 7-month-old infant with pruritus and progressive intrahepatic cholestasis. His liver biopsy showed portal and perivenular fibrosis with bland bilirubinostasis. His parents were asymptomatic heterozygous for the same mutation. He is currently on vitamin supplements and cholestyramine and his family has also been counseled for liver transplantation. Our report confirms that patients with biallelic mutation in USP53 develop cholestatic liver disease.

Congenital Bile Acid Synthesis Defect Type 3 With Severe Neonatal Cholestasis.

Congenital bile acid synthesis defect type 3 is a rare metabolic liver disease with only eight patients reported in literature. We describe clinical, pathological and molecular features for a ninth patient. A 4-month-old infant presented to us with conjugated hyperbilirubinemia. His liver biopsy revealed giant cell change, steatosis, and activity with diffuse fibrosis. Immunostaining with bile salt export pump showed preserved canalicular pattern and γ-glutamyl transferase 1 staining showed unusual complete membranous pattern. Genetic workup revealed homozygous single base pair duplication in exon 3 of the CYP7B1 gene. He succumbed to liver disease at 7 months of age.

Compound Heterozygous Myosin 5B (Myo5b) Mutation with Early Onset Progressive Cholestasis and No Intestinal Failure.

Background: Exome sequencing studies have recently identified novel genes implicated in normal or low GGT pediatric cholestasis including myosin 5B (MYO5B). Case report: We identified novel compound heterozygote mutations in exon 14 and exon 19 of the MYO5B gene in an 18-month-old Indian child with history of fluctuating jaundice and severe pruritus. His liver biopsy showed portal and perivenular fibrosis with focal bridging septa and mild activity. He is currently on UDCA, cholestyramine and vitamin supplements. There is no history of diarrhea. His asymptomatic mother showed heterozygous mutation in exon 19 of the MYO5B gene and his asymptomatic father showed heterozygous mutation in exon 14 of the MYO5B gene. Conclusion: Our report confirms that patients with compound heterozygote mutations in MYO5B develop progressive cholestasis with no intestinal disease.

CARMIL2 Immunodeficiency with Epstein Barr Virus Associated Smooth Muscle Tumor (EBV-SMT). Report of a Case with Comprehensive Review of Literature.

Background: Primary immunodeficiency (PID) having defects related to lymphocyte cytotoxic pathway or T-cell dysfunction are well known for developing opportunistic infections and Epstein-Barr virus (EBV)-associated diseases. CARMIL2 deficiency is a recently described combined immunodeficiency (CID) disorder characterized by defective CD28-mediated T cell co-stimulation, altered cytoskeletal dynamics, susceptibility to various infections and Epstein Barr Virus smooth muscle tumor (EBV-SMT). Case report: We report a homozygous CARMIL2 pathogenic variant presenting with recurrent infections and EBV associated smooth muscle tumor (SMT) in a child. Conclusion: The present study reports that EBV SMT may occur in a child with CARMIL2 deficiency.

Auto-immune hepatitis following COVID vaccination.

Unprecedented loss of life due to the COVID pandemic has necessitated the development of several vaccines in record time. Most of these vaccines have received approval without being extensively whetted for their adverse effect and efficacy profiles. Most adverse effects have been mild, nonetheless, more serious thromboembolic events have also been reported. Autoimmune hepatitis (AIH) can occur in predisposed individuals where an immune mediated reaction against hepatocytes is triggered by environmental factors. Vaccines are a very rare cause of AIH. We report two such cases of AIH triggered by COVID (Covishield) vaccination. While one patient made an uneventful recovery, another succumbed to the liver disease. Ours is the first report of Covishield vaccination related AIH and second ever after any form of COVID vaccination. We hope that our report does not deter COVID vaccination drives. However, we also hope to raise awareness of its potential side effects and the increased role of pharmacovigilance in guiding treatment.

Primary tuberculosis of the graft masquerading pyogenic liver abscess in a pediatric liver recipient.

Primary tuberculosis (TB) of the graft presenting as multiple liver abscesses is previously unreported. A 14-month-old male child in the early post liver transplant (LT) period presented with high-grade fever spikes and on evaluation was found to have multiple pyogenic liver abscesses (PLA) in the CT abdomen. His fever was not responding to intravenous antibiotics and liver biopsy was done which showed numerous acid fast bacilli. Genetic analysis confirmed the bacilli as Mycobacterium tuberculosis (MTB). Timely diagnosis and prompt introduction of antituberculosis therapy were lifesaving.

RNA-seq reveals outcome-specific gene expression of MMP7 and PCK1 in biliary atresia.

The disease phenotype in biliary atresia (BA) is caused by a fibro-inflammatory process leading to destruction of cholangiocytes, obstruction of ductular pathways and eventual progression to liver cirrhosis. The first line of management is a Kasai portoenterostomy (KPE) followed by liver transplantation (LT) in some children. Several factors have been postulated to affect the outcome of KPE and/or the subsequent progression of liver disease. However, no biomarkers have been identified in the liver for BA. We aimed to address this deficit. Whole transcriptome mRNA sequencing was performed for 29 samples (25 BA and 4 Controls) to identify the candidate genes predicting the prognosis of KPE. These results were further confirmed with quantitative Realtime PCR (qPCR). Analysis from RNA-sequencing data identified matrix metalloproteinase7 (MMP7) and phosphoenolpyruvate carboxykinase (PCK1) as potential determinants of the outcome of KPE. MMP7 expression was significantly elevated in patients who failed to clear jaundice after KPE as well as in patients with End Stage Liver Disease (ESLD). In contrast, PCK1 level was upregulated in patients who had successful KPE, while there was a significant down regulation in patients who failed KPE. MMP7 and PCK1 expression patterns had an inverse relation to the outcome of KPE and hence could potentially be used as biomarkers to predict KPE outcome and disease progression, enabling clinicians to design new treatment strategies for BA.

Living donor liver transplantation in Alagille syndrome-Single center experience from south Asia.

To analyze the clinical characteristics and the outcomes of living donor liver transplantation in children with Alagille syndrome (AGS). Clinical data of children with AGS who underwent liver transplantation between July 2009 and May 2019 in our unit were retrospectively analyzed. Primary end-points were patient and graft survival. Ten children with AGS underwent living donor liver transplantation at a median age of 28 months (range, 12-84 months). Jaundice was the most common initial symptom and was noted after a median duration of 20 days after birth (range, 7-60 days). Two patients had undergone Kasai porto-enterostomy for misdiagnosis of biliary atresia. The most common indication for transplantation was severe pruritus with poor quality of life. Explant livers in three children showed cirrhosis with early well-differentiated hepatocellular carcinoma. We have 100% patient and graft survival at a mean follow-up of 32 months (range 3-72 months). The median z-score for weight and height at liver transplantation was -2.66 (range: -6.44 to -0.9) and -3.6 (range: -7.96 to -0.93) while at follow-up was -1.7 (range: -3.4 to -0.35) and -2.1 (range: -3.9 to -1.4), respectively. The estimated glomerular filtration rate was normal pretransplant and follow-up. This is the first series of LDLT for Alagille syndrome in the Indian sub-continent. We report excellent post-transplant outcomes in contrast to outcomes reported from Western literature.

Age Is Not a Criterion in Patient Selection for Kasai Portoenterostomy.

AIMS: The aim of our study was to compare the outcome of Kasai portoenterostomy (KPE) in children with biliary atresia (BA) older than 90 days to children less than 90 days and to study its safety and efficacy in children older than 90 days. SUBJECTS AND METHODS: Relevant data were collected from our prospectively maintained database of all children with BA who underwent KPE over a 5-year period. Children were divided into two groups: Group 1 ≤90 days and Group 2 >90 days. Data analyzed and compared included total and direct bilirubin, aspartate aminotransferase-to-platelet ratio index (APRI), and the outcome of procedure which was defined as a serum direct bilirubin <2 mg/dl within 6 months after surgery. Standard statistical tests were used for analysis. RESULTS: Out of 62 children, 45 children were in Group 1 and 17 children were in Group 2. Children in Group 2 had similar total and direct bilirubin compared to children in Group 1. APRI, an indicator of fibrosis, was significantly increased in Group 2 (P = 0.08). About 47% of children in Group 2 had Stage III fibrosis on liver histology compared to 22% of children in Group 1. None of the children in Group 2 had synthetic liver failure (refractory ascites, hypoalbuminemia, or coagulopathy unresponsive to Vitamin K) or portal hypertension. KPE was successful in 29.4% of children in Group 2 and 44% in children in Group 1. There was no perioperative mortality in our group. CONCLUSIONS: KPE was successful in a third of children over 90 days of age and can be safely performed in this group. In the absence of synthetic liver failure, age should not be a disqualification for performing KPE.

Melanotic intracranial epidermoid: Case report and description of a new subtype.

Intracranial epidermoids are generally seen as hypodense nonenhancing lesions on computed tomography scans; and, as T1 hypointense and T2 hyperintense lesions on magnetic resonance imaging (MRI). Unusual radiological findings have been reported earlier. The authors present the case of a 54-year old male patient who had prior intracranial surgery. On MRI, there was a thick peripheral mantle of diffusion restriction with a central core of brilliant T1 hyperintensity and very black T2 hypointensity. The peripheral mantle showing diffusion restriction was heterogeneously T1 hypointense and T2 hyperintense. At surgery, there was a typical pearly white epidermoid peripherally with a greenish-brown centre. Pathology showed abundant extracellular melanin which was also found in the basal layer. The authors present the first case of a melanin pigmented intracranial epidermoid in literature, describing a new histological subtype.