Graft-versus-host disease is locally maintained in target tissues by resident progenitor-like T cells.

In allogeneic hematopoietic stem cell transplantation, donor αß T cells attack recipient tissues, causing graft-versus-host disease (GVHD), a major cause of morbidity and mortality. A central question has been how GVHD is sustained despite T cell exhaustion from chronic antigen stimulation. The current model for GVHD holds that disease is maintained through the continued recruitment of alloreactive effectors from blood into affected tissues. Here, we show, using multiple approaches including parabiosis of mice with GVHD, that GVHD is instead primarily maintained locally within diseased tissues. By tracking 1,203 alloreactive T cell clones, we fitted a mathematical model predicting that within each tissue a small number of progenitor T cells maintain a larger effector pool. Consistent with this, we identified a tissue-resident TCF-1+ subpopulation that preferentially engrafted, expanded, and differentiated into effectors upon adoptive transfer. These results suggest that therapies targeting affected tissues and progenitor T cells within them would be effective.

Critical updates in neuroendocrine tumors: Version 9 American Joint Committee on Cancer staging system for gastroenteropancreatic neuroendocrine tumors.

The American Joint Committee on Cancer (AJCC) staging system for all cancer sites, including gastroenteropancreatic neuroendocrine tumors (GEP-NETs), is meant to be dynamic, requiring periodic updates to optimize AJCC staging definitions. This entails the collaboration of experts charged with evaluating new evidence that supports changes to each staging system. GEP-NETs are the second most prevalent neoplasm of gastrointestinal origin after colorectal cancer. Since publication of the AJCC eighth edition, the World Health Organization has updated the classification and separates grade 3 GEP-NETs from poorly differentiated neuroendocrine carcinoma. In addition, because of major advancements in diagnostic and therapeutic technologies for GEP-NETs, AJCC version 9 advocates against the use of serum chromogranin A for the diagnosis and monitoring of GEP-NETs. Furthermore, AJCC version 9 recognizes the increasing role of endoscopy and endoscopic resection in the diagnosis and management of NETs, particularly in the stomach, duodenum, and colorectum. Finally, T1NXM0 has been added to stage I in these disease sites as well as in the appendix.

Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche.

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts1. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types2,3. However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE2 drives the expansion οf a population of Sca-1+ reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1+ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE2 promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1+ cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE2-Ptger4. Analyses of patient-derived organoids established that PGE2-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE2-Ptger4-Yap signalling axis.

TCF7L2 transcriptionally regulates Fgf15 to maintain bile acid and lipid homeostasis through gut-liver crosstalk.

FGF19/FGF15 is an endocrine regulator of hepatic bile salt and lipid metabolism, which has shown promising effects in the treatment of NASH in clinical trials. FGF19/15 is transcribed and released from enterocytes of the small intestine into enterohepatic circulation in response to bile-induced FXR activation. Previously, the TSS of FGF19 was identified to bind Wnt-regulated TCF7L2/encoded transcription factor TCF4 in colorectal cancer cells. Impaired Wnt signaling and specifical loss of function of its coreceptor LRP6 have been associated with NASH. We, therefore, examined if TCF7L2/TCF4 upregulates Fgf19 in the small intestine and restrains NASH through gut-liver crosstalk. We examined the mice globally overexpressing, haploinsufficient, and conditional knockout models of TCF7L2 in the intestinal epithelium. The TCF7L2+/- mice exhibited increased plasma bile salts and lipids and developed diet-induced fatty liver disease while mice globally overexpressing TCF7L2 were protected against these traits. Comprehensive in vivo analysis revealed that TCF7L2 transcriptionally upregulates FGF15 in the gut, leading to reduced bile synthesis and diminished intestinal lipid uptake. Accordingly, VilinCreert2 ; Tcf7L2fl/fl mice showed reduced Fgf19 in the ileum, and increased plasma bile. The global overexpression of TCF7L2 in mice with metabolic syndrome-linked LRP6R611C substitution rescued the fatty liver and fibrosis in the latter. Strikingly, the hepatic levels of TCF4 were reduced and CYP7a1 was increased in human NASH, indicating the relevance of TCF4-dependent regulation of bile synthesis to human disease. These studies identify the critical role of TCF4 as an upstream regulator of the FGF15-mediated gut-liver crosstalk that maintains bile and liver triglyceride homeostasis.

Hepatocellular Adenoma: Report of 2 Cases That Highlight the Relevance of Phenotype-Genotype Correlation in the Pediatric Population.

BACKGROUND: Hepatocellular adenoma (HCA) in the pediatric population is very rare and there are only limited studies, especially with molecular characterization of the tumors. Main HCA subtypes recognized in the current WHO classification include HNF1A-inactivated HCA (H-HCA), inflammatory HCA (IHCA), ß-catenin-activated HCA (b-HCA), and ß-catenin-activated IHCA (b-IHCA) and sonic hedgehog HCA (shHCA) is reported as an emerging subtype. METHODS: Clinical history, pathological information, and molecular studies for a series of 2 cases of pediatric HCA were reviewed. RESULTS: Case 1 was a b-HCA characterized by somatic CTNNB1 S45 mutation in a 11-year-old male with Abernethy malformation. Case 2 was a H-HCA characterized by germline HNF1A variant (c.526+1G>A) in a 15-year-old male associated with maturity-onset diabetes of the young type 3 (MODY3). CONCLUSION: Our findings highlight the rarity of these 2 cases associated with adenomatosis, and the contribution of molecular/genetic analysis for proper sub-typing, prognosis and family surveillance.

Microangiopathic Hemolytic Anemia Is a Late and Fatal Complication of Gastric Signet Ring Cell Carcinoma: A Systematic Review and Case-Control Study.

BACKGROUND: Microangiopathic hemolytic anemia (MAHA) is a rare paraneoplastic syndrome that has been reported in patients with gastric signet ring cell carcinoma (SRCC). Clinical and prognostic features of MAHA in this setting have been poorly described. MATERIALS AND METHODS: We conducted a systematic review in 8 databases of gastric SRCC complicated by MAHA and performed a case-control study assessing factors associated with survival in patients with gastric SRCC and MAHA in our pooled cohort compared with age-, sex-, and stage-matched cases of gastric SRCC from the Surveillance, Epidemiology, and End Results (SEER) database. Descriptive analyses were performed and multivariable Cox-proportional hazards regression modeling was used to determine factors associated with overall survival. RESULTS: All identified patients (n = 47) were symptomatic at index presentation, commonly with back/bone pain, and dyspnea. Microangiopathic hemolytic anemia was the first manifestation of gastric SRCC in 94% of patients. Laboratory studies were notable for anemia (median 7.7 g/dL), thrombocytopenia (median 45.5 × 103/µL), and hyperbilirubinemia (median 2.3 mg/dL). All patients with MAHA had metastatic disease at presentation, most often to the bone, bone marrow, and lymph nodes. Median survival in patients with gastric SRCC and MAHA was significantly shorter than a matched SEER-derived cohort with metastatic gastric SRCC (7 weeks vs 28 weeks, P < .01). In multivariate analysis, patients with MAHA were at significantly increased risk of mortality (HR 3.28, 95% CI 2.11-5.12). CONCLUSION: Microangiopathic hemolytic anemia is a rare, late-stage complication of metastatic gastric SRCC and is associated with significantly decreased survival compared with metastatic gastric SRCC alone.

An Expert, Multidisciplinary Perspective on Best Practices in Biomarker Testing in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a rare and aggressive malignancy that arises from the intrahepatic biliary tree and is associated with a poor prognosis. Until recently, the treatment landscape of advanced/metastatic iCCA has been limited primarily to chemotherapy. In recent years, the advent of biomarker testing has identified actionable genetic alterations in 40%-50% of patients with iCCA, heralding an era of precision medicine for these patients. Biomarker testing using next-generation sequencing (NGS) has since become increasingly relevant in iCCA; however, several challenges and gaps in standard image-guided liver biopsy and processing have been identified. These include variability in tissue acquisition relating to the imaging modality used for biopsy guidance, the biopsy method used, number of passes, needle choice, specimen preparation methods, the desmoplastic nature of the tumor, as well as the lack of communication among the multidisciplinary team. Recognizing these challenges and the lack of evidence-based guidelines for biomarker testing in iCCA, a multidisciplinary team of experts including interventional oncologists, a gastroenterologist, medical oncologists, and pathologists suggest best practices for optimizing tissue collection and biomarker testing in iCCA.

Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: Results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: Results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.

BACKGROUND & AIMS: Diagnostic histological criteria for autoimmune hepatitis (AIH) have not been clearly established. Previously published criteria focused mainly on chronic AIH, in which inflammatory changes mainly occur in portal/periportal regions and may not be applicable to acute presentation of AIH, in which inflammatory changes are typically predominantly lobular in location. International consensus criteria for the diagnosis and assessment of disease severity in both acute and chronic AIH are thus urgently needed. METHODS: Seventeen expert liver pathologists convened at an international workshop and subsequently used a modified Delphi panel approach to establish consensus criteria for the histopathological diagnosis of AIH. RESULTS: The consensus view is that liver biopsy should remain standard for diagnosing AIH. AIH is considered likely, if there is a predominantly portal lymphoplasmacytic hepatitis with more than mild interface activity and/or more than mild lobular hepatitis in the absence of histological features suggestive of another liver disease. AIH is also considered likely if there is predominantly lobular hepatitis with or without centrilobular necroinflammation and at least one of the following features: portal lymphoplasmacytic hepatitis, interface hepatitis or portal-based fibrosis, in the absence of histological features suggestive of another liver disease. Emperipolesis and hepatocellular rosettes are not regarded as being specific for AIH. CONCLUSIONS: The criteria proposed in this consensus statement provide a uniform approach to the histological diagnosis of AIH, which is relevant for patients with an acute as well as a chronic presentation and to more accurately reflect the current understanding of liver pathology in AIH.

α-Fetoprotein mRNA in situ hybridisation is a highly specific marker of hepatocellular carcinoma: a multi-centre study.

BACKGROUND: Pathologic diagnosis of hepatocellular carcinoma (HCC) can be challenging in differentiating from benign and non-hepatocytic malignancy lesions. The aim of this study was to investigate the potential utility of α-fetoprotein (AFP) mRNA RNAscope, a sensitive and specific method, in the diagnosis of HCC. METHODS: Three independent retrospective cohorts containing 2216 patients with HCC, benign liver lesions, and non-hepatocytic tumours were examined. AFP was detected using ELISA, IHC (Immunohistochemistry), and RNAscope. Glypican3 (GPC3), hepatocyte paraffin-1 (HepPar-1), and arginase-1 (Arg-1) proteins were detected using IHC. RESULTS: AFP RNAscope improved the HCC detection sensitivity by 24.7-32.7% compared with IHC. In two surgical cohorts, a panel of AFP RNAscope and GPC3 provided the best diagnostic value in differentiating HCC from benign hepatocytic lesions (AUC = 0.905 and 0.811), and a panel including AFP RNAscope, GPC3, HepPar-1, and Arg-1 yielded the best AUC (0.971 and 0.977) when distinguishing HCC from non-hepatocytic malignancies. The results from the liver biopsy cohort were similar, and additional application of AFP RNAscope improved the sensitivity by 18% when distinguishing HCC from benign hepatocytic lesions. CONCLUSIONS: AFP mRNA detected by RNAscope is highly specific for hepatocytic malignancy and may serve as a novel diagnostic biomarker for HCC.

Type 3 Inositol 1,4,5-Trisphosphate Receptor Is Increased and Enhances Malignant Properties in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the second most common malignancy arising in the liver. It carries a poor prognosis, in part because its pathogenesis is not well understood. The type 3 inositol 1,4,5-trisphosphate receptor (ITPR3) is the principal intracellular calcium ion (Ca2+ ) release channel in cholangiocytes, and its increased expression has been related to the pathogenesis of malignancies in other types of tissues, so we investigated its role in CCA. ITPR3 expression was increased in both hilar and intrahepatic CCA samples as well as in CCA cell lines. Deletion of ITPR3 from CCA cells impaired proliferation and cell migration. A bioinformatic analysis suggested that overexpression of ITPR3 in CCA would have a mitochondrial phenotype, so this was also examined. ITPR3 normally is concentrated in a subapical region of endoplasmic reticulum (ER) in cholangiocytes, but both immunogold electron microscopy and super-resolution microscopy showed that ITPR3 in CCA cells was also in regions of ER in close association with mitochondria. Deletion of ITPR3 from these cells impaired mitochondrial Ca2+ signaling and led to cell death. Conclusion: ITPR3 expression in cholangiocytes becomes enhanced in CCA. This contributes to malignant features, including cell proliferation and migration and enhanced mitochondrial Ca2+ signaling.

Morphological spectrum of immune check-point inhibitor therapy-associated gastritis.

AIMS: Immune check-point inhibitors are frequently used in the treatment of a variety of solid tumours. The mechanism of action of these drugs involves up-regulation of cytotoxic T cells, which can lead to a lack of self-tolerance and immune-related adverse events, including those involving the gastrointestinal tract. This study was performed to characterise the histological features of immune check-point inhibitor therapy-associated gastritis. METHODS AND RESULTS: Gastric biopsies from patients on immune check-point inhibitor therapy with clinical suspicion of drug-associated gastrointestinal injury were identified. The predominant histological pattern of injury, distribution of injury, degree of tissue eosinophilia and prominence of apoptosis were recorded. Presenting symptoms, treatment and follow-up data were obtained by medical chart review. The 12 patients included in the study group were treated with ipilimumab, nivolumab or pembrolizumab for a variety of tumours. Symptoms at presentation included nausea, vomiting and diarrhoea. Chronic active gastritis with intra-epithelial lymphocytosis and prominent apoptosis was seen in eight of 12 patients, and was the most useful combination for the diagnosis of drug-induced gastritis in these patients. Four patients showed focal enhancing gastritis with a lymphohistiocytic cuff around inflamed glands reminiscent of Crohn's disease. One of those four patients was homozygous for the ATG16L1 Crohn's disease-associated gene variant, but had no history of inflammatory bowel disease. Ten patients responded to medication withdrawal and steroid therapy, while two required treatment with infliximab. CONCLUSIONS: Awareness of the morphological spectrum of immune check-point inhibitor therapy-associated gastritis is important for the accurate diagnosis and prompt management of these patients.

Clinical utility of genomic analysis in adults with idiopathic liver disease.

BACKGROUND & AIMS: Adult patients suffering from liver disease of unknown cause represent an understudied and underserved population. The use of whole-exome sequencing (WES) for the assessment of a broader spectrum of non-oncological diseases, among adults, remains poorly studied. We assessed the utility of WES in the diagnosis and management of adults with unexplained liver disease despite comprehensive evaluation by a hepatologist and with no history of alcohol overuse. METHODS: We performed WES and deep phenotyping of 19 unrelated adult patients with idiopathic liver disease recruited at a tertiary academic health care center in the US. RESULTS: Analysis of the exome in 19 cases identified 4 monogenic disorders in 5 unrelated adults. Patient 1 suffered for 18 years from devastating complications of undiagnosed type 3 familial partial lipodystrophy due to a deleterious heterozygous variant in PPARG. Molecular diagnosis enabled initiation of leptin replacement therapy with subsequent normalization of liver aminotransferases, amelioration of dyslipidemia, and decreases in daily insulin requirements. Patients 2 and 3 were diagnosed with MDR3 deficiency due to recessive mutations in ABCB4. Patient 4 with a prior diagnosis of non-alcoholic steatohepatitis was found to harbor a mitochondrial disorder due to a homozygous pathogenic variant in NDUFB3; this finding enabled initiation of disease preventive measures including supplementation with antioxidants. Patient 5 is a lean patient with hepatic steatosis of unknown etiology who was found to have a damaging heterozygous variant in APOB. CONCLUSIONS: Genomic analysis yielded an actionable diagnosis in a substantial number (∼25%) of selected adult patients with chronic liver disease of unknown etiology. This study supports the use of WES in the evaluation and management of adults with idiopathic liver disease in clinical practice. LAY SUMMARY: We performed whole-exome sequencing in 19 adult patients with unexplained liver disease after an unrevealing conventional work-up performed by a hepatologist. In 5 cases, genomic analysis led to a diagnosis and informed treatment and management of the disease. Therefore, we suggest using whole-exome sequencing in the evaluation and management of adults with unexplained liver disease.

Immunohistochemical and molecular features of cholangiolocellular carcinoma are similar to well-differentiated intrahepatic cholangiocarcinoma.

Cholangiolocellular carcinoma is characterized by low grade cytologic atypia, and anastomosing cords and glands resembling cholangioles or canals of Hering. Cholangiolocellular carcinoma has been variously regarded as a subtype of intrahepatic cholangiocarcinoma (World Health Organization 2000), combined hepatocellular-cholangiocarcinoma of stem cell subtype (World Health Organization 2010) and a distinct type of primary liver carcinoma. Capture-based next generation sequencing targeting the coding regions of 479 cancer genes and select introns was performed on 17 cases (5 cholangiolocellular carcinomas, 7 intrahepatic cholangiocarcinomas, 5 mixed cholangiolocellular-intrahepatic cholangiocarcinomas) along with immunohistochemistry for CK19, SALL4, CD56, CD117, and EMA. For 5 mixed cholangiolocellular-intrahepatic cholangiocarcinoma, the individual areas were micro-dissected prior to sequencing. CK19 and EMA were positive in all cases; both luminal and cytoplasmic EMA was seen in 3/5 cholangiolocellular carcinoma and 3/6 intrahepatic cholangiocarcinomas. CD117 and SALL4 were negative in all cases. CD56 was positive in 2/5 cholangiolocellular carcinoma, 4/6 intrahepatic cholangiocarcinoma and 2/5 mixed cases. Mutations typical of intrahepatic cholangiocarcinoma (IDH1/2, PBRM1, FGFR2) were present in 90% of cases with cholangiolocellular carcinoma component. The genomic profile (IDH1/2 mutations, FGFR2 fusions, chromatin-remodeling gene mutations such as ARID1A, PBRM1) and copy number alterations were similar in cholangiolocellular carcinoma, intrahepatic cholangiocarcinoma and mixed cholangiolocellular-intrahepatic cholangiocarcinoma. In all mixed cases, the immunohistochemistry results, mutational profile and copy number alterations in both components were similar. Cholangiolocellular carcinoma should be categorized as a histologic subtype of well-differentiated intrahepatic cholangiocarcinoma, and should not be considered a distinct entity, or combined hepatocellular-cholangiocarcinoma unless a distinct hepatocellular component is also present.

Knowledge gaps in the appendix: a multi-institutional study from seven academic centers.

Appendix pathology represents uncommonly encountered specimens with unique diagnostic challenges. To delineate common knowledge gaps, extramural consults submitted to seven institutions between 2016-2017 were reviewed. All appendix consults were resections (100%, n = 43), and the majority were directed for consultation by the originating pathologist (95%, n = 41) with no additional studies performed by the consultant (65%, n = 28). This study was dominated by inquiries related to low grade appendiceal mucinous neoplasms (44%, n = 19) and goblet cell carcinoid related neoplasms (19%, n = 8). Of the 43 appendiceal consults, 19 were submitted by the contributing pathologist as low grade appendiceal mucinous neoplasm, but only half of these were diagnosed by the consultant as such (n = 9). Low grade appendiceal mucinous neoplasm-related consultation themes included diverticular disease, criteria for invasion, high grade atypia, extra-appendiceal mucin, and staging. Examples of major disagreements that were downgraded included consults submitted as low grade appendiceal mucinous neoplasm and diagnosed by the consultant as serrated polyp (n = 3), appendicitis (n = 1), and benign appendix (n = 1). Examples of major disagreements-upgraded included cases submitted as low grade appendiceal mucinous neoplasm and diagnosed by the consultant as low grade appendiceal mucinous neoplasm with high-risk features (n = 2) and mucinous adenocarcinoma (n = 2). One case contained both a major disagreement-upgrade (low grade appendiceal mucinous neoplasm changed to high grade appendiceal mucinous neoplasm) and a major disagreement-downgrade (pT3 changed to Tis). Of the 15 cases diagnosed by the consultants as low grade appendiceal mucinous neoplasm, submitted diagnoses included low grade appendiceal mucinous neoplasm (n = 9), adenocarcinoma (n = 5), and one case was submitted without a diagnosis. For goblet cell carcinoid-related consults, the usual inquiry related to distinguishing goblet cell carcinoid from goblet cell carcinoid with adenocarcinoma (adenocarcinoma ex-goblet cell carcinoid). Of the 38 overall consults with a submitted diagnosis, 53% (n = 20) were disagreements, and most of these were major disagreements-downgraded (n = 13).

Von Meyenburg Complex: Current Concepts and Imaging Misconceptions.

Von Meyenburg complexes, also known as biliary hamartomas, are ductal plate malformations that are usually diagnosed on imaging studies when there are multiple as small hepatic cysts. However, because of variations in histology, they actually have a wide spectrum of imaging appearances ranging from solid, to mixed solid and cystic, to cystic lesions. The objective of the review is to provide up-to-date information about the embryopathogenesis of von Meyenburg complexes, their imaging appearances, diagnostic pitfalls, and clinical significance.

Cryptogenic cirrhosis: Old and new perspectives in the era of molecular and genomic medicine.

Cryptogenic cirrhosis (CC) is defined as cirrhosis of unknown etiology despite extensive clinical, laboratory and pathologic work-up, and constitutes approximately 5-10% of all cirrhosis cases. Histologic examination can provide important clues and help identify the potential etiology of CC. Most CC cases can still be classified into four histologic patterns: hepatitic, steatotic, biliary, and patternless (bland). The use of genetic testing has significantly improved diagnostic ability and treatment, especially in pediatric patients with acute and chronic liver diseases. More recently, whole exome sequencing has been used for identifying genetic alterations that lead to a diagnosis in adults with liver disease of unknown etiology. Recent advances in genomic analysis has allowed the unraveling of the underlying etiology in a subset of CC cases, and also helped identify new disorders. Providing a diagnosis for these patients has several important implications for treatment, possible genetic counseling, and transplant eligibility. However, detailed clinical and histologic characterization of the patients still remains an important part of the CC work-up, since clinicopathologic and genomic correlation is crucial in making a diagnosis, or in some cases, discovery of a new entity. This article summarizes the main histologic findings that can be observed in CC cases, potential causes of CC, and recent advances in the field.

ACOX2 deficiency: A disorder of bile acid synthesis with transaminase elevation, liver fibrosis, ataxia, and cognitive impairment.

Acyl CoA Oxidase 2 (ACOX2) encodes branched-chain acyl-CoA oxidase, a peroxisomal enzyme believed to be involved in the metabolism of branched-chain fatty acids and bile acid intermediates. Deficiency of this enzyme has not been described previously. We report an 8-y-old male with intermittently elevated transaminase levels, liver fibrosis, mild ataxia, and cognitive impairment. Exome sequencing revealed a previously unidentified homozygous premature termination mutation (p.Y69*) in ACOX2 Immunohistochemistry confirmed the absence of ACOX2 expression in the patient's liver, and biochemical analysis showed marked elevation of intermediate bile acids upstream of ACOX2. These findings define a potentially treatable inborn error of bile acid biosynthesis caused by ACOX2 deficiency.

Prototheca zopfii Colitis in Inherited CARD9 Deficiency.

Human protothecosis is a rare microalgae infection, and its dissemination typically occurs in immunocompromised individuals, but no specific immune defect has been reported. Here, we describe an 8-year-old daughter of a consanguineous union with abdominal pain and bloody diarrhea for 3 months who was found to have pancolitis with numerous microalgae identified as Prototheca zopfii. In the absence of a known immunodeficiency, exome sequencing was performed, which uncovered a novel recessive frameshift mutation in CARD9 (p.V261fs). This report highlights that CARD9 deficiency should be investigated in patients with unexplained systemic/visceral protothecosis and suggests a new mechanistic insight into anti-Prototheca immunity.

Molecular testing for the clinical diagnosis of fibrolamellar carcinoma.

Fibrolamellar carcinoma has a distinctive morphology and immunophenotype, including cytokeratin 7 and CD68 co-expression. Despite the distinct findings, accurate diagnosis of fibrolamellar carcinoma continues to be a challenge. Recently, fibrolamellar carcinomas were found to harbor a characteristic somatic gene fusion, DNAJB1-PRKACA. A break-apart fluorescence in situ hybridization (FISH) assay was designed to detect this fusion event and to examine its diagnostic performance in a large, multicenter, multinational study. Cases initially classified as fibrolamellar carcinoma based on histological features were reviewed from 124 patients. Upon central review, 104 of the 124 cases were classified histologically as typical of fibrolamellar carcinoma, 12 cases as 'possible fibrolamellar carcinoma' and 8 cases as 'unlikely to be fibrolamellar carcinoma'. PRKACA FISH was positive for rearrangement in 102 of 103 (99%) typical fibrolamellar carcinomas, 9 of 12 'possible fibrolamellar carcinomas' and 0 of 8 cases 'unlikely to be fibrolamellar carcinomas'. Within the morphologically typical group of fibrolamellar carcinomas, two tumors with unusual FISH patterns were also identified. Both cases had the fusion gene DNAJB1-PRKACA, but one also had amplification of the fusion gene and one had heterozygous deletion of the normal PRKACA locus. In addition, 88 conventional hepatocellular carcinomas were evaluated with PRKACA FISH and all were negative. These findings demonstrate that FISH for the PRKACA rearrangement is a clinically useful tool to confirm the diagnosis of fibrolamellar carcinoma, with high sensitivity and specificity. A diagnosis of fibrolamellar carcinoma is more accurate when based on morphology plus confirmatory testing than when based on morphology alone.