Identification of FGFR4 as a regulator of myofibroblast differentiation in Pulmonary Fibrosis.

Introduction IPF is a devastating lung disease with limited therapeutic options. FGFR4 is a known receptor for several paracrine Fibroblast growth factors (FGFs). FGFR4 is also the main receptor for FGF19, an endocrine FGF that was demonstrated by our group to have anti-fibrotic properties in the lung. We aimed to determine whether FGFR4 could modulate pulmonary fibrogenesis. Methods We assessed FGFR4 mRNA and protein levels in IPF and control lungs. In vitro, we determined the effect of TGF-b, Endothelin-1 and PDGF on FGFR4 expression in human lung fibroblasts. We determined the effect FGFR4 inhibition, using a specific pharmacological inhibitor (FGF401), or genetic deletion in murine embryonic fibroblasts (MEFs) on TGF-b-induced myofibroblastic differentiation. In vivo, we evaluated the development of bleomycin-induced lung fibrosis in Fgfr4-deficient (Fgfr4-/-) mice compared to Wild Type littermates (WT), and after FGF401 treatment in WT mice compared to a control group receiving the solvent only. Results FGFR4 was decreased in IPF lungs as compared to control lungs, at mRNA and protein levels. In vitro, FGFR4 was downregulated after treatment by TGF- ß, Endothelin-1 and PDGF. In vitro, FGFR4 inhibition by FGF401 prevented TGF-b1-induced collagen and ACTA2 increase in lung fibroblasts. Similar results were observed in Fgfr4-/- MEFs. In vivo, FGFR4 genetic deficiency or FGFR4 pharmacological inhibition did not modulate bleomycin-induced pulmonary fibrosis. Conclusion Our data suggest that FGFR4 exerts pro-fibrotic properties by enhancing TGF- ß signaling in vitro. However, the inhibition of FGFR4 is not sufficient to prevent the development of pulmonary fibrosis in vivo.

Borderline Hepatocellular Adenomas: A Practical Diagnostic Approach Based on Pathologic and Molecular Features.

Borderline hepatocellular adenomas (BL-HCA) are characterized by focal architectural/cytologic atypia and reticulin loss, features that are insufficient for a definitive diagnosis of hepatocellular carcinoma (HCC). The diagnosis and management of BL-HCA are challenging as their biological behavior, especially in terms of malignant potential, is still debated. We aimed to compare the clinicopathologic and molecular features of BL-HCA with those of typical HCA (T-HCA), HCA with malignant transformation (HCC on HCA), and HCC to assess the risk of malignancy. One hundred six liver resection specimens were retrospectively selected from 2 reference centers, including 39 BL-HCA, 42 T-HCA, 12 HCC on HCA, and 13 HCC specimens. Somatic mutations, including TERT promoter mutations associated with HCA malignant transformation and the gene expression levels of 96 genes, were investigated in 93 frozen samples. Additionally, TERT promoter mutations were investigated in 44 formalin-fixed, paraffin-embedded samples. The clinical features of patients with BL-HCA were similar to those of patients with T-HCA, patients being mainly women (69%) with a median age of 37 years. The median tumor size was 7.5 cm, 64% of patients had a single nodule, and no recurrence was observed. Compared with T-HCA, BL-HCA was significantly enriched in ß-catenin-mutated HCA in exon 3 (41% vs 6%; P < .001). Unsupervised statistical analysis based on gene expression showed that BL-HCA overlapped with T-HCA and HCC on HCA, favoring a molecular continuum of the tumors. TERT promoter mutations were observed only in HCC on HCA (42%) and in HCC (38%). In conclusion, these results suggest that despite their worrisome morphologic features, the clinicopathologic and molecular features of BL-HCA are much closer to those of T-HCA than those of HCC on HCA or HCC. This strongly supports the usefulness of combining morphologic and molecular analyses in a practical diagnostic approach for guiding the management of BL-HCA.

Molecular deciphering of primary liver neuroendocrine neoplasms confirms their distinct existence with foregut-like profile.

Isolated hepatic localizations of neuroendocrine tumors (NETs) are generally considered as metastatic NETs of unknown primary but could correspond to primary hepatic NETs (PHNETs), a poorly explored entity. We aimed to describe the clinicopathological and molecular features of PHNETs and compare them with other primary NETs. We assembled a retrospective cohort of patients managed for hepatic localization of NET without extra-hepatic primary tumor after exhaustive clinical, imaging, and immunohistochemical characterization. We performed whole-exome sequencing with mutational and copy number analysis. Transcriptomic profiles were compared with pancreatic (n = 31), small-bowel (n = 22), and lung (n = 15) NETs using principal component analysis, unsupervised clustering, and gene set enrichment analysis. Among 27 screened patients, 16 had PHNET (solitary tumor in 63%, median size 11 cm, G2 NETs in 81%) following clinical and pathological review. DNA analyses showed 'foregut-like' genomic profiles with frequent alterations in pathways of Fanconi DNA repair (75%), histone modifiers (58%), adherens junctions (58%), and cell cycle control (50%). The most frequently involved genes were KMT2A (58%), ATM (42%), CDH1, CDKN2C, FANCF, and MEN1 (33% each). Transcriptomic analyses showed that PHNETs clustered closer to foregut (pancreatic, lung) NETs than to midgut (small-bowel) NETs, while remaining a distinct entity with a specific profile. Assessment of potentially predictive biomarkers suggested efficacy of treatments usually active in foregut NETs. In conclusion, PHNETs display a foregut-like molecular profile distinct from other types of NETs, with recurrent molecular alterations. Upon exhaustive work-up to exclude an unrecognized primary tumor, PHNETs should not be considered metastatic NETs from an unknown primary. © 2022 The Pathological Society of Great Britain and Ireland.

[Lymphocytic " itis", from esophagus to large bowel]. / « ites ¼ lymphocytaires, de l'œsophage au côlon.

Intra-epithelial lymphocytosis is an elementary lesion frequently observed in the gastrointestinal tract, which can be found from the esophagus to the colon. Many conditions of a varied nature (dysimmunitary diseases, drugs, infections…) are associated with intra-epithelial lymphocytosis, and the etiological diagnosis most often requires an anatomo-clinical correlation. The pathologist will have to identify histological lesions associated with intra-epithelial lymphocytosis allowing the diagnosis to be oriented in order to propose appropriate treatment. In this review, the main entities associated with digestive intra-epithelial lymphocytosis will be presented, detailing the key elements allowing their diagnosis.

[Digestive infectious pathology: Diagnoses not to be missed]. / Pathologie infectieuse du tube digestif : diagnostics à ne pas manquer.

The recent context of COVID-19 has renewed the interest of pathologists in diseases of infectious origin. This interest is even stronger in the gastrointestinal tract where symptoms are aspecific, often frustrating with a normal endoscopic appearance sometimes leading to diagnostic erraticity. In this context, systematic biopsies performed by the clinician are sometimes the only way to reach a diagnosis. Nevertheless, the precise diagnosis of these pathologies requires a good knowledge of the context in which they occur, the histopathological aspect and a rigorous analysis using special stains and/or immunohistochemical analyses. Some infectious diseases of the gastrointestinal tract are well known to pathologists who are widely called upon to diagnose them (Helicobacter pylori gastritis, Candida albicans oesophagitis or CMV colitis), but others are more difficult to diagnose. In this article, we will present, after having recalled the various useful special stains, rare or difficult to diagnose bacterial or parasitic pathologies "not to be missed" in the digestive tract.

Gene expression signature as a surrogate marker of microvascular invasion on routine hepatocellular carcinoma biopsies.

BACKGROUND & AIMS: Microvascular invasion (MVI), a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma (HCC), is only detectable by microscopic examination of the surgical specimen. We aimed to define a transcriptomic signature associated with MVI in HCC than can be applied to formalin-fixed paraffin-embedded (FFPE) biopsies for use in clinical practice. METHODS: To identify a gene expression signature related to MVI by using NanoString technology, we selected a set of 200 genes according to the literature and RNA-sequencing data obtained from a cohort of 150 frozen HCC samples previously published. We used 178 FFPE-archived HCC samples, including 109 surgical samples for the training set and 69 paired pre-operative biopsies for the validation set. In 14 cases of the training set, a paired biopsy was available and was also analyzed. RESULTS: We identified a 6-gene signature (ROS1, UGT2B7, FAS, ANGPTL7, GMNN, MKI67) strongly associated with MVI in the training set of FFPE surgical HCC samples, with 82% accuracy (sensitivity 82%, specificity 81%, AUC 0.82). The NanoString gene expression was highly correlated in 14 paired surgical/biopsy HCC samples (mean R: 0.97). In the validation set of 69 FFPE HCC biopsies, the 6-gene NanoString signature predicted MVI with 74% accuracy (sensitivity 73%, specificity 76%, AUC 0.74). Moreover, on multivariate analysis, the MVI signature was associated with overall survival in both sets (hazard ratio 2.29; 95% CI 1.03-5.07; p = 0.041). CONCLUSION: We defined a 6-gene signature that can accurately predict MVI in FFPE HCC biopsy samples, which is also associated with overall survival, although its survival impact must be confirmed by extensive study with further clinical data. LAY SUMMARY: Microvascular invasion, a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma, is only detectable by microscopic examination of a surgical specimen. In this study, we defined a relevant surrogate signature of microvascular invasion in hepatocellular carcinoma that may be applied in clinical practice with routine tumor biopsy and integrated into the therapeutic strategy.

Autofluorescence imaging within the liver: a promising tool for the detection and characterization of primary liver tumors.

OBJECTIVES: To assess the performance of 405 nm-induced autofluorescence for the characterization of primary liver nodules on ex vivo resected specimens. MATERIALS AND METHODS: Forty resected liver specimens bearing 53 primary liver nodules were included in this IRB-approved prospective study. Intratissular spectroscopic measurements were performed using a 25-G fibered-needle on all ex vivo specimens: 5 autofluorescence measurements were performed in both nodules and adjacent parenchyma. The spectra derivatives of the 635 and 670 nm autofluorescence peaks observed in nodules and in adjacent liver parenchyma were compared (Kruskal-Wallis and Mann-Whitney when appropriate). RESULTS: A total of 42 potentially evolutive primary liver nodules-34 hepatocellular carcinomas, 4 intrahepatic cholangiocarcinomas, 4 hepatocellular adenomas-and 11 benign nodules-5 focal nodular hyperplasias, 6 regenerative nodules-were included. Both 635 and 670 nm Δderivatives were significantly higher in benign as compared to potentially evolutive (PEV) nodules (respectively 32.9 ± 4.5 vs 15.3 ± 1.4; p < 0.0001 and 5.7 ± 0.6 vs 2.5 ± 0.1; p < 0.0001) with respective sensitivity and specificity of 78% and 91% for distinguishing PEV from benign nodules. CONCLUSION: 405 nm-induced autofluorescence enables the discrimination of benign from PEV primary liver nodules, suggesting that autofluorescence imaging could be used to optimize US targeted liver biopsies. KEY POINTS: • 405 nm-induced autofluorescence can distinguish liver tumors from the adjacent liver parenchyma. • The analysis of autofluorescence imaging observed within primary liver tumors can discriminate benign tumors from those requiring follow-up or targeted liver biopsy. • In current practice, autofluorescence imaging could be embedded within biopsy needle, to enable, in addition to ultrasound guidance, optimal targeting of liver nodules which could optimize tissue sampling.

Blood fibrocytes are associated with severity and prognosis in COVID-19 pneumonia.

Increased blood fibrocytes are associated with a poor prognosis in fibrotic lung diseases. We aimed to determine whether the percentage of circulating fibrocytes could be predictive of severity and prognosis during coronavirus disease 2019 (COVID-19) pneumonia. Blood fibrocytes were quantified by flow cytometry as CD45+/CD15-/CD34+/collagen-1+ cells in patients hospitalized for COVID-19 pneumonia. In a subgroup of patients admitted in an intensive care unit (ICU), fibrocytes were quantified in blood and bronchoalveolar lavage (BAL). Serum amyloid P (SAP), transforming growth factor-ß1 (TGF-ß1), CXCL12, CCL2, and FGF2 concentrations were measured. We included 57 patients in the hospitalized group (median age = 59 yr [23-87]) and 16 individuals as healthy controls. The median percentage of circulating fibrocytes was higher in the patients compared with the controls (3.6% [0.2-9.2] vs. 2.1% [0.9-5.1], P = 0.04). Blood fibrocyte count was lower in the six patients who died compared with the survivors (1.6% [0.2-4.4] vs. 3.7% [0.6-9.2], P = 0.02). Initial fibrocyte count was higher in patients showing a complete lung computed tomography (CT) resolution at 3 mo. Circulating fibrocyte count was decreased in the ICU group (0.8% [0.1-2.0]), whereas BAL fibrocyte count was 6.7% (2.2-15.4). Serum SAP and TGF-ß1 concentrations were increased in hospitalized patients. SAP was also increased in ICU patients. CXCL12 and CCL2 were increased in ICU patients and negatively correlated with circulating fibrocyte count. We conclude that circulating fibrocytes were increased in patients hospitalized for COVID-19 pneumonia, and a lower fibrocyte count was associated with an increased risk of death and a slower resolution of lung CT opacities.

Smooth muscular layer: A new helpful criterion to reclassify tumor deposits into metastatic lymph nodes in patients with colo-rectal adenocarcinoma.

CONTEXT: The origin of tumor deposit in colorectal cancer is still unknown, and currently there is no single morphological feature to distinguish a metastatic lymph node from a tumor deposit. Histologically, the normal lymph node capsule and trabeculae contain a smooth muscular layer, which when present in extramural deposits would strongly suggest their lymph node origin. OBJECTIVE: We analyze the value of the smooth muscular layer criterion in reclassifying tumor deposit into metastatic lymph node. DESIGN: A total of 458 colo-rectal carcinomas surgical specimens treated or not by neoadjuvant (radio)chemotherapy were retrospectively included. Harvested tumor deposits were analyzed by Hematoxylin and Eosin and elastin staining on 10 consecutive serial sections and by α- smooth muscle actin immunostaining. RESULTS: A total of 129 tumor deposits were identified. 77 (60%) tumor deposits were reclassified into metastatic lymph node, of which 63 (49%) presented a smooth muscular layer on the initial Hematein Eosin staining and/or after serial tissue sections, confirmed by positive α-smooth muscle actin immunostaining in 43 out of 45 cases (90%). Fourteen (18%) additional tumor deposits were reclassified into metastatic lymph node by the appearance of lymphoid tissue after serial sections. CONCLUSIONS: The presence of a smooth muscular layer in a presumable tumor deposit is helpful in pointing out its lymph node origin in patients with colo-rectal carcinomas. This criterion could improve the inter-observer agreement of tumor deposit identification, allowing accurate nodal staging and better assessment of patient's prognosis.

Use of Artificial Intelligence as an Innovative Method for Liver Graft Macrosteatosis Assessment.

The worldwide implementation of a liver graft pool using marginal livers (ie, grafts with a high risk of technical complications and impaired function or with a risk of transmitting infection or malignancy to the recipient) has led to a growing interest in developing methods for accurate evaluation of graft quality. Liver steatosis is associated with a higher risk of primary nonfunction, early graft dysfunction, and poor graft survival rate. The present study aimed to analyze the value of artificial intelligence (AI) in the assessment of liver steatosis during procurement compared with liver biopsy evaluation. A total of 117 consecutive liver grafts from brain-dead donors were included and classified into 2 cohorts: ≥30 versus <30% hepatic steatosis. AI analysis required the presence of an intraoperative smartphone liver picture as well as a graft biopsy and donor data. First, a new algorithm arising from current visual recognition methods was developed, trained, and validated to obtain automatic liver graft segmentation from smartphone images. Second, a fully automated texture analysis and classification of the liver graft was performed by machine-learning algorithms. Automatic liver graft segmentation from smartphone images achieved an accuracy (Acc) of 98%, whereas the analysis of the liver graft features (cropped picture and donor data) showed an Acc of 89% in graft classification (≥30 versus <30%). This study demonstrates that AI has the potential to assess steatosis in a handy and noninvasive way to reliably identify potential nontransplantable liver grafts and to avoid improper graft utilization.

Comparison of pulsed and oscillating gradient diffusion-weighted MRI for characterizing hepatocellular nodules in liver cirrhosis: ex vivo study in a rat model.

BACKGROUND: In contrast to classical pulsed gradient diffusion-weighted MRI, oscillating gradient diffusion-weighted MR imaging (DWI) is sensitive to short distance diffusion changes at the intracellular level. PURPOSE: To compare the diagnostic performance of pulsed and oscillating DWI for characterizing hepatocellular nodules in a rat model of hepatic cirrhosis. STUDY TYPE: Prospective, experimental study. ANIMAL MODEL: Cirrhosis was induced by weekly intraperitoneal injection of diethylnitrosamine in Wistar rats. FIELD STRENGTH/SEQUENCE: Ex vivo liver MRI was performed at 7T with T1 -weighted, T2 -weighted, pulsed, and oscillating gradient diffusion-weighted sequences. ASSESSMENT: Apparent diffusion coefficient from pulsed (ADCpulsed ) and oscillating gradient (ADCoscillating ) sequences was calculated in 82 nodules identified on the T1 /T2 -weighted images and on pathological examination. Two pathologists classified the nodules in three categories: benign (regenerative and low-grade dysplastic nodules), with intermediate malignancy (high-grade dysplastic nodules and early hepatocellular carcinomas) and overtly malignant (progressed hepatocellular carcinomas). STATISTICAL TESTS: Differences between groups were assessed with Kruskal-Wallis and Mann-Whitney tests. RESULTS: ADC, mainly ADCoscillating , increased in the group of nodules with intermediate malignancy (ADCpulsed : 0.75 ± 0.25 × 10-3 mm2 /s vs. 0.64 ± 0.07 × 10-3 mm2 /s in benign nodules, P = 0.025; ADCoscillating : 0.81 ± 0.20 × 10-3 mm2 /s vs. 0.65 ± 0.13 × 10-3 mm2 /s, P = 0.0008) and ADCpulsed decreased in the group of progressed hepatocellular carcinomas (ADCpulsed : 0.60 ± 0.08 × 10-3 mm2 /s, P = 0.042; ADCoscillating : 0.68 ± 0.08 × 10-3 mm2 /s, P = 0.1). DATA CONCLUSION: ADC during hepatocarcinogenesis in rats increased in nodules with intermediate malignancy and decreased in progressed hepatocellular carcinomas. Our results suggest that oscillating gradient DWI is more sensitive to the early steps of hepatocarcinogenesis and might be useful for differentiating between high-grade dysplastic nodules / early hepatocellular carcinomas and regenerating nodules / low-grade dysplastic nodules. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2020;51:1065-1074.

The histone acetyltransferase hMOF promotes vascular invasion in hepatocellular carcinoma.

BACKGROUND & AIMS: Vascular invasion is a major prognostic factor in hepatocellular carcinoma (HCC). We previously identified histone H4 acetylated at lysine 16 (H4K16ac), a histone modification involved in transcription activation, as a biomarker of microvascular invasion (mVI) in HCC. This study aimed to investigate the role of hMOF, the histone acetyltransferase responsible for H4K16 acetylation, in the process of vascular invasion in HCC. METHODS: hMOF expression was assessed by RT-qPCR and immunohistochemistry in a retrospective series of HCC surgical samples, and correlated with the presence of mVI. The functional role of hMOF in HCC vascular invasion was investigated in vitro in HCC cell lines using siRNA, transcriptomic analysis and transwell invasion assay, and in vivo using a Zebrafish embryo xenograft model. RESULTS: We found that hMOF was significantly upregulated at the protein level in HCC with mVI, compared with HCC without mVI (P < .01). Transcriptomic analysis showed that hMOF downregulation in HCC cell line lead to significant downregulation of key genes and pathways involved in vascular invasion. These results were confirmed by transwell invasion assay, where hMOF downregulation significantly reduced HCC cells invasion. Finally, hMOF downregulation significantly reduced tumour cell intravasation and metastasis in vivo. CONCLUSIONS: Altogether, these results underpin a critical role for hMOF in vascular invasion in HCC, via transcription activation of key genes involved in this process. These data confirm the major role of epigenetic alterations in HCC progression, and pave the way for future therapies targeting hMOF in HCC.

Macrotrabecular-massive hepatocellular carcinoma: A distinctive histological subtype with clinical relevance.

We recently identified a histological subtype of hepatocellular carcinoma (HCC), designated as "macrotrabecular-massive" (MTM-HCC) and associated with specific molecular features. In order to assess the clinical relevance of this variant, we investigated its prognostic value in two large series of patients with HCC treated by either surgical resection or radiofrequency ablation (RFA). We retrospectively included 237 HCC surgical samples and 284 HCC liver biopsies from patients treated by surgical resection and RFA, respectively. Histological slides were reviewed by pathologists specialized in liver disease, and the MTM-HCC subtype was defined by the presence of a predominant (>50%) macrotrabecular architecture (more than six cells thick). The main clinical and biological features were recorded at baseline. Clinical endpoints were early and overall recurrence. The MTM-HCC subtype was identified in 12% of the whole cohort (16% of surgically resected samples, 8.5% of liver biopsy samples). It was associated at baseline with known poor prognostic factors (tumor size, alpha-fetoprotein level, satellite nodules, and vascular invasion). Multivariate analysis showed that MTM-HCC subtype was an independent predictor of early and overall recurrence (surgical series: hazard ratio, 3.03; 95% confidence interval, 1.38-6.65; P = 0.006; and 2.76; 1.63-4.67; P < 0.001; RFA series: 2.37; 1.36-4.13; P = 0.002; and 2.19; 1.35-3.54; P = 0.001, respectively). Its prognostic value was retained even after patient stratification according to common clinical, biological, and pathological features of aggressiveness. No other baseline parameter was independently associated with recurrence in the RFA series. CONCLUSION: The MTM-HCC subtype, reliably observed in 12% of patients eligible for curative treatment, represents an aggressive form of HCC that may require more specific therapeutic strategies. (Hepatology 2018;68:103-112).

Short- and Long-Term Outcomes of Liver Resection for Intrahepatic Cholangiocarcinoma Associated with the Metabolic Syndrome.

BACKGROUND: While the metabolic syndrome (MS) is being recognized as an important risk factor for intrahepatic cholangiocarcinoma (ICC), the outcomes of liver resection in this context remain poorly described. This study aims to report the short- and long-term results of hepatectomy for patients with MS as risk factor for the development of ICC (MS+). METHODS: All patients undergoing hepatectomy for ICC between 2000 and 2016 at a single center were retrospectively analyzed. The perioperative outcomes of MS+ and ICC patients without MS (MS-) were compared. RESULTS: Among 115 resected ICC patients, 40 (34.8%) were MS+ and 75 (65.2%) were MS-. MS+ exhibited an increased Charlson comorbidity index (5 ± 2 vs. 2 ± 2, p < 0.001) than MS- patients. While operative characteristics did not differ significantly between the 2 groups, MS+ experienced higher rate of major complications (62.5 vs. 29.3%, p = 0.001). On multivariate analysis, MS+ was an independent risk factor of major complication (HR 2.86, 95% CI 1.07-7.60, p = 0.036) and major cardiorespiratory complication (HR 4.35, 95% CI 1.50-12.62, p = 0.007). Pathological analysis revealed that MS+ displayed higher rates of non-alcoholic fatty liver disease (60.0 vs. 31.1%, p = 0.003) and non-alcoholic steatohepatitis (25 vs. 5.4%, p = 0.005). MS+ was independently associated with decreased risk of recurrence (HR 0.47, 95% CI 0.26-0.85, p = 0.001). CONCLUSIONS: MS+ accounts for 35% of resected ICC patients. The existence of significant cardiovascular comorbidities increases postoperative morbidity and requires specific management.

[Large scale teaching in pathology]. / Pédagogie à grande échelle en ACP.

Medical education is currently facing great changes that affect all medical specialties, including anatomical pathology. Due to rapidly increasing medical knowledge and diagnostic complexity, we are living an era of teaching resources mutualization. We present different tools that allow large numbers of students to access courses, self-evaluations, and competencies assessments. MOOC platforms and e-learning platforms are central to these new online tools, which include the French National Platform of Medical Specialties, dedicated to the teaching of 50,000 medical residents in France. We also discuss "serious games" and the use of images and virtual slides in anatomical pathology teaching. These new modalities can deliver essential knowledge to large student populations, but they must be used in conjunction with adapted teacher-led courses focusing on competencies and professional skills in order to be fully effective.

Contribution of virtual biopsy to the screening of microvascular invasion in hepatocellular carcinoma: A pilot study.

BACKGROUND & AIMS: Microvascular invasion (mVI) is a major prognostic factor in hepatocellular carcinoma (HCC) that cannot be detected before surgery. Predictive biomarkers of mVI are thus urgently needed. We have developed an original approach of virtual biopsy to assess the performance of an immunohistochemical panel comprising three biomarkers of mVI (H4K16ac, H4K20me2, PIVKA-II) for the prediction of mVI in HCC core needle biopsies (CNB). METHODS: A test set of HCC surgical specimens (n = 64) and an independent validation set of HCC CNB (n = 42) were retrospectively constituted. Immunostainings were first quantified in the test set on the whole tissue section, to determine optimal cut-off values for each marker. From the digitised image of the whole section, three virtual biopsies were provided. Immunostainings and accuracy of the panel for the prediction of mVI were further assessed in virtual biopsies and in the validation set of CNB. RESULTS: In virtual biopsies, PIVKA-II/H4K16ac had the best performance for prediction of mVI, with sensitivity, specificity, predictive positive value (PPV), and predictive negative value (PNV) of 30%, 97%, 91%, 56%, respectively. In CNB, PIVKA-II/H4K20me2 showed the best accuracy for prediction of mVI, with sensitivity, specificity, PPV, and NPV of 43%, 95%, 90%, and 62%, respectively. The two panels were independent predictive factors of mVI (PIVKA-II/H4K16ac, P = .037; PIVKA-II/H4K20me2, P = .026). CONCLUSION: This study shows that a panel of two markers is able to predict mVI in HCC CNB, and pave the way for the future development of prognostic biomarkers in HCC that could guide the therapeutic strategy.

Ultra-short bowel is an independent risk factor for liver fibrosis in adults with home parenteral nutrition.

BACKGROUND & AIMS: Intestinal failure-associated liver disease is rare in adults and risk factors are unclear. The aim of this study was to determine risk factors of liver fibrosis in adults receiving home parenteral nutrition for intestinal failure and its impact on survival. METHODS: We retrospectively analysed patients with irreversible intestinal failure who underwent a liver biopsy between 2000 and 2013. Significant liver fibrosis was defined as ≥F2 according to NASH-CRN score. RESULTS: Thirty-two patients (46 years [29-60]) underwent liver biopsy 55 months (9-201) after beginning parenteral nutrition. Twenty-six patients (81%) had a short bowel (gut < 200 cm), including 12 (37%) with an ultra-short bowel (gut < 20 cm). Eighteen patients (56%) had liver fibrosis (4 F2, 10 F3, 4 F4), associated with steatohepatitis (72%) and/or cholestasis (17%). Factors associated with occurrence of liver fibrosis included ultra-short bowel (83% vs 13% at 60 months; P < .001), alcohol consumption (73% vs 33% at 60 months; P < .001) and diabetes (80% vs 34% at 60 months; P = .01). Home parenteral nutrition composition, quantity, or duration, episodes of sepsis, abandoned bowel segment were not associated with fibrosis. Ultra-short bowel [risk ratio 12.4, P < .001] and alcohol consumption [risk ratio 7.4, P = .009] independently predicted the development of liver fibrosis on multivariate analysis. After a median follow-up of 118 months (72-155), survival was poorer in patients who developed liver fibrosis than in those without (59% vs 92% at 120 months; P = .02). CONCLUSION: An ultra-short bowel and alcohol consumption are independent risk factors for liver fibrosis in adults requiring HPN.

18F-FDG Uptake in Well-Differentiated Neuroendocrine Tumors Correlates with Both Ki-67 and VHL Pathway Inactivation.

BACKGROUND: 18F-FDG-PET scan positivity correlates with poor prognosis in neuroendocrine neoplasms (NEN). Glucose transporter 1 (GLUT1) and carbonic anhydrase 9 (CA9) are markers of aggressiveness in tumors. Together with von Hippel-Lindau protein (pVHL), they are involved in tumor cell metabolism via the hypoxia-inducible factor signaling pathway. The aim of this study was to compare, in a series of well-differentiated neuroendocrine tumors (NET), the 18F-FDG uptake and expression of the proliferation markers Ki-67, GLUT1, CA9, and pVHL. PATIENTS AND METHODS: This retrospective study included 27 patients with well-differentiated NET. 18F-FDG-PET images were evaluated by the maximum standardized uptake value (SUVmax). GLUT1, CA9, and pVHL were analyzed by immunohistochemistry. RESULTS: The NET were of pancreatic (n = 19), midgut (n = 4), duodenal (n = 1), esophageal (n = 1), rectal (n = 1), and pulmonary (n = 1) origin. Eight, 11, and 8 tumors were grade 1, 2, and 3, respectively. The mean/median Ki-67 index was 15/10% (1-60). The mean/median SUVmax was 6.2/5.2 (1.4-18.7). SUVmax correlated with greater tumor size (p = 0.03), higher expression of Ki-67 (p = 0.04), and lower expression of pVHL (p = 0.008). In the group of 16 NET with a low proliferative index (Ki-67 index <10%), 5/6 (83%) of the tumors with a high SUVmax had decreased pVHL expression (p = 0.0013). CONCLUSION: This study confirms that 18F-FDG-PET uptake correlates with both tumor size and proliferation in well-differentiated NET, and it highlights a subset of low-grade but 18F-FDG-PET-positive NET related to sporadic inactivation of the VHL pathway.

Clinical and Biomarker Evaluations of Sunitinib in Patients with Grade 3 Digestive Neuroendocrine Neoplasms.

BACKGROUND/AIMS: Angiogenesis is extensively developed in well-differentiated pancreatic neuroendocrine tumours (PanNET) where sunitinib was shown to prolong progression-free survival, leading to nationwide approval. However, clinical experience in patients with grade 3 gastroenteropancreatic neuroendocrine neoplasms (GEPNEN-G3) remains limited. This prospective phase II trial evaluated potential predictive biomarkers of sunitinib activity in patients with advanced GEPNEN-G3. METHODS: Sunitinib was given at a dose of 37.5 mg/day as a continuous daily dosing until progression or unacceptable toxicity. Evaluation of activity was based on RECIST1.1. Safety was evaluated according to NCI-CTCAE v4. Pharmacokinetics of sunitinib and its main active metabolite SU12662 were evaluated. All tumour samples were reviewed histologically for tumour differentiation. PDGFRß, carbonic anhydrase 9, Ki-67, VEGFR2, and p-AKT were quantified using immunohistochemistry and their expression correlated with response by RECIST1.1. RESULTS: Thirty-one patients were included and 26 had available histological tissue. Six and 20 patients presented well-differentiated tumours (NET-G3) and neuroendocrine carcinoma (NEC), respectively. Eighteen patients responded to sunitinib (4 experienced partial responses and 14 tumour stabilization). A high p-AKT expression correlated with lower response to sunitinib (OR 0.94, 95% CI 0.89-0.99, p = 0.04). Safety and PK exposure to sunitinib and SU12662 in these patients were consistent with that reported in PanNET. CONCLUSION: Sunitinib showed evidence of activity in patients with GEPNEN-G3 with expected toxicity profile. In the NET-G3 and NEC groups, 4/6 and 11/20 patients were responders, respectively. High p-AKT expression predicted a lower response to sunitinib. Our study allowed the identification of a potential biomarker of resistance/sensitivity to sunitinib in aggressive GEPNEN-G3.