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The adoption of comprehensive genomic profiling in oncology has rapidly increased the demand for standardized tumor sample processing in diagnostic laboratories. Automation of DNA and RNA library preparation workflows offers the possibility to scale-up and standardize sample processing. We report on the clinical implementation of the automated TruSight Oncology 500 High-Throughput library preparation workflow from formalin-fixed, paraffin-embedded tumor samples using the Biomek i7 hybrid Workstation. Using the same input amount, the automated workflow was validated against manual library preparation. Quality control metrics (total and mapped reads, median insert size, and median exon coverage) and the detection of tumor mutational burden, a complex biomarker, were concordant between the manual and automated workflows. The automated workflow was implemented on a total of 2997 pan-cancer clinical samples to detect genomic variants and complex biomarkers. Workflow automation resulted in a 4-fold reduction in hands-on time and a 1.7-fold reduction in total runtime compared with manual library preparation (6 hours vs. 23 hours; 24 hours vs. 42.5 hours, respectively) for a 48 DNA + 48 RNA sample batch. The automated workflow required one technician versus three technicians to manually prepare the same number of libraries. This study shows that implementation of the automated TruSight Oncology 500 High-Throughput workflow significantly reduced hands-on time and processing time per sample compared with manual library preparation., Competing Interests: Disclosure Statement M.K. has received personal fees for speaker honoraria and support for attending meetings or travel from Veracyte Inc., outside the submitted work. O.N. reports personal fees from Novartis outside the submitted work. A.-L.V. reports personal fees from AstraZeneca outside the submitted work. M.A. received speaker honoraria from Boehringer Ingelheim. P.S. reports personal fees for speaker honoraria BMS; grants from BMS, AstraZeneca, and MSD; and personal fees for advisory board participation from BMS, AstraZeneca, and MSD, outside the submitted work. J.B. reports grants from German Cancer Aid and consulting from MSD, outside the submitted work. A.S. has received personal fees for advisory board participation from Agilent, Aignostics, Amgen, AstraZeneca, Bayer, BMS, Eli Lilly, Illumina, Incyte, Janssen, MSD, Novartis, Pfizer, Qlucore, Roche, Seagen, Takeda, and Thermo Fisher; and grants from Bayer, BMS, Chugai, and Incyte, outside the submitted work. D.K. reports personal fees for speaker honoraria from AstraZeneca and Pfizer and personal fees for advisory board participation from Bristol Myers Squibb, outside the submitted work., (Copyright © 2025 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background and Aims: RORc-expressing immune cells play important roles in inflammation, autoimmune disease and cancer. They are required for lymphoid organogenesis and have been implicated in tertiary lymphoid structure (TLS) formation. TLSs are formed in many cancer types and have been correlated with better prognosis and response to immunotherapy. In liver cancer, some TLSs are pro-tumorigenic as they harbor tumor progenitor cells and support their growth. The processes involved in TLS development and acquisition of pro- or anti-tumorigenic roles are largely unknown. This study aims to explore the role of RORc-expressing cells in TLS development in the context of inflammation-associated liver cancer., Methods: IKKβ(EE) Hep mice, exhibiting chronic liver inflammation, TLS formation and liver cancer, were crossed with RORc knockout mice to explore RORc's effect on TLS and tumor formation. TLS phenotypes were analyzed using transcriptional, proteomic, and immunohistochemical techniques. CD4, CD8, and B cell depletions were used to assess their contribution to liver TLS and tumor formation., Results: RORc-expressing cells are detected within TLSs of both human patients and mice developing intrahepatic cholangiocarcinoma. In mice, these cells negatively regulate TLS formation, as excess TLSs form in their absence. CD4 cells are essential for liver TLS formation, while B cells are required for TLS formation specifically in the absence of RORc-expressing cells. Importantly, in chronically inflamed livers lacking RORc-expressing cells, TLSs become anti-tumorigenic, reducing tumor load. Anti-tumorigenic TLSs revealed enrichment of exhausted CD8 cells with effector functions, germinal center B cells and plasma cells. B cells are key in limiting tumor development, possibly via tumor-directed antibodies., Conclusions: RORc-expressing cells negatively regulate B cell responses and facilitate the pro-tumorigenic functions of hepatic TLSs., Competing Interests: Declaration of Competing Interest: Authors declare no conflict interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Targeted tumor only sequencing has become a standard practice in cancer diagnostics. This study aims to develop an approach for robust copy number variant calling in tumor samples using only off-target region (OTR) reads. We also established a clinical use case for homologous recombination deficiency (HRD) score estimation (HRDest) using the sum of telomeric-allelic imbalance and large-scale state transition scores without the need for loss of heterozygosity information. A strong correlation was found between HRD score and the sum of telomeric-allelic imbalance + large-scale state transition in The Cancer Genome Atlas cohort (ρ = 0.99, P < 2.2 × 10 -16 ) and in a clinical in-house cohort of 34 tumors (ρ = 0.9, P = 5.1 × 10 -13 ) comparing whole-exome sequencing and targeted sequencing data. HRDest scores from 1086 clinical cases were compared with The Cancer Genome Atlas data set. There were no significant differences in HRD score distribution within the analyzed tumor types. As a control, commercially available HRD standards were also sequenced, and the HRDest scores obtained from the OTR reads were well within the HRD reference range provided by the manufacturer. In conclusion, OTR reads of tumor-only panel sequencing can be used to determine genome-wide copy number variant profiles and to approximate HRD scores., Competing Interests: Disclosure Statement O.N. reports personal fees from Novartis outside the submitted work. A.-L.V. reports personal fees from Astra Zeneca outside the submitted work. J.B. reports a grant from the German Cancer Aid outside the submitted work. S.F. reports consulting or advisory board membership for Bayer, Illumina, and Roche; honoraria from Amgen, Eli Lilly, PharmaMar, and Roche; research funding from AstraZeneca, Pfizer, PharmaMar, and Roche; and travel or accommodation expenses from Amgen, Eli Lilly, Illumina, PharmaMar, and Roche. P.S. reports personal fees from Bristol Myers Squibb, Merck Sharp & Dohme (MSD), Incyte, Janssen, Amgen, Novartis, Roche, and AstraZeneca outside the submitted work. A.S. reports grants and personal fees from Bayer and Bristol Myers Squibb; grants from Chugai; and personal fees from AstraZeneca, MSD, Takeda, Seattle Genetics, Novartis, Illumina, Thermo Fisher, Eli Lilly, and Takeda outside the submitted work. D.K. reports personal fees from AstraZeneca, Bristol Myers Squibb, Pfizer, Lilly, Agilent, and Takeda outside the submitted work. The remaining authors declare no conflict of interest., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Germline variant evaluation in precision oncology opens new paths toward the identification of patients with genetic tumor risk syndromes and the exploration of therapeutic relevance. Here, we present the results of germline variant analysis and their clinical implications in a precision oncology study for patients with predominantly rare cancers., Patients and Methods: Matched tumor and control genome/exome and RNA sequencing was carried out for 1485 patients with rare cancers (79%) and/or young adults (77% younger than 51 years) in the National Center for Tumor Diseases/German Cancer Consortium (NCT/DKTK) Molecularly Aided Stratification for Tumor Eradication Research (MASTER) trial, a German multicenter, prospective, observational precision oncology study. Clinical and therapeutic relevance of prospective pathogenic germline variant (PGV) evaluation was analyzed and compared to other precision oncology studies., Results: Ten percent of patients (n = 157) harbored PGVs in 35 genes associated with autosomal dominant cancer predisposition, whereof up to 75% were unknown before study participation. Another 5% of patients (n = 75) were heterozygous carriers for recessive genetic tumor risk syndromes. Particularly, high PGV yields were found in patients with gastrointestinal stromal tumors (GISTs) (28%, n = 11/40), and more specifically in wild-type GISTs (50%, n = 10/20), leiomyosarcomas (21%, n = 19/89), and hepatopancreaticobiliary cancers (16%, n = 16/97). Forty-five percent of PGVs (n = 100/221) supported treatment recommendations, and its implementation led to a clinical benefit in 40% of patients (n = 10/25). A comparison of different precision oncology studies revealed variable PGV yields and considerable differences in germline variant analysis workflows. We therefore propose a detailed workflow for germline variant evaluation., Conclusions: Genetic germline testing in patients with rare cancers can identify the very first patient in a hereditary cancer family and can lead to clinical benefit in a broad range of entities. Its routine implementation in precision oncology accompanied by the harmonization of germline variant evaluation workflows will increase clinical benefit and boost research., Competing Interests: Disclosure AJ: Honoraria: AstraZeneca. CH: Honoraria: Roche, Novartis; research funding: Boehringer Ingelheim; consulting or advisory board: Boehringer Ingelheim. CVG: Employed by Illumina since August 2021. LM reports non-financial support from Celgene outside the submitted work (travel expenses). MAu: Consulting or advisory board membership: Bristol-Myers Squibb, Ipsen, Merck KGaA, MSD Sharp & Dohme, Novartis, Pfizer, Roche, PharmaMar; research funding: AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Exelixis, Ipsen, Merck KGaA, Novartis, Pfizer, PharmaMar, Roche; honoraria: Blueprint Medicines, Bristol-Myers Squibb, Ipsen, Janssen-Cilag, Merck KGaA, Pfizer, PharmaMar. PHoh: Advisory board membership: Roche, Deciphera, PharmaMar, Pfizer, BluMedicine, GSK; honoraria: Bristol-Meyers-Squibb, Astrazeneca; research funding: Novartis, Siemens. MK: Honoraria: Bayer, MSD, Eisai, Ipsen, Eli Lilly; research funding: Eli Lilly, Ipsen, Loxo Oncology. RFS: Consulting or advisory board membership: Astellas, Bristol-Myers Squibb, Celgene, Pfizer; research funding: AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Pfizer, PharmaMar, Roche; honoraria: Daiichi Sankyo, Pfizer. GF: Honoraria: Amgen, Armo Biosciences, Bayer, Bristol-Myers Squibb, Eli Lilly, HRA Pharma, Merck KGaA, MSD Sharp & Dohme, Pierre Fabre, Roche, Sanofi, Servier, Shire; research funding: Merck KGaA. SB: Consulting or advisory board membership: ADC Therapeutics, Blueprint Medicines, Daichii-Sankyo, Deciphera, Eli Lilly, Exelixis, Janssen-Cilag, Mundipharma, Novartis, Plexxikon; honoraria: Bayer, Eli Lilly, GlaxoSmithKline, Novartis, Pfizer, PharmaMar; research funding: Blueprint Medicines, Incyte, Novartis. JTS: Consulting or advisory board membership: AstraZeneca, Bayer, Bristol Myers Squibb, Celgene, Immunocore, Novartis, Roche, Shire; honoraria: AstraZeneca, Aurikamed, Baxalta, Bristol Myers Squibb, Celgene, Falk Foundation, iomedico, Immunocore, Novartis, Roche, Shire; research funding: Bristol-Myers Squibb, Celgene, Roche; minor equity in iTheranostics and Pharma15; member of the Board of Directors for Pharma15, all outside the submitted work. ALI: Honoraria: Bristol-Myers Squibb, Janssen-Cilag, Takeda, Roche. NvB: Consulting or advisory board membership: Novartis; honoraria: Novartis, Takeda. PJJ: Consulting or advisory board membership, honoraria, research funding, travel or accommodation expenses: Abbvie, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Novartis, Pfizer, Roche, Servier. KHM: Consulting: Celgene/BMS, Novartis, Jazz Pharmaceuticals, Pfizer; honoraria: Celgene/BMS, Daiichi Sankyo, Astellas, AbbVie, Novartis; research funding: Celgene. AS: Consulting or advisory board membership, honoraria: AGCT, AstraZeneca, Bayer, Bristol-Myers Squibb, Chugai, Eli Lilly, Illumina, Janssen, MSD Sharp & Dohme, Novartis, Pfizer, Roche, Seattle Genetics, Takeda, Thermo Fisher; research funding: Bayer, Bristol-Myers Squibb, Chugai. WW: Consulting or advisory board membership, honoraria: Agilent, Amgen, Astellas, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, Illumina, Merck, MSD Sharp & Dohme, Pfizer, NewOncology, Novartis, Roche, Takeda; research funding: AstraZeneca, Bristol-Myers Squibb, MSD Sharp & Dohme, Roche. SF: Consulting or advisory board membership: Bayer, Illumina, Roche; honoraria: Amgen, Eli Lilly, PharmaMar, Roche; research funding: AstraZeneca, Pfizer, PharmaMar, Roche; travel or accommodation expenses: Amgen, Eli Lilly, Illumina, PharmaMar, Roche. ES: Honoraria: AstraZeneca, Illumina. All other authors have declared no conflicts of interest. Data sharing All evaluated germline variants can be found in Supplementary Table S5, available at https://doi.org/10.1016/j.annonc.2022.07.008. Sequencing data have been deposited in the European Genome-phenome Archive (https://www.ebi.ac.uk/ega/datasets) under accession EGAS00001005537., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Folate-mediated 1-carbon metabolism requires several nutrients, including vitamin B6. Circulating biomarker concentrations indicating high vitamin B6 status are associated with a reduced risk of colorectal cancer (CRC). However, little is known about the effect of B6 status in relation to clinical outcomes in CRC patients., Objectives: We investigated survival outcomes in relation to vitamin B6 status in prospectively followed CRC patients., Methods: A total of 2031 patients with stage I-III CRC participated in 6 prospective patient cohorts in the international FOCUS (folate-dependent 1-carbon metabolism in colorectal cancer recurrence and survival) Consortium. Preoperative blood samples were used to measure vitamin B6 status by the direct marker pyridoxal 5'-phosphate (PLP), as well as the functional marker HK-ratio (HKr)[3'-hydroxykynurenine: (kynurenic acid + xanthurenic acid + 3'-hydroxy anthranilic acid + anthranilic acid)]. Using Cox proportional hazards regression, we examined associations of vitamin B6 status with overall survival (OS), disease-free survival (DFS), and risk of recurrence, adjusted for patient age, sex, circulating creatinine concentrations, tumor site, stage, and cohort., Results: After a median follow-up of 3.2 y for OS, higher preoperative vitamin B6 status as assessed by PLP and the functional marker HKr was associated with 16-32% higher all-cause and disease-free survival, although there was no significant association with disease recurrence (doubling in PLP concentration: HROS, 0.68; 95% CI: 0.59, 0.79; HRDFS, 0.84; 95% CI: 0.75, 0.94; HRRecurrence, 0.96; 95% CI: 0.84, 1.09; HKr: HROS, 2.04; 95% CI: 1.67, 2.49; HRDFS, 1.56; 95% CI: 1.31, 1.85; HRRecurrence, 1.21; 95% CI: 0.96,1. 52). The association of PLP with improved OS was consistent across colorectal tumor site (right-sided colon: HROS, 0.75; 95% CI: 0.59, 0.96; left-sided colon: HROS, 0.71; 95% CI: 0.55, 0.92; rectosigmoid junction and rectum: HROS, 0.61; 95% CI: 0.47, 0.78)., Conclusion: Higher preoperative vitamin B6 status is associated with improved OS among stage I-III CRC patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Background: Mutations in DNA damage repair genes, in particular genes involved in homology-directed repair, define a subgroup of men with prostate cancer with a more unfavorable prognosis but a therapeutic vulnerability to PARP inhibition. In current practice, mutational testing of prostate cancer patients is commonly done late i.e., when the tumor is castration resistant. In addition, most sequencing panels do not include TP53, one of the most crucial tumor suppressor genes in human cancer. In this proof-of-concept study, we sought to extend the clinical use of these molecular markers by exploring the early prognostic impact of mutations in TP53 and DNA damage repair genes in men with primary, nonmetastatic prostate cancer undergoing radical prostatectomy (RPX)., Methods: Tumor specimens from a cohort of 68 RPX patients with intermediate (n = 11, 16.2%) or high-risk (n = 57, 83.8%) disease were analyzed by targeted next generation sequencing using a 37 DNA damage repair and checkpoint gene panel including TP53. Sequencing results were correlated to clinicopathologic variables as well as PSA persistence or time to PSA failure. In addition, the distribution of TP53 and DNA damage repair gene mutations was analyzed in three large publicly available datasets (TCGA, MSKCC and SU2C)., Results: Of 68 primary prostate cancers analyzed, 23 (33.8%) were found to harbor a mutation in either TP53 (n = 12, 17.6%) or a DNA damage repair gene (n = 11, 16.2%). The vast majority of these mutations (22 of 23, 95.7%) were detected in primary tumors from patients with high-risk features. These mutations were mutually exclusive in our cohort and additional data mining suggests an enrichment of DNA damage repair gene mutations in TP53 wild-type tumors. Mutations in either TP53 or a DNA damage repair gene were associated with a significantly worse prognosis after RPX. Importantly, the presence of TP53/DNA damage repair gene mutations was an independent risk factor for PSA failure or PSA persistence in multivariate Cox regression models., Conclusion: TP53 or DNA damage repair gene mutations are frequently detected in primary prostate cancer with high-risk features and define a subgroup of patients with an increased risk for PSA failure or persistence after RPX. The significant adverse impact of these alterations on patient prognosis may be exploited to identify men with prostate cancer who may benefit from a more intensified treatment., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. SAK reports grant support from Viewray, Inc., outside the work reported., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background & Aims: The SALVE Histopathology Group (SHG) developed and validated a grading and staging system for the clinical and full histological spectrum of alcohol-related liver disease (ALD) and evaluated its prognostic utility in a multinational cohort of 445 patients., Methods: SALVE grade was described by semiquantitative scores for steatosis, activity (hepatocellular injury and lobular neutrophils) and cholestasis. The histological diagnosis of steatohepatitis due to ALD (histological ASH, hASH) was based on the presence of hepatocellular ballooning and lobular neutrophils. Fibrosis staging was adapted from the Clinical Research Network staging system for non-alcoholic fatty liver disease and the Laennec staging system and reflects the pattern and extent of ALD fibrosis. There are 7 SALVE fibrosis stages (SFS) ranging from no fibrosis to severe cirrhosis., Results: Interobserver κ-value for each grading and staging parameter was >0.6. In the whole study cohort, long-term outcome was associated with activity grade and cholestasis, as well as cirrhosis with very broad septa (severe cirrhosis) (p <0.001 for all parameters). In decompensated ALD, adverse short-term outcome was associated with activity grade, hASH and cholestasis (p = 0.038, 0.012 and 0.001, respectively), whereas in compensated ALD, hASH and severe fibrosis/cirrhosis were associated with decompensation-free survival (p = 0.011 and 0.001, respectively). On multivariable analysis, severe cirrhosis emerged as an independent histological predictor of long-term survival in the whole study cohort. Severe cirrhosis and hASH were identified as independent predictors of short-term survival in decompensated ALD, and also as independent predictors of decompensation-free survival in compensated ALD., Conclusion: The SALVE grading and staging system is a reproducible and prognostically relevant method for the histological assessment of disease activity and fibrosis in ALD., Lay Summary: Patients with alcohol-related liver disease (ALD) may undergo liver biopsy to assess disease severity. We developed a system to classify ALD under the microscope by grading ALD activity and staging the extent of liver scarring. We validated the prognostic performance of this system in 445 patients from 4 European centers., Competing Interests: Conflicts of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Targeted therapies have improved survival and quality of life for patients with non-small-cell lung cancer with actionable driver mutations. However, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 gene (HER2, also known as ERBB2) exon 20 insertions (Ex20mut) are characterized by a poor response to currently approved tyrosine kinase inhibitors and immunotherapies. The underlying immune biology is not well understood., Materials and Methods: We carried out messenger RNA expression profiling of lung adenocarcinomas (ADCs) with ERBB2 (n = 19) and EGFR exon 20-insertion mutations (n = 13) and compared these to tumors with classical EGFR mutations (n = 40, affecting EGFR exons 18, 19 or 21) and EGFR/ERBB2 mutation-negative lung ADC (EGFR/ERBB2wt, n = 26) focusing on immunologically relevant transcripts. Tumor-infiltrating immune cells were estimated from gene expression profiles., Results: Cytotoxic cells were significantly lower in EGFR-mutated tumors regardless of the affected exon, while Th1 cells were significantly lower in EGFR-Ex20mut compared to EGFR/ERBB2wt tumors. We assessed the differentially expressed genes of ERBB2-Ex20mut and EGFR-Ex20mut tumors compared to EGFR-Ex18/19/21mut and EGFR/ERBB2wt tumors. Of these, the genes GUSB, HDAC11, IFNGR2, PUM1, RASGRF1 and RBL2 were up-regulated, while a lower expression of CBLC, GBP1, GBP2, GBP4 and MYC was observed in all three comparison groups. The omnibus test revealed 185 significantly (FDR = 5%) differentially expressed genes and we found these four most significant gene expression changes in the study cohort: VHL and JAK1 were overexpressed in ERBB2-Ex20mut and EGFR-Ex20mut tumors compared to both EGFR-Ex18/19/21mut and EGFR/ERBB2wt tumors. RIPK1 and STK11IP showed the highest expression in ERBB2-Ex20mut tumors., Conclusions: Targeted gene expression profiling is a promising tool to read out the characteristics of the tumor microenvironment from routine diagnostic lung cancer biopsies. Significant immune reactivity and specific immunosuppressive characteristics in ERBB2-Ex20mut and EGFR-Ex20mut lung ADC with at least some degree of immune infiltration support further clinical evaluation of immune-modulators as partners of immune checkpoint inhibitors in such tumors., Competing Interests: Disclosure MKi reports grants from QuIP, outside the submitted work. DK reports personal fees from AstraZeneca, Bristol-Myers Squibb GmbH and Pfizer Pharma GmbH, outside the submitted work. VE reports personal fees from AstraZeneca, Bayer, Lilly, BMS, MSD Sharp, Novartis and Thermo Fisher, outside the submitted work. TM reports grants and non-financial support from Roche Diagnostics GmbH, Penzberg, Germany, outside the submitted work; in addition, TM has a patent WO2019158460 pending, a patent WO2019211418 pending, a patent WO2019215223 pending, a patent EP3391053 issued and a patent EP3365679 pending. NF reports personal fees and travel grants from AbbVie, AstraZeneca, Boehringer Ingelheim, BMS and Takeda, outside the submitted work, and personal fees from Amgen, BerlinChemie, BeiGene, MSD, Novartis, Pfizer, Roche and Sanofi, outside the submitted work. MR reports personal fees from Amgen, AstraZeneca, BMS, Boehringer Ingelheim, Lilly, Merck, MSD, Novartis, Pfizer, Roche and Samsung, outside the submitted work. SF reports personal fees from Amgen, Bayer, Eli Lilly and Roche; grants from AstraZeneca and Pfizer; and grants and personal fees from PharmaMar, outside the submitted work. PS reports grants from QuIP, during the conduct of the study; grants and personal fees from BMS, MSD, Roche, AstraZeneca, Novartis and Pfizer; personal fees from Chugai, AbbVie and Ipsen; and grants from Sanofi-Aventis, Illumina and Thermo Fisher, outside the submitted work. MT reports personal fees from AbbVie, Lilly and Takeda; grants, personal fees and non-financial support from BMS; personal fees and non-financial support from Boehringer, MSD and Novartis; grants and personal fees from Celgene and Roche; and grants from AstraZeneca, outside the submitted work. JB reports grants from German Cancer Aid, outside the submitted work. PC reports grants and personal fees from AstraZeneca, Novartis, Roche and Takeda and personal fees from Boehringer Ingelheim, Chugai and Pfizer, outside the submitted work. AS reports personal fees from AstraZeneca, MSD, Takeda, Seattle Genetics, Novartis, Illumina, Thermo Fisher, Eli Lily and Takeda; grants and personal fees from Bayer and BMS; and grants from Chugai, outside the submitted work. All other authors have declared no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background and Aims: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies., Methods: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays., Results: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-β proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved., Conclusions: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature., Lay Summary: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC., Competing Interests: Conflict of interest J.M.L. receives research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb, Boehringer-Ingelheim and Ipsen, and consulting fees from Eli Lilly, Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Exelixis, Merck, Ipsen, Genentech, Roche, Glycotest, Leerink Swann LLC, Fortress Biotech, Nucleix, Can-Fite Biopharma, Sirtex, Mina Alpha Ltd and AstraZeneca. S.L.F consults for the following companies: 89 Bio, Amgen, Axcella Health, Blade Therapeutics, Bristol Myers Squibb, Can-Fite Biopharma, ChemomAb, Escient Pharmaceuticals, Forbion, Foresite laboratories, Galmed, Gordian Biotechnology, Glycotest, Glympse Bio, Hepgene, In sitro, Morphic Therapeutics, North Sea Therapeutics, Novartis, Ono Pharmaceuticals, Pfizer Pharmaceuticals, Scholar Rock Surrozen. He has stock options in the following companies: Blade Therapeutics, Escient, Galectin, Galmed, Genfit, Glympse, Hepgene, Lifemax, Metacrine, Morphic Therapeutics, Nimbus, North Sea Therapeutics, Scholar Rock, Surrozen. He receives research support from Morphic Therapeutics, Novo Nordisk, and Galmed, and has an SBIR grant with Abalone Bio. A.L. is a consultant for Neuwave and Histosonics. C.P.M.S.O. consults for Bayer, Novartis, Novonordisk, Allergan, Pfizer, Roche and Zambon. P.S. is receiving research grants from BMS, Roche, Incyte, Chugai and consulting fees from BMS, MSD, Incyte, Janssen, Roche, AstraZeneca and Amgen. H.W., P.T., and A.V.U. are or were salaried employees of Sema4 at the time of the study. H.W., P.T., and A.V.U. hold Sema4 stock options. B.M. received consultancy fees from Bayer-Shering Pharma, and speaker fees from Eisai and MSD. The rest of authors have nothing to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: This study evaluated the consequences in Europe of the COVID-19 outbreak on pathology laboratories orientated toward the diagnosis of thoracic diseases., Materials and Methods: A survey was sent to 71 pathology laboratories from 21 European countries. The questionnaire requested information concerning the organization of biosafety, the clinical and molecular pathology, the biobanking, the workload, the associated research into COVID-19, and the organization of education and training during the COVID-19 crisis, from 15 March to 31 May 2020, compared with the same period in 2019., Results: Questionnaires were returned from 53/71 (75%) laboratories from 18 European countries. The biosafety procedures were heterogeneous. The workload in clinical and molecular pathology decreased dramatically by 31% (range, 3%-55%) and 26% (range, 7%-62%), respectively. According to the professional category, between 28% and 41% of the staff members were not present in the laboratories but did teleworking. A total of 70% of the laboratories developed virtual meetings for the training of residents and junior pathologists. During the period of study, none of the staff members with confirmed COVID-19 became infected as a result of handling samples., Conclusions: The COVID-19 pandemic has had a strong impact on most of the European pathology laboratories included in this study. Urgent implementation of several changes to the organization of most of these laboratories, notably to better harmonize biosafety procedures, was noted at the onset of the pandemic and maintained in the event of a new wave of infection occurring in Europe., Competing Interests: Disclosure The authors have declared no conflicts of interest. Data sharing Anonymized participant-level data are available for investigators. Ethical approval The study complied with the law of January 1978 (78-17) relating to computing, data, and freedom known as ‘computing and freedoms’ and with rules 2016/679 of the European Parliament (of 25 May 2018) concerning the general data protection regulation (GDPR; MR004). The study was registered by the Nice University Hospital (R04-39) and filed on the Health Data Hub website (7 July 2020) (www.health-data-hub.fr/depot)., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Defects in DNA damage repair genes characterize a subset of men with prostate cancer and provide an attractive opportunity for precision oncology approaches. The prevalence of such perturbations in newly diagnosed, treatment-naïve patients with a high risk for lethal disease outcome, however, has not been sufficiently explored., Patients and Methods: Prostate cancer specimens from 67 men with newly diagnosed early onset, localized high-risk/locally advanced or metastatic prostate cancer were included in this prospective pilot study. Tumor samples, including 30 prostate biopsies, were analyzed by targeted next generation sequencing using a formalin-fixed, paraffin-embedded tissue-optimized 37 DNA damage repair and checkpoint gene panel., Results: The drop-out rate due to an insufficient quantity of DNA was 4.5% (3 of 67 patients). In the remaining 64 patients, the rate of pathogenic DNA damage repair gene mutations was 26.6%. The highest rate of pathogenic DNA damage repair and checkpoint gene mutations was found in men with treatment-naïve metastatic prostate cancer (38.9%). In addition, a high number of likely pathogenic mutations and gene deletions were detected. Altogether, one or more pathogenic mutation, likely pathogenic mutation or gene deletion affected 43 of 64 patients (67.2%) including 29 of 36 patients (80.6%) with treatment-naïve metastatic prostate cancer. Men with metastatic prostate cancer showed a high prevalence of alterations in TP53 (36.1%)., Conclusions: This pilot study demonstrates the feasibility, performance and clinical relevance of somatic targeted next generation sequencing using a unique 37 DNA damage repair and checkpoint gene panel under routine conditions. Our results indicate that this approach can detect actionable DNA repair gene alterations, uncommon mutations as well as mutations associated with therapy resistance in a high number of patients, in particular patients with treatment-naïve metastatic prostate cancer., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Tumor mutational burden (TMB) is a quantitative assessment of the number of somatic mutations within a tumor genome. Immunotherapy benefit has been associated with TMB assessed by whole-exome sequencing (wesTMB) and gene panel sequencing (psTMB). The initiatives of Quality in Pathology (QuIP) and Friends of Cancer Research have jointly addressed the need for harmonization among TMB testing options in tissues. This QuIP study identifies critical sources of variation in psTMB assessment., Methods: A total of 20 samples from three tumor types (lung adenocarcinoma, head and neck squamous cell carcinoma, and colon adenocarcinoma) with available WES data were analyzed for psTMB using six panels across 15 testing centers. Interlaboratory and interplatform variation, including agreement on variant calling and TMB classification, were investigated. Bridging factors to transform psTMB to wesTMB values were empirically derived. The impact of germline filtering was evaluated., Results: Sixteen samples had low interlaboratory and interpanel psTMB variation, with 87.7% of pairwise comparisons revealing a Spearman's ρ greater than 0.6. A wesTMB cut point of 199 missense mutations projected to psTMB cut points between 7.8 and 12.6 mutations per megabase pair; the corresponding psTMB and wesTMB classifications agreed in 74.9% of cases. For three-tier classification with cut points of 100 and 300 mutations, agreement was observed in 76.7%, weak misclassification in 21.8%, and strong misclassification in 1.5% of cases. Confounders of psTMB estimation included fixation artifacts, DNA input, sequencing depth, genome coverage, and variant allele frequency cut points., Conclusions: This study provides real-world evidence that all evaluated panels can be used to estimate TMB in a routine diagnostic setting and identifies important parameters for reliable tissue TMB assessment that require careful control. As complex or composite biomarkers beyond TMB are likely playing an increasing role in therapy prediction, the efforts by QuIP and Friends of Cancer Research also delineate a general framework and blueprint for the evaluation of such assays., (Copyright © 2020 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Background: Tumor mutational burden (TMB) is an emerging biomarker used to identify patients who are more likely to benefit from immuno-oncology therapy. Aside from various unsettled technical aspects, biological variables such as tumor cell content and intratumor heterogeneity may play an important role in determining TMB., Methods: TMB estimates were determined applying the TruSight Oncology 500 targeted sequencing panel. Spatial and temporal heterogeneity was analyzed by multiregion sequencing (two to six samples) of 24 pulmonary adenocarcinomas and by sequencing a set of matched primary tumors, locoregional lymph node metastases, and distant metastases in five patients., Results: On average, a coding region of 1.28 Mbp was covered with a mean read depth of 609x. Manual validation of the mutation-calls confirmed a good performance, but revealed noticeable misclassification during germline filtering. Different regions within a tumor showed considerable spatial TMB variance in 30% (7 of 24) of the cases (maximum difference, 14.13 mut/Mbp). Lymph node-derived TMB was significantly lower (p = 0.016). In 13 cases, distinct mutational profiles were exclusive to different regions of a tumor, leading to higher values for simulated aggregated TMB. Combined, intratumor heterogeneity and the aggregated TMB could result in divergent TMB designation in 17% of the analyzed patients. TMB variation between primary tumor and distant metastases existed but was not profound., Conclusions: Our data show that, in addition to technical aspects such as germline filtering, the tumor content and spatially divergent mutational profiles within a tumor are relevant factors influencing TMB estimation, revealing limitations of single-sample-based TMB estimations in a clinical context., (Copyright © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Background: Panel sequencing based estimates of tumor mutational burden (psTMB) are increasingly replacing whole exome sequencing (WES) tumor mutational burden as predictive biomarker of immune checkpoint blockade (ICB)., Design: A mathematical law describing psTMB variability was derived using a random mutation model and complemented by the contributions of non-randomly mutated real-world cancer genomes and intratumoral heterogeneity through simulations in publicly available datasets., Results: The coefficient of variation (CV) of psTMB decreased inversely proportional with the square root of the panel size and the square root of the TMB level. In silico simulations of all major commercially available panels in the TCGA pan-cancer cohort confirmed the validity of this mathematical law and demonstrated that the CV was 35% for TMB = 10 muts/Mbp for the largest panels of size 1.1-1.4 Mbp. Accordingly, misclassification rates (gold standard: WES) to separate 'TMBhigh' from 'TMBlow' using a cut-point of 199 mutations were 10%-12% in TCGA-LUAD and 17%-19% in TCGA-LUSC. A novel three-tier psTMB classification scheme which accounts for the likelihood of misclassification is proposed. Simulations in two WES datasets of immunotherapy treated patients revealed that small gene panels were poor predictors of ICB response. Moreover, we noted substantial intratumoral variance of psTMB scores in the TRACERx 100 cohort and identified indel burden as independent marker complementing missense mutation burden., Conclusions: A universal mathematical law describes accuracy limitations inherent to psTMB, which result in substantial misclassification rates. This scenario can be controlled by two measures: (i) a panel design that is based on the mathematical law described in this article: halving the CV requires a fourfold increase in panel size, (ii) a novel three-tier TMB classification scheme. Moreover, inclusion of indel burden can complement TMB reports. This work has substantial implications for panel design, TMB testing, clinical trials and patient management., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Lipid droplets, a morphologic feature of adipocytic tumors, are strongly regulated by associated proteins of the perilipin/PAT (perilipin, adipophilin, and tail-interacting protein of 47 kD) family. So far, the use of perilipins as markers for differential diagnosis of soft tissue tumors has only been studied in a few cases. The aim of this study was to investigate the expression of perilipins in 478 human soft tissue tumors and 60 respective normal tissues. Perilipin 1 was immunohistochemically positive in all studied cases of well-differentiated liposarcomas, >90% of myxoid round cell liposarcomas, and >70% of pleomorphic liposarcomas, whereas only the differentiated components of dedifferentiated liposarcomas were immunohistochemically positive for perilipin 1. All other types of soft tissue sarcomas were negative for perilipin 1. Perilipin 2 was more prominent in dedifferentiated and pleomorphic liposarcomas and nearly all other high-grade sarcomas. In well-differentiated liposarcomas, lipomas, or normal adipose tissue, perilipin 2 was virtually absent. In addition, long-term stimulation of adipogenesis in the liposarcoma cell line LiSa-2 restored perilipin 1 expression, as exhibited in the source tumor. Furthermore, knockdown of perilipin 2 or perilipin 3 in LiSa-2 cells influenced lipid droplet number and size as well as cell vitality. In summary, perilipin 1 is a promising marker for the differential diagnosis of liposarcomas from other soft tissue sarcomas, whereas perilipin 2 correlates negatively with tumor grade and may be therapeutically useful., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is among the most prevalent human malignancies worldwide with rising incidence in industrialised countries, few therapeutic options and poor prognosis. To expand and improve therapeutic strategies, identification of drug targets involved in several liver cancer-related pathways is crucial. Virtually all signal transduction cascades cross the nuclear envelope and therefore require components of the nuclear transport system (NTS), including nuclear transport receptors (e.g. importins and exportins) and the nuclear pore complex. Accordingly, members of the NTS represent promising targets for therapeutic intervention. Selective inhibitors of nuclear export have already entered clinical trials for various malignancies. Herein, we review the current knowledge regarding alterations of the NTS and their potential for targeted therapy in HCC., (Copyright © 2017. Published by Elsevier B.V.)

Background & Aims: Modulation of microRNA expression is a potential treatment for hepatocellular carcinoma (HCC). Therefore, the epigenetically regulated microRNA-449 family (miR-449a, miR-449b, miR-449c) was characterized with regards to its functional effects and target genes in HCC., Methods: After transfection of miR-449a, miR-449b, and/or miR-449c, tumor-relevant functional effects were analyzed using in vitro assays and a xenograft mouse model. Binding specificities, target genes, and regulated pathways of each miRNA were identified by microarray analyses. Target genes were validated by luciferase reporter assays and expression analyses in vitro. Furthermore, target gene expression was analyzed in 61 primary human HCCs compared to normal liver tissue., Results: Tumor suppressive effects, binding specificities, target genes, and regulated pathways of miR-449a and miR-449b differed from those of miR-449c. Transfection of miR-449a, miR-449b, and/or miR-449c inhibited cell proliferation and migration, induced apoptosis, and reduced tumor growth to different extents. Importantly, miR-449a, miR-449b, and, to a lesser degree, miR-449c directly targeted SOX4, which codes for a transcription factor involved in epithelial-mesenchymal transition and HCC metastasis, and thereby inhibited TGF-β-mediated cell migration., Conclusions: This study provides detailed insights into the regulatory network of the epigenetically regulated miRNA-449 family and, for the first time, describes distinct tumor suppressive effects and target specificities of miR-449a, miR-449b, and miR-449c. Our results indicate that particularly miR-449a and miR-449b may be considered for miRNA replacement therapy to prevent HCC progression and metastasis., Lay Summary: In this study, we demonstrated that the microRNA-449 family acts as a tumor suppressor in liver cancer by causing cell death and inhibiting cell migration. These effects are caused by downregulation of the oncogene SOX4, which is frequently overexpressed in liver cancer. We conclude that the microRNA-449 family may be a target for liver cancer therapy., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Disruption of the tumor-suppressive p53 network is a key event in human malignancies, including primary liver cancer. In response to different types of stress, p53 mediates several antiproliferative cellular outcomes, such as cell cycle arrest, apoptosis, and senescence, by activation or repression of its target genes. Metabolic alterations initiating or being part of the p53 response have become an actively studied research area in the p53 field, with several aspects that still remain to be elucidated. Herein, we identified GMP synthetase (GMPS), a key enzyme of de novo purine biosynthesis, as an important p53 repression target using a large-scale proteomics approach. This p53-mediated repression of GMPS could be validated by immunoblotting in Sk-Hep1, HepG2, and HuH6 cells. Moreover, we found GMPS transcriptionally repressed in a p21-dependent manner and its repression maintained in the context of p53-mediated cellular senescence. More important, direct knockdown of GMPS by RNA interference resulted in reduced cell viability and was sufficient to trigger cellular senescence. Finally, by comparing murine hepatocellular carcinomas, which developed in p53 wild-type ( +/+ ) versus p53 null ( -/- ) mice, we observed higher GMPS expression in the latter, supporting the in vivo relevance of our findings. We conclude that repression of GMPS by p53 through p21 is a functionally relevant part of the p53-mediated senescence program limiting tumor cell growth in liver cancer., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Sinonasal carcinomas (SNCs) comprise various rare tumor types that are characterized by marked histologic diversity and largely unknown molecular profiles, yet share an overall poor prognosis owing to an aggressive clinical course and frequent late-stage diagnosis. The lack of effective systemic therapies for locally advanced or metastatic SNC poses a major challenge to therapeutic decision making for individual patients. We here aimed to identify actionable genetic alterations in a patient with metastatic SNC whose tumor, despite all diagnostic efforts, could not be assigned to any known SNC category and was refractory to multimodal therapy., Patients and Methods: We used whole-exome and transcriptome sequencing to identify a KIT exon 11 mutation (c.1733&#95;1735del, p.D579del) as potentially druggable target in this patient and carried out cancer hotspot panel sequencing to detect secondary resistance-conferring mutations in KIT. Furthermore, as a step towards clinical exploitation of the recently described signatures of mutational processes in cancer genomes, we established and applied a novel bioinformatics algorithm that enables supervised analysis of the mutational catalogs of individual tumors., Results: Molecularly guided treatment with imatinib in analogy to the management of gastrointestinal stromal tumor (GIST) resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor. KIT dependency was further validated by a secondary KIT exon 17 mutation (c.2459&#95;2462delATTCinsG, p.D820&#95;S821delinsG) that was detected upon tumor progression after 10 months of imatinib treatment and provided a rationale for salvage therapy with regorafenib, which has activity against KIT exon 11/17 mutant GIST., Conclusions: These observations highlight the potential of unbiased genomic profiling for uncovering the vulnerabilities of individual malignancies, particularly in rare and unclassifiable tumors, and underscore that KIT exon 11 mutations represent tractable therapeutic targets across different histologies., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Benign liver tumours represent a challenge in clinical management. There is considerable controversy with respect to the indications for surgery as the evidence for surgical treatment is variable. The aim of this retrospective study was to analyse the indication and outcome after resection of benign, solid liver lesions., Methods: Data of 79 patients, who underwent liver resection between 2001 and 2012, were analysed for demographic and outcome parameters., Results: Thirty-eight patients with focal nodular hyperplasia (48%), 23 patients with haemangioma (29%) and 18 patients with hepatocellular adenoma (23%) underwent a hepatic resection. A major hepatic resection was performed in 23 patients (29%) and a minor resection in 56 patients (71%). The post-operative mortality rate was zero and the 30-day morbidity rate 13.9%. After a median follow-up of 64 months, 75 patients (95%) were alive, and no patient had developed recurrent disease. Fifty-four patients (68%) were pre-operatively symptomatic, of which, 87% had complete or partial relief of symptoms after a liver resection. The incidence of symptoms increased with the lesions' size., Discussion: The management of benign liver lesions necessitates an individualized therapy within a multidisciplinary, evidence-based, treatment algorithm. Resection of benign liver lesions can be performed safely in well-selected patients without mortality and low post-operative morbidity., (© 2015 International Hepato-Pancreato-Biliary Association.)

Background & Aims: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response., Methods: In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways., Results: Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2., Conclusions: Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Metabolic and transcriptomic differences between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) compartments, particularly in the context of obesity, may play a role in colorectal carcinogenesis. We investigated the differential functions of their metabolic compositions., Objectives: Biochemical differences between adipose tissues (VAT compared with SAT) in patients with colorectal carcinoma (CRC) were investigated by using mass spectrometry metabolomics and gene expression profiling. Metabolite compositions were compared between VAT, SAT, and serum metabolites. The relation between patients' tumor stage and metabolic profiles was assessed., Design: Presurgery blood and paired VAT and SAT samples during tumor surgery were obtained from 59 CRC patients (tumor stages I-IV) of the ColoCare cohort. Gas chromatography time-of-flight mass spectrometry and liquid chromatography quadrupole time-of-flight mass spectrometry were used to measure 1065 metabolites in adipose tissue (333 identified compounds) and 1810 metabolites in serum (467 identified compounds). Adipose tissue gene expression was measured by using Illumina's HumanHT-12 Expression BeadChips., Results: Compared with SAT, VAT displayed elevated markers of inflammatory lipid metabolism, free arachidonic acid, phospholipases (PLA2G10), and prostaglandin synthesis-related enzymes (PTGD/PTGS2S). Plasmalogen concentrations were lower in VAT than in SAT, which was supported by lower gene expression of FAR1, the rate-limiting enzyme for ether-lipid synthesis in VAT. Serum sphingomyelin concentrations were inversely correlated (P = 0.0001) with SAT adipose triglycerides. Logistic regression identified lipids in patients' adipose tissues, which were associated with CRC tumor stage., Conclusions: As one of the first studies, we comprehensively assessed differences in metabolic, lipidomic, and transcriptomic profiles between paired human VAT and SAT and their association with CRC tumor stage. We identified markers of inflammation in VAT, which supports prior evidence regarding the role of visceral adiposity and cancer., (© 2015 American Society for Nutrition.)

Background: A novel classification of pulmonary adenocarcinoma (ADC) distinguishing five growth patterns has been established by the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society. There is evidence that an additional cribriform pattern associates with a distinct clinical behavior., Methods: We evaluated the predominant growth pattern of 674 resected ADC as recommended by the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society, including the cribriform pattern. The predominant pattern type was correlated with clinical, molecular, and survival data., Results: Two hundred forty-eight (36.8%) of the pulmonary ADC were solid, 207 (30.6%) were acinar, 101 (15%) were papillary, 55 (8.2%) were micropapillary, 35 (5.2%) were lepidic, and 28 cases (4.2%) were cribriform predominant (cpADC). Minor cribriform components were frequently observed (28.6% of all cases). cpADC showed the second highest proliferative capacity of all patterns, no somatic mutations in the epidermal growth factor receptor (p = 0.001) and a high rate of KRAS mutations. Overall survival (OS) of patients with cpADC (mean OS: 62.7 months) ranged in between survival times of patients with acinar (mean OS: 71.3 months) and solid predominant ADC (mean OS: 54.5 months); cpADC was associated with the worst disease-free survival (DFS) of all patterns (mean DFS: 36.9 months). Both associations were confirmed by multivariate analysis (p < 0.01 for both OS and DFS). Hazard ratios for cpADC were 1.72 for OS and 2.99 for DFS, with lepidic predominant ADC set as reference (hazard ratio: 1)., Conclusions: Our data support the introduction of cpADC as a novel category into future morphology based on pulmonary ADC classifications. Further international studies are required to validate the reported clinicopathological associations of the cribriform pattern.

Introduction: Detection of anaplastic lymphoma kinase (ALK)-gene rearrangements in non-small-cell lung cancer (NSCLC) is mainly performed by fluorescence in-situ hybridization (FISH). The question was raised if FISH might be replaced by immunohistochemistry (IHC) in a reliable and reproducible manner across different laboratories., Methods: After calibration of the staining instruments and training of the observers to binary interpretation (positive versus negative), 15 NSCLC were independently tested for ALK protein expression by IHC only in a multicenter setting (16 institutes). Each laboratory utilized the VENTANA ALK-D5F3 IHC assay. As demonstrated by FISH the samples displayed unequivocal ALK break-positivity (6×) and negativity (7×), as well as ALK positive-"borderline" character (2×), which is challenging for FISH diagnosis and thus was RT-PCR-confirmed., Results: All seven ALK FISH-negative cases were homogenously scored as ALK-IHC negative. All 16 participants scored the two ALK positive-"borderline" samples as unequivocally positive according to their protein expression. Concordant IHC interpretation was also noticed in four of six unequivocal ALK break positive cases. In two of six some observers described a weak/heterogeneous ALK-IHC staining. This would have resulted in a subsequent ALK-testing (FISH/PCR) in a routine diagnostic setting., Conclusions: This so-called "ALK-Harmonization-Study" shows for the first time that predictive semiquantitative IHC reveals reliable and reproducible results across several labs when methodology and interpretation are strictly defined and the pathologists are uniquely trained. The application of validated ALK IHC assays and its comparison to ALK-FISH is highly needed in future clinical trials. This might answer the question if ALK-IHC cannot only serve as a prescreening tool, but as a stand-alone test at least in cases displaying an unequivocally staining pattern as well as an alternative predictive test in samples with reduced FISH interpretability.

Background: The anaplastic lymphoma kinase (ALK) inhibitor crizotinib has recently received approval for the treatment of patients with locally advanced or metastatic ALK-positive non-small-cell lung cancer (NSCLC). As the therapeutic prescription postulates the detection of ALK rearrangements, reliable diagnostic approaches are of utmost importance. With this study, we present the data of the first German ALK-round robin test based on genomic DNA in situ hybridization (ISH). The application of immunohistochemistry (IHC) for ALK protein detection was optional and not required for certification., Methods: Two tissue microarrays, each consisting of the same 10 NSCLC but in different arrangement of the cases, were generated (five unequivocally ALK-positive and five unequivocally ALK-negative cases). ISH-based results and optional IHC data had to be submitted within 10 working days. A successful participation (certification) was reached if at least 19 of the 20 possible points were scored (2 points for a correct case classification)., Results: Fifty-three of 59 participants (89.8%) provided their data for ISH-ALK detection within the submission period. Thirty-two of 53 participants (60.3%) received at least 19 points required for certification. Remarkably, the range of cells with aberrant ALK signal configuration was broad in ALK-negative (0-13%) and in ALK-positive cases (15-95%). Thirty-five participants supported the round robin test with optional ALK IHC results, which displayed a great heterogeneity in the ALK ISH-positive cases., Conclusion: In essence, our ALK ISH round robin test clearly demonstrates that there is accumulating need for improvement of ALK testing. Although ISH may be regarded as a well-established procedure, its broad application in a diagnostic setting is challenging and requires standardized methods and harmonized interpretation to achieve sound results for therapeutic decisions. The same is true for ALK IHC which, however if standardized, might improve the diagnostic approach.

Background & Aims: Hepatocellular steatosis is the most frequent liver disease in the western world and may develop further to steatohepatitis, liver cirrhosis and hepatocellular carcinoma. We have previously shown that lipid droplet (LD)-associated proteins of the perilipin/PAT-family are differentially expressed in hepatocyte steatosis and that perilipin is expressed de novo. The aim of this study was to determine the conditions for the temporal regulation of de novo synthesis of perilipin in vitro and in vivo., Methods: Immunohistochemical PAT-analysis was performed with over 120 liver biopsies of different etiology and duration of steatosis. Steatosis was induced in cultured hepatocytic cells with combinations of lipids, steatogenic substances and DMSO for up to 40 days under conditions of stable down-regulation of adipophilin and/or TIP47., Results: Whereas perilipin and adipophilin were expressed in human chronic liver disease irrespective of the underlying etiology, in acute/microvesicular steatosis TIP47, and MLDP were recruited from the cytoplasm to LDs, adipophilin was strongly increased, but perilipin was virtually absent. In long-term steatosis models in vitro, TIP47, MLDP, adipophilin, and finally perilipin were gradually induced. Perilipin and associated formation of LDs were intricately regulated on the transcriptional (PPARs, C/EBPs, SREBP), post-transcriptional, and post-translational level (TAG-amount, LD-fusion, phosphorylation-dependent lipolysis). In long-term steatosis models under stable down-regulation of adipophilin and/or TIP47, MLDP substituted for TIP47, and perilipin for adipophilin., Conclusions: LD-maturation in hepatocytes in vivo and in vitro involves sequential expression of TIP47, MLDP, adipophilin and finally perilipin. Thus, perilipin might be used for the differential diagnosis of chronic vs. acute steatosis., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Allelic variants of fucosyltransferases 2 and 3 (FUT2/3) influence serum levels of CA19-9, a screening parameter commonly used for detection of biliary malignancy in PSC. We aimed at improving diagnostic accuracy of CA19-9 by determining the impact of FUT2/3 genotypes., Methods: CA19-9 levels were measured in 433 PSC patients, 41 of whom had biliary malignancy. Genotypes for FUT3 and FUT2 were used to assign patients to one of three groups: A, no FUT3 activity regardless of FUT2 activity; B, both FUT2 and FUT3 activity and C, no FUT2 activity without loss of FUT3 activity. Group-specific cut-off values were determined by Youden's index., Results: The median CA19-9 values of cancer-free patients were significantly different (p<0.001) in Groups A (2.0U/ml), B (17.0U/ml), and C (37.0U/ml). Biliary malignancy patients in Groups B and C had significantly higher CA19-9 values than cancer-free patients (p<0.001). The optimal cut-off, as determined by ROC analysis, for all patients was 88.5U/ml. Optimal cut-off values in Groups A, B, and C were 4.0U/ml, 74.5U/ml, and 106.8U/ml, respectively. Use of these values improved sensitivity of CA19-9 in Groups B and C. Further, use of group-dependent cut-off values with 90% sensitivity resulted in a 42.9% reduction of false positive results., Conclusions: Use of FUT2/3 genotype-dependent cut-off values for CA19-9 improved sensitivity and reduced the number of false positive results., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

In the context of personalized oncology, screening for somatic tumor mutations is crucial for prediction of an individual patient's response to therapy. Massive parallel sequencing (MPS) has been suggested for routine diagnostics, but this technology has not been sufficiently evaluated with respect to feasibility, reliability, and cost effectiveness with routine diagnostic formalin-fixed, paraffin-embedded material. We performed ultradeep targeted semiconductor-based MPS (190 amplicons covering hotspot mutations in 46 genes) in a variety of formalin-fixed, paraffin-embedded diagnostic samples of lung adenocarcinoma tissue with known EGFR mutations (n = 28). The samples reflected the typical spectrum of tissue material for diagnostics, including small biopsies and samples with low tumor-cell content. Using MPS, we successfully sequenced all samples, with a mean read depth of 2947 reads per amplicon. High-quality sequence reads were obtained from samples containing ≥10% tumor material. In all but one sample, variant calling identified the same EGFR mutations as were detected by conventional Sanger sequencing. Moreover, we identified 43 additional mutations in 17 genes and detected amplifications in the EGFR and ERBB2 genes. MPS performance was reliable and independent of the type of material, as well as of the fixation and extraction methods, but was influenced by tumor-cell content and the degree of DNA degradation. Using sample multiplexing, focused MPS approached diagnostically acceptable cost rates., (Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies with poor prognosis and increasing incidence in the Western world. Only for a minority of HCC patients, surgical treatment options offer potential cure and therapeutic success of pharmacological approaches is limited. Highly specific approaches (e.g., kinase inhibitors) did not significantly improve the situation so far, possibly due to functional compensation, genetic heterogeneity of HCC, and development of resistance under selective pressure. In contrast, transcriptional regulators (especially transcription factors and co-factors) may integrate and process input signals of different (oncogenic) pathways and therefore represent cellular bottlenecks that regulate tumor cell biology. In this review, we want to summarize the current knowledge about central transcriptional regulators in human hepatocarcinogenesis and their potential as therapeutic target structures. Genomic and transcriptomic data of primary human HCC revealed that many of these factors showed up in subgroups of HCCs with a more aggressive phenotype, suggesting that aberrant activity of transcriptional regulators collect input information to promote tumor initiation and progression. Therefore, expression and dysfunction of transcription factors and co-factors may gain relevance for diagnostics and therapy of HCC., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Introduction: Cytotoxic chemotherapy remains the mainstay of treatment for most patients with advanced disease. Recently, anaplastic lymphoma kinase (ALK) expression as a major target for successful treatment with ALK inhibitors was detected in a subset of non-small-cell lung carcinomas, usually as a result of echinoderm microtubule-associated protein-like 4 (EML4)-ALK rearrangements. Although the chromosomal breakpoint within the EML4 gene varied, the breakpoint within ALK was most frequently reported within intron 19 or rarely in exon 20. Therefore, the different EML4-ALK variants so far contain the same 3' portion of ALK starting with exon 20., Methods: Here, we report a novel EML4-ALK variant detected by reverse transcription polymerase chain reaction analysis., Results: Subsequent sequencing revealed an EML4-ALK fusion variant in which exon 6 of EML4 was fused to exon 19 of ALK. It occurred in a predominant solid pulmonary adenocarcinoma of a 65-year-old woman with a clear split signal of ALK in fluorescence in situ hybridization analysis and a weakly homogeneous ALK expression in immunohistochemical staining., Conclusions: Because of the growing number of fusion variants a primary reverse transcription polymerase chain reaction-based screening for ALK-positive non-small-cell lung carcinoma patients may not be sufficient for predictive diagnostics but transcript-based approaches and sequencing of ALK fusion variants might finally contribute to an optimized selection of patients.

Background & Aims: Differential expression of tumor-relevant proteins based on aberrant proteasomal degradation may contribute to human (hepato)carcinogenesis. Recently, we identified the E3 ubiquitin ligase seven in absentia homolog (SIAH)-1 as frequently dysregulated in human hepatocellular carcinoma (HCC). We therefore systematically analyzed the expression, functional relevance, as well as possible downstream effectors of SIAH-1 in human liver carcinogenesis., Methods: SIAH-1 expression was analyzed at the transcript and protein levels in human hepatocarcinogenesis and in HCC cells. Proliferation, apoptosis, and migration of different HCC cell lines were examined after siRNA-mediated inhibition of SIAH-1. In order to identify downstream effectors that mediate SIAH-1 effects, correlative analyses of protein expression profiles were performed., Results: In HCC tissues both reduction of cytoplasmic SIAH-1 and especially its nuclear accumulation positively correlated with HCC progression. RNA interference revealed that nuclear expression of SIAH-1 predominantly supported HCC cell proliferation and migration while only moderately affecting anti-apoptosis. In de-differentiated human HCCs, nuclear SIAH-1 accumulation significantly correlated with the expression of the transcription factor far-upstream element (FUSE)-binding protein (FBP)-3. In vitro, SIAH-1 positively and indirectly regulated FBP-3 which itself primarily supported HCC cell proliferation. Indeed, high level expression of FBP-3 in human HCCs significantly correlated with reduced overall survival of patients., Conclusions: Nuclear accumulation of the E3 ubiquitin ligase SIAH-1 supports different pro-tumorigenic cellular processes associated with tumor growth and tumor cell dissemination in human hepatocarcinogenesis. It promotes HCC cell proliferation by at least partly employing the transcription factor FBP-3. Therefore, interference with SIAH-1 activity represents a promising approach to suppress HCC growth., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Introduction: Activating somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have recently been characterized in a subset of patients with advanced non-small cell lung cancer (NSCLC). Patients harboring these mutations in their tumors show excellent response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The EGFR-TKI gefitinib has been approved in Europe for the treatment of adult patients with locally advanced or metastatic NSCLC with activating mutations of the EGFR TK. Because EGFR mutation testing is not yet well established across Europe, biomarker-directed therapy only slowly emerges for the subset of NSCLC patients most likely to benefit: those with EGFR mutations., Methods: The "EGFR testing in NSCLC: from biology to clinical practice" International Association for the Study of Lung Cancer-European Thoracic Oncology Platform multidisciplinary workshop aimed at facilitating the implementation of EGFR mutation testing. Recommendations for high-quality EGFR mutation testing were formulated based on the opinion of the workshop expert group., Results: Co-operation and communication flow between the various disciplines was considered to be of most importance. Participants agreed that the decision to request EGFR mutation testing should be made by the treating physician, and results should be available within 7 working days. There was agreement on the importance of appropriate sampling techniques and the necessity for the standardization of tumor specimen handling including fixation. Although there was no consensus on which laboratory test should be preferred for clinical decision making, all stressed the importance of standardization and validation of these tests., Conclusion: The recommendations of the workshop will help implement EGFR mutation testing in Europe and, thereby, optimize the use of EGFR-TKIs in clinical practice.

Introduction: This study was designed to develop a both sensitive and efficient algorithm for detection of lymph node micrometastases and to determine its prognostic impact in patients with early stage non-small cell lung cancer (NSCLC)., Methods: One hundred seventy patients with NSCLC p stage I and II were included in this study, of which n = 5299 lymph nodes were obtained and submitted to histopathologic analysis. From each patient, a median number of 31 lymph nodes was received (N-1 position: median n = 16; N-2 position: median n = 15). Immunohistochemistry was performed to detect micrometastases unobvious by conventional microscopy using antibodies against cytokeratins (CK) (pan-CK: KL-1, CK 5/6, CK 7) and the epithelial marker Ber-EP4., Results: In 82 patients (48.2%), micrometastases were revealed in immunohistochemistry staining. KL-1 detected micrometastases in 201 (99.5%) of 202 positive lymph nodes. Subsequently, this resulted in an upstaging in 39 patients (20.5%). Detection of micrometastases in otherwise tumor-free N2-lymph nodes had a significant prognostic impact (mean disease-free survival 21.4 versus 45.3 months, p = 0.022), affecting 4.7% of patients. Survival differences between patients who were upstaged into stage II (N0>N1) and those remaining in stage I were not statistically significant (p = 0.537)., Conclusion: Extended workup of N2-lymph nodes using one broad-spectrum keratin marker in otherwise N2-negative lymph nodes may represent a both efficient and sensitive approach to the identification of micrometastases in dissected lymph nodes of patients with early stage NSCLC.

Keratinocyte migration is essential for the rapid closure of the epidermis in the process of wound healing. Mesenchymal cell-derived hepatocyte growth factor (HGF) is a central regulator of this process. However, the molecular mechanisms and relevant genes that facilitate this cellular response are still poorly defined. We used heterologous cocultures combining primary human keratinocytes and genetically modified murine fibroblasts to identify key factors mediating HGF-induced epidermal cell migration. The absence of c-Jun activity in fibroblasts completely abolished the expression of HGF in these cells and consequently altered the behavior of keratinocytes. Time-resolved expression series of keratinocytes stimulated with HGF disclosed target genes regulating HGF-dependent motility. In addition to well-established HGF-dependent wound healing-associated genes, carcinoembryogenic antigen-related cell adhesion molecule (CEACAM)-1 and the urokinase plasminogen activator (uPA)/uPA-receptor (uPAR) pathway were identified as possible mediators in HGF-induced keratinocyte migration. The functional relevance of CEACAM-1 and uPA/uPAR on epidermal cell motility was demonstrated using the HaCaT cell culture model. In conclusion, the distinct spatiotemporal regulation of genes by HGF is essential for proper epidermal cell migration in cutaneous wound healing.

Background: Decoy receptor 3 (DcR3) is a soluble protein that binds to and inactivates the death ligand CD95L. Here, we studied a possible association between DcR3 expression and prognosis in patients with renal cell carcinomas (RCCs)., Methods: A tissue microarray containing RCC tumor tissue samples and corresponding normal tissue samples was generated. Decoy receptor 3 expression in tumors of 560 patients was examined by immunohistochemistry. The effect of DcR3 expression on disease-specific survival and progression-free survival was assessed using univariate analysis and multivariate Cox regression analysis. Decoy receptor 3 serum levels were determined by ELISA., Findings: High DcR3 expression was associated with high-grade (P = .005) and high-stage (P = .048) RCCs. The incidence of distant metastasis (P = .03) and lymph node metastasis (P = .002) was significantly higher in the group with high DcR3 expression. Decoy receptor 3 expression correlated negatively with disease-specific survival (P < .001) and progression-free survival (P < .001) in univariate analyses. A multivariate Cox regression analysis retained DcR3 expression as an independent prognostic factor that outperformed the Karnofsky performance status. In patients with high-stage RCCs expressing DcR3, the 2-year survival probability was 25%, whereas in patients with DcR3-negative tumors, the survival probability was 65% (P < .001). Moreover, DcR3 serum levels were significantly higher in patients with high-stage localized disease (P = .007) and metastatic disease (P = .001)., Interpretation: DcR3 expression is an independent prognostic factor of RCC progression and mortality. Therefore, the assessment of DcR3 expression levels offers valuable prognostic information that could be used to select patients for adjuvant therapy studies.

Cholangiocarcinoma (CC) arises from the biliary epithelium and in most cases represents adenocarcinoma. Pathomorphological evaluation is of decisive impact for the prognosis and management of CC. Morphological subtyping (histotype; hilar vs peripheral type), TNM classification, lymphatic spread, and resection margin status are of prognostic relevance. Distinction from hepatic metastases may be aided by immunohistology and clinico-pathological correlation. There is convincing evidence of the development of CC via premalignant lesions, especially biliary intraepithelial neoplasia, although further knowledge about the biology and diagnostic definition of these lesions has to be accumulated. Currently, there are no established molecular markers of prognosis or therapeutic target structures to be evaluated at the tissue level. Future progress is needed and expected in novel differential diagnostic and predictive markers, in uniform definition of resection margin status and further understanding of molecular and morphological changes in the development of CC.

SPARC-like protein 1 (SPARCL1), a member of the SPARC family, is downregulated in various tumors. In the present study, the expression and localization of SPARCL1 were analyzed in a wide range of nontumorous and neoplastic pancreatic tissues by quantitative reverse transcription-polymerase chain reaction, laser capture microdissection, microarray analysis, and immunohistochemistry. For functional analysis, proliferation and invasion assays were used in cultured pancreatic cancer cells. Pancreatic ductal adenocarcinoma (PDAC) and other pancreatic neoplasms exhibited increased SPARCL1 mRNA levels compared to those of the normal pancreas. SPARCL1 mRNA levels were low to absent in microdissected and cultured pancreatic cancer cells, and promoter demethylation increased SPARCL1 levels only slightly in three of eight cell lines. SPARCL1 was observed in small capillaries in areas of inflammation/tumor growth and in some islet cells. In PDAC, 15.4% of vessels were SPARCL1-positive. In contrast, the percentage of SPARCL1-positive vessels was higher in chronic pancreatitis and benign and borderline pancreatic tumors. Recombinant SPARCL1 inhibited pancreatic cancer cell invasion and exerted moderate growth-inhibitory effects. In conclusion, SPARCL1 expression in pancreatic tissues is highly correlated with level of vascularity. Its anti-invasive effects and reduced expression in metastasis indicate tumor-suppressor function.

The recent elucidation of the human type I keratin gene domain allowed the completion of the so far only partially characterized subcluster of type I keratin genes, KRT25-KRT28 (formerly KRT25A-KRT25D), representing the counterparts of the type II inner root sheath (IRS) keratin genes, KRT71-KRT74 (encoding proteins K71-K74, formerly K6irs1-K6irs4). Here, we describe the expression patterns of the type I IRS keratin proteins K25-K28 (formerly K25irs1-K25irs4) and their mRNAs. We found that K25 (K25irs1), K27 (K25irs3), and K28 (K25irs4) occur in the Henle layer, the Huxley layer, and in the IRS cuticle. Their expression extends from the bulb region up to the points of terminal differentiation of the three layers. In contrast, K26 (K25irs2) is restricted to the upper IRS cuticle. Apart from the three IRS layers, K25 (K25irs1), K27 (K25irs3), and K28 (K25irs4) are also present in the hair medulla. Based on previous, although controversial claims of the occurrence in the IRS of various "classical" epithelial keratins, we undertook a systematic study using antibodies against the presently described human epithelial and hair keratins and show that the type I keratins K25-K28 (K25irs1-K25irs4) and the type II keratins K71-K74 (K6irs1-K6irs4) represent the IRS keratins of the human hair follicle.

Background/aims: In hepatic fibrogenesis, stellate cells are activated leading to production and deposition of extracellular matrix. To clarify the role of PDGF-B in liver fibrogenesis, we overexpressed PDGF-B in the liver of transgenic mice., Methods: Transgenic mice for the conditional overexpression of PDGF-B in the liver under control of an albumin promoter were generated utilising the Cre/loxP system. Constitutive PDGF-B expression was achieved after breeding with mice expressing Cre-recombinase under actin promoter control. Tamoxifen inducible expression was achieved after breeding with mice expressing Cre under transthyretin receptor promoter control. Levels of fibrosis were assessed and the expression of regulators of matrix remodelling was measured., Results: PDGF-B expression caused hepatic stellate cell and myofibroblast activation marked by alpha-smooth muscle actin and PDGFR-beta expression. Liver fibrosis was verified macroscopically, histologically and by collagen I mRNA quantification in 4-6 week-old animals. MMP-2, MMP-9 and TIMP-1 were upregulated whereas TGF-beta expression was unchanged., Conclusions: We identified PDGF-B as a proliferative and profibrogenic stimulus and potential inducer of stellate cell transdifferentiation in vivo. PDGF-B overexpression causes liver fibrosis without significantly upregulating TGF-beta1, suggesting a TGF-beta-independent mechanism. The established model provides a tool for testing anti-PDGF-B therapeutic strategies in liver fibrosis in vivo.