Prevalence of Molecular Alterations in a Swiss Cohort of 512 Colorectal Carcinoma Patients by Targeted Next-Generation Sequencing Analysis in Routine Diagnostics.

INTRODUCTION: Colorectal carcinoma (CRC) is among the most common carcinomas in women and men. In the advanced stage, patients are treated based on the RAS status. Recent studies indicate that in the future, in addition to KRAS and NRAS, alterations in other genes, such as PIK3CA or TP53, will be considered for therapy. Therefore, it is important to know the mutational landscape of routinely diagnosed CRC. METHOD: We report the molecular profile of 512 Swiss CRC patients analyzed by targeted next-generation sequencing as part of routine diagnostics at our institute. RESULTS: Pathogenic and likely pathogenic variants were found in 462 (90%) CRC patients. Variants were detected in TP53 (54.3%), KRAS (48.2%), PIK3CA (15.6%), BRAF (13.5%), SMAD4 (10.5%), FBXW7 (7.8%), NRAS (3.5%), PTEN (2.7%), ERBB2 (1.6%), AKT1 (1.5%), and CTNNB1 (0.9%). The remaining pathogenic alterations were found in the genes ATM(n= 1), MAP2K1(n= 1), and IDH2(n= 1). DISCUSSION/CONCLUSIONS: Our analysis revealed the prevalence of potential predictive markers in a large cohort of CRC patients obtained during routine diagnostic analysis. Furthermore, our study is the first of this size to uncover the molecular landscape of CRC in Switzerland.

The Genomic Landscape of Serrated Lesion of the Colorectum: Similarities and Differences With Tubular and Tubulovillous Adenomas.

Serrated lesions of the colorectum are the precursors of 15-30% of colorectal cancers (CRCs). These lesions have a peculiar morphological appearance, and they are more difficult to detect than conventional adenomatous polyps. In this study, we sought to define the genomic landscape of these lesions using high-depth targeted sequencing. Eight sessile serrated lesions without dysplasia (SSL), three sessile serrated lesions with dysplasia (SSL/D), two traditional serrated adenomas (TSA), and three tubular adenomas (TA) were retrieved from the files of the Institute of Pathology of the University Hospital Basel and from the GILAB AG, Allschwil, Switzerland. Samples were microdissected together with the matched normal counterpart, and DNA was extracted for library preparation. Library preparation was performed using the Oncomine Comprehensive Assay targeting 161 common cancer driver genes. Somatic genetic alterations were defined using state-of-the-art bioinformatic analysis. Most SSLs, as well as all SSL/Ds and TSAs, showed the classical BRAF p.V600E mutation. The BRAF-mutant TSAs showed additional alterations in CTNNB1, NF1, TP53, NRAS, PIK3CA, while TA showed a consistently different profile, with mutations in ARID1A (two cases), SMAD4, CDK12, ERBB3, and KRAS. In conclusion, our results provide evidence that SSL/D and TSA are similar in somatic mutations with the BRAF hotspot somatic mutation as a major driver of the disease. On the other hand, TAs show a different constellation of somatic mutations such as ARID1A loss of function.

Nestin and CD34 expression in colorectal cancer predicts improved overall survival.

BACKGROUND: Nestin, a class VI intermediate filament protein of the cytoskeleton, and CD34, a transmembrane phosphoglycoprotein, are markers of progenitor cells. This study aimed to evaluate their expression and clinical significance in colorectal cancer. METHODS: A clinically annotated tissue microarray, including 599 patients with colorectal cancer, was analyzed by immunohistochemistry. Furthermore, nestin and CD34 correlations with HIF-1a and a panel of cytokines and chemokines were assessed using quantitative reverse transcription PCR and The Cancer Genome Atlas dataset. RESULTS: Expression of nestin and CD34 was observed only in the tumor stroma. Patients displaying high expression of nestin and CD34 demonstrated higher rates of T1 and T2 tumors (p = .020), lower vascular invasion (p < .001) and improved 5-year overall survival (65%; 95% CI = 55-73 vs 45%; 95% CI = 37-53) after adjusting for clinicopathological characteristics (HR: 0.67; 95% CI = 0.46-0.96). A moderate to strong correlation (r = 0.37-0.78, p < .03) of nestin and CD34 was demonstrated for the following markers; HIF-1α, CD4, CD8, FOXP3, IRF1, GATA3, CCL2, CCL3, CXCL12 and CCL21. CONCLUSIONS: Combined expression of nestin and CD34 expression is associated with better overall survival possibly by modulating a favorable immune response.

Infiltration by IL22-Producing T Cells Promotes Neutrophil Recruitment and Predicts Favorable Clinical Outcome in Human Colorectal Cancer.

Immune cell infiltration in colorectal cancer effectively predicts clinical outcome. IL22, produced by immune cells, plays an important role in inflammatory bowel disease, but its relevance in colorectal cancer remains unclear. Here, we addressed the prognostic significance of IL22+ cell infiltration in colorectal cancer and its effects on the composition of tumor microenvironment. Tissue microarrays (TMA) were stained with an IL22-specific mAb, and positive immune cells were counted by expert pathologists. Results were correlated with clinicopathologic data and overall survival (OS). Phenotypes of IL22-producing cells were assessed by flow cytometry on cell suspensions from digested specimens. Chemokine production was evaluated in vitro upon colorectal cancer cell exposure to IL22, and culture supernatants were used to assess neutrophil migration in vitro Evaluation of a testing (n = 425) and a validation TMA (n = 89) revealed that high numbers of IL22 tumor-infiltrating immune cells were associated with improved OS in colorectal cancer. Ex vivo analysis indicated that IL22 was produced by CD4+ and CD8+ polyfunctional T cells, which also produced IL17 and IFNγ. Exposure of colorectal cancer cells to IL22 promoted the release of the neutrophil-recruiting chemokines CXCL1, CXCL2, and CXCL3 and enhanced neutrophil migration in vitro Combined survival analysis revealed that the favorable prognostic significance of IL22 in colorectal cancer relied on the presence of neutrophils and was enhanced by T-cell infiltration. Altogether, colorectal cancer-infiltrating IL22-producing T cells promoted a favorable clinical outcome by recruiting beneficial neutrophils capable of enhancing T-cell responses.

Low Expression of Programmed Death 1 (PD-1), PD-1 Ligand 1 (PD-L1), and Low CD8+ T Lymphocyte Infiltration Identify a Subgroup of Patients With Gastric and Esophageal Adenocarcinoma With Severe Prognosis.

Prognosis of gastric and esophageal cancer is poor and treatment improvements are needed. Programmed cell death 1 receptor (PD-1) interaction with its ligand PD-L1 in tumor micro-environment promotes immune tolerance and blocking monoclonal antibodies have entered clinical practice. However, clinical significance of PD-1 and PD-L1 expression in gastric and esophageal adenocarcinomas, particularly in non-Asian patients, is still unclear. Three tissue microarrays including 190 clinically annotated esophageal (n = 31) and gastric (n = 159) adenocarcinomas and 58 paired mucosa specimens, were stained with PD-1, PD-L1, and CD8-specific reagents in indirect immunohistochemistry assays. PD-L1 expression was detectable in 23.2% of cancer specimens. High PD-1 expression was detectable in 37.3% of cases and high CD8+ infiltration in 76%. PD-L1 and high PD1 expression significantly correlated with each other (r s = 0.404, P < 0.0001) and both significantly correlated with CD8+ infiltration (r s = 0.435, P = 0.0003, and r s = 0.444; P = 0.0004, respectively). CD8+ lymphocyte infiltration correlated with improved survival in univariate (P = 0.009), but not multivariate analysis. Most interestingly, multivariate analysis and Kaplan-Meier curves indicate that combined low PD-1/PD-L1 expression and low CD8+ lymphocyte infiltration significantly correlate with poor prognosis. Our data document the clinical significance of a microenvironmental signature including PD-1/PD-L1 expression and CD8+ lymphocyte infiltration in gastric and esophageal adenocarcinomas and contribute to identify a patients' subset requiring more aggressive peri-operative treatments.

Sensitive detection methods are key to identify secondary EGFR c.2369C>T p.(Thr790Met) in non-small cell lung cancer tissue samples.

BACKGROUND: Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. METHODS: Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). RESULTS: In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. CONCLUSIONS: A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.

Molecular Profile of Gastrointestinal Stromal Tumors in Sixty-Eight Patients from a Single Swiss Institution.

INTRODUCTION: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. It has distinct molecular features and primarily affects the KIT and PDGFRA genes. OBJECTIVE: We wanted to assess the molecular profile of 68 GIST patients who were sequenced consecutively between 2014 and 2019 at our institute of pathology. METHODS: Our cohort comprised 60 primary and 8 metastatic GIST patients; 43 and 57% of the cases, respectively, were analyzed by Sanger sequencing or next-generation sequencing (NGS). RESULTS: Of the 60 primary GIST patients, 47 (78%) showed a KIT mutation; 2 cases showed a double KIT mutation, and 1 of these was a therapy-naive GIST. Nine (15%) patients harbored a PDGFRA mutation, 2 (3%) had a BRAF mutation, 1 (2%) had a PIK3CA mutation, and 1 (2%) did not show any mutation. One BRAF and the PIK3CA mutation have not been described in GIST before. All metastatic GIST harbored exclusively KIT mutations. CONCLUSION: A retrospective analysis of GIST sequenced at our institute revealed incidences of KIT and PDGFRA mutations comparable to those in other cohorts from Europe. Interestingly, we found 2 previously undescribed mutations in the BRAF and PIK3CA genes as well as 1 treatment-naive case with a double KIT mutation in exon 11.

DNA methylation instability by BRAF-mediated TET silencing and lifestyle-exposure divides colon cancer pathways.

BACKGROUND: Aberrations in DNA methylation are widespread in colon cancer (CC). Understanding origin and progression of DNA methylation aberrations is essential to develop effective preventive and therapeutic strategies. Here, we aimed to dissect CC subtype-specific methylation instability to understand underlying mechanisms and functions. METHODS: We have assessed genome-wide DNA methylation in the healthy normal colon mucosa (HNM), precursor lesions and CCs in a first comprehensive study to delineate epigenetic change along the process of colon carcinogenesis. Mechanistically, we used stable cell lines, genetically engineered mouse model of mutant BRAFV600E and molecular biology analysis to establish the role of BRAFV600E-mediated-TET inhibition in CpG-island methylator phenotype (CIMP) inititation. RESULTS: We identified two distinct patterns of CpG methylation instability, determined either by age-lifestyle (CC-neutral CpGs) or genetically (CIMP-CpGs). CC-neutral-CpGs showed age-dependent hypermethylation in HNM, all precursors, and CCs, while CIMP-CpGs showed hypermethylation specifically in sessile serrated adenomas/polyps (SSA/Ps) and CIMP-CCs. BRAFV600E-mutated CCs and precursors showed a significant downregulation of TET1 and TET2 DNA demethylases. Stable expression of BRAFV600E in nonCIMP CC cells and in a genetic mouse model was sufficient to repress TET1/TET2 and initiate hypermethylation at CIMP-CpGs, reversible by BRAFV600E inhibition. BRAFV600E-driven CIMP-CpG hypermethylation occurred at genes associated with established CC pathways, effecting functional changes otherwise achieved by genetic mutation in carcinogenesis. CONCLUSIONS: Hence, while age-lifestyle-driven hypermethylation occurs generally in colon carcinogenesis, BRAFV600E-driven hypermethylation is specific for the "serrated" pathway. This knowledge will advance the use of epigenetic biomarkers to assess subgroup-specific CC risk and disease progression.

Hepatocellular Carcinoma Xenografts Established From Needle Biopsies Preserve the Characteristics of the Originating Tumors.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Treatment options for patients with advanced-stage disease are limited. A major obstacle in drug development is the lack of an in vivo model that accurately reflects the broad spectrum of human HCC. Patient-derived xenograft (PDX) tumor mouse models could overcome the limitations of cancer cell lines. PDX tumors maintain the genetic and histologic heterogeneity of the originating tumors and are used for preclinical drug development in various cancers. Controversy exists about their genetic and molecular stability through serial passaging in mice. We aimed to establish PDX models from human HCC biopsies and to characterize their histologic and molecular stability during serial passaging. A total of 54 human HCC needle biopsies that were derived from patients with various underlying liver diseases and tumor stages were transplanted subcutaneously into immunodeficient, nonobese, diabetic/severe combined immunodeficiency gamma-c mice; 11 successfully engrafted. All successfully transplanted HCCs were Edmondson grade III or IV. HCC PDX tumors retained the histopathologic, transcriptomic, and genomic characteristics of the original HCC biopsies over 6 generations of retransplantation. These characteristics included Edmondson grade, expression of tumor markers, tumor gene signature, tumor-associated mutations, and copy number alterations. Conclusion: PDX mouse models can be established from undifferentiated HCCs, with an overall success rate of approximately 20%. The transplanted tumors represent the entire spectrum of the molecular landscape of HCCs and preserve the characteristics of the originating tumors through serial passaging. HCC PDX models are a promising tool for preclinical personalized drug development.

PipeIT: A Singularity Container for Molecular Diagnostic Somatic Variant Calling on the Ion Torrent Next-Generation Sequencing Platform.

The accurate identification of somatic mutations has become a pivotal component of tumor profiling and precision medicine. In molecular diagnostics laboratories, somatic mutation analyses on the Ion Torrent sequencing platform are typically performed on the Ion Reporter platform, which requires extensive manual review of the results and lacks optimized analysis workflows for custom targeted sequencing panels. Alternative solutions that involve custom bioinformatics pipelines involve the sequential execution of software tools with numerous parameters, leading to poor reproducibility and portability. We describe PipeIT, a stand-alone Singularity container of a somatic mutation calling and filtering pipeline for matched tumor-normal Ion Torrent sequencing data. PipeIT is able to identify pathogenic variants in BRAF, KRAS, PIK3CA, CTNNB1, TP53, and other cancer genes that the clinical-grade Oncomine workflow identified. In addition, PipeIT analysis of tumor-normal paired data generated on a custom targeted sequencing panel achieved 100% positive predictive value and 99% sensitivity compared with the 68% to 80% positive predictive value and 92% to 96% sensitivity using the default tumor-normal paired Ion Reporter workflow, substantially reducing the need for manual curation of the results. PipeIT can be rapidly deployed to and ensures reproducible results in any laboratory and can be executed with a single command with minimal input files from the users.

A stitch in time saves nine: external quality assessment rounds demonstrate improved quality of biomarker analysis in lung cancer.

Biomarker analysis has become routine practice in the treatment of non-small cell lung cancer (NSCLC). To ensure high quality testing, participation to external quality assessment (EQA) schemes is essential. This article provides a longitudinal overview of the EQA performance for EGFR, ALK, and ROS1 analyses in NSCLC between 2012 and 2015. The four scheme years were organized by the European Society of Pathology according to the ISO 17043 standard. Participants were asked to analyze the provided tissue using their routine procedures. Analysis scores improved for individual laboratories upon participation to more EQA schemes, except for ROS1 immunohistochemistry (IHC). For EGFR analysis, scheme error rates were 18.8%, 14.1% and 7.5% in 2013, 2014 and 2015 respectively. For ALK testing, error rates decreased between 2012 and 2015 by 5.2%, 3.2% and 11.8% for the fluorescence in situ hybridization (FISH), FISH digital, and IHC subschemes, respectively. In contrast, for ROS1 error rates increased between 2014 and 2015 for FISH and IHC by 3.2% and 9.3%. Technical failures decreased over the years for all three markers. Results show that EQA contributes to an ameliorated performance for most predictive biomarkers in NSCLC. Room for improvement is still present, especially for ROS1 analysis.

High expression of HOXA13 correlates with poorly differentiated hepatocellular carcinomas and modulates sorafenib response in in vitro models.

Hepatocellular carcinoma (HCC) represents the fifth and ninth cause of mortality among male and female cancer patients, respectively and typically arises on a background of a cirrhotic liver. HCC develops in a multi-step process, often encompassing chronic liver injury, steatosis and cirrhosis eventually leading to the malignant transformation of hepatocytes. Aberrant expression of the class I homeobox gene family (HOX), a group of genes crucial in embryogenesis, has been reported in a variety of malignancies including solid tumors. Among HOX genes, HOXA13 is most overexpressed in HCC and is known to be directly regulated by the long non-coding RNA HOTTIP. In this study, taking advantage of a tissue microarray containing 305 tissue specimens, we found that HOXA13 protein expression increased monotonically from normal liver to cirrhotic liver to HCC and that HOXA13-positive HCCs were preferentially poorly differentiated and had fewer E-cadherin-positive cells. In two independent cohorts, patients with HOXA13-positive HCC had worse overall survival than those with HOXA13-negative HCC. Using HOXA13 immunohistochemistry and HOTTIP RNA in situ hybridization on consecutive sections of 16 resected HCCs, we demonstrated that HOXA13 and HOTTIP were expressed in the same neoplastic hepatocyte populations. Stable overexpression of HOXA13 in liver cancer cell lines resulted in increased colony formation on soft agar and migration potential as well as reduced sensitivity to sorafenib in vitro. Our results provide compelling evidence of a role for HOXA13 in HCC development and highlight for the first time its ability to modulate response to sorafenib.

Vascular endothelial growth factor A amplification in colorectal cancer is associated with reduced M1 and M2 macrophages and diminished PD-1-expressing lymphocytes.

VEGFA is an angiogenic factor secreted by tumors, in particular those with VEGFA amplification, as well as by macrophages and lymphocytes in the tumor microenvironment. Here we sought to define the presence of M1/M2 macrophages, PD-1-positive lymphocytes and PD-L1 tumoral and stromal expression in colorectal cancers harboring VEGFA amplification or chromosome 6 polysomy. 38 CRCs of which 13 harbored VEGFA amplification, 6 with Chr6 polysomy and 19 with neutral VEGFA copy number were assessed by immunohistochemistry for CD68 (marker for M1/M2 macrophages), CD163 (M2 macrophages), programmed death 1(PD-1)- tumor infiltrating and stromal lymphocytes as well as tumoral and stromal PD-1 ligand (PD-L1) expression. CRCs with VEGFA amplification or Chr6 polysomy were associated with decreased M1/M2 macrophages, reduced PD-1-expressing lymphocyte infiltration, as well as reduced stromal expression of PD-L1 at the tumor front. Compared to intermediate-grade CRCs, high-grade CRCs were associated with increased M1/M2 macrophages and increased tumoral expression of PD-L1. Our results suggest that VEGFA amplification or Chr6 polysomy is associated with an altered tumor immune microenvironment.

MAGE-A Antigens and Cancer Immunotherapy.

MAGE-A antigens are expressed in a variety of cancers of diverse histological origin and germinal cells. Due to their relatively high tumor specificity, they represent attractive targets for active specific and adoptive cancer immunotherapies. Here, we (i) review past and ongoing clinical studies targeting these antigens, (ii) analyze advantages and disadvantages of different therapeutic approaches, and (iii) discuss possible improvements in MAGE-A-specific immunotherapies.

The Interplay Between Neutrophils and CD8+ T Cells Improves Survival in Human Colorectal Cancer.

Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer. Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes, and functional features of colorectal cancer-infiltrating CD66b+ neutrophils and their crosstalk with CD8+ T cells.Experimental Design: CD66b+ and CD8+ cell infiltration was analyzed by IHC on a tissue microarray including >650 evaluable colorectal cancer samples. Phenotypic profiles of tissue-infiltrating and peripheral blood CD66b+ cells were evaluated by flow cytometry. CD66b+/CD8+ cells crosstalk was investigated by in vitro experiments.Results: CD66b+ cell infiltration in colorectal cancer is significantly associated with increased survival. Interestingly, neutrophils frequently colocalize with CD8+ T cells in colorectal cancer. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, coculture with peripheral blood or tumor-associated neutrophils (TAN) enhances CD8+ T-cell activation, proliferation, and cytokine release induced by suboptimal concentrations of anti-CD3 mAb. Moreover, under optimal activation conditions, CD8+ cell stimulation in the presence of CD66b+ cells results in increasing numbers of cells expressing CD45RO/CD62L "central memory" phenotype. Importantly, combined tumor infiltration by CD66b+ and CD8+ T lymphocytes is associated with significantly better prognosis, as compared with CD8+ T-cell infiltration alone.Conclusions: Neutrophils enhance the responsiveness of CD8+ T cells to T-cell receptor triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of colorectal cancer infiltration by CD8+ T cells, suggesting that they might effectively promote antitumor immunity. Clin Cancer Res; 23(14); 3847-58. ©2017 AACR.

Human Papillomavirus (HPV) Detection in Cytologic Specimens: Similarities and Differences of Available Methodology.

Accumulating evidence regarding the causative role of human papillomavirus (HPV) in a wide range of malignant and nonmalignant diseases highlights the importance of HPV testing. This study describes and discusses the efficacy and characteristics of 4 well-established and commercially available tests. Here, 181 cytologic specimens from cervical smears were analyzed using the HPV SIGN PQ (Diatech) and the Linear Array (Roche) method. Discrepant results were further studied with the Real Time High-Risk HPV (Abbott) method and the INNO-LiPA (Fujirebio) method. Of 181 cytologic specimens, 61 (34%) showed discrepant results. High-risk HPV was not detected in 9 cases by HPV SIGN PQ, in 16 cases by Linear Array, in 10 cases by Real Time High-Risk HPV, and in 6 cases by INNO-LiPA, respectively. Lack of DNA detection or problems in interpreting the result were seen in 9 cases with HPV SIGN PQ, 8 cases with Linear Array, 3 cases with Real Time High-Risk HPV, and 3 cases with INNO-LiPA, respectively. This study indicates that the choice of HPV detection method has a substantial influence on the HPV risk classification of tested PAP smears and clinical follow-up decisions.

Hands-On Experience: Accreditation of Pathology Laboratories according to ISO 15189.

Accreditation is a procedure by which an authoritative body gives formal recognition that an organization is competent to carry out specific tasks according to certain standards. Accreditation of pathology laboratories according to ISO 15189 is now becoming more and more a matter of course in continental Europe. This review describes some practical experience aspects with our own pathology laboratory accreditation according to ISO 15189, and outlines the advantages, addresses critical points, and discusses certain caveats of this process.

HMGA1 Expression in Human Hepatocellular Carcinoma Correlates with Poor Prognosis and Promotes Tumor Growth and Migration in in vitro Models.

BACKGROUND: HMGA1 is a non-histone nuclear protein that regulates cellular proliferation, invasion and apoptosis and is overexpressed in many carcinomas. In this study we sought to explore the expression of HMGA1 in HCCs and cirrhotic tissues, and its effect in in vitro models. METHODS: We evaluated HMGA1 expression using gene expression microarrays (59 HCCs, of which 37 were matched with their corresponding cirrhotic tissue and 5 normal liver donors) and tissue microarray (192 HCCs, 108 cirrhotic tissues and 79 normal liver samples). HMGA1 expression was correlated with clinicopathologic features and patient outcome. Four liver cancer cell lines with stable induced or knockdown expression of HMGA1 were characterized using in vitro assays, including proliferation, migration and anchorage-independent growth. RESULTS: HMGA1 expression increased monotonically from normal liver tissues to cirrhotic tissue to HCC (P<.01) and was associated with Edmondson grade (P<.01). Overall, 51% and 42% of HCCs and cirrhotic tissues expressed HMGA1, respectively. Patients with HMGA1-positive HCCs had earlier disease progression and worse overall survival. Forced expression of HMGA1 in liver cancer models resulted in increased cell growth and migration, and vice versa. Soft agar assay showed that forced expression of HMGA1 led to increased foci formation, suggesting an oncogenic role of HMGA1 in hepatocarcinogenesis. CONCLUSIONS: HMGA1 is frequently expressed in cirrhotic tissues and HCCs and its expression is associated with high Edmondson grade and worse prognosis in HCC. Our results suggest that HMGA1 may act as oncogenic driver of progression, implicating it in tumor growth and migration potential in liver carcinogenesis.

Hepatic Notch1 deletion predisposes to diabetes and steatosis via glucose-6-phosphatase and perilipin-5 upregulation.

Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH.