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Telomerase activation is the earliest event in hepatocellular carcinoma (HCC) development. Thus, we aimed to elucidate the role of telomere length maintenance during liver carcinogenesis.Telomere length was measured in the tumor and non-tumor liver tissues of 1,502 patients (978 with HCC) and integrated with TERT alterations and expression, as well as clinical and molecular (analyzed by genome, exome, targeted and/or RNA-sequencing) features of HCC. The preclinical efficacy of anti-TERT antisense oligonucleotides (ASO) was assessed in vitro in 26 cell lines and in vivo in a xenograft mouse model.Aging, liver fibrosis, male sex and excessive alcohol consumption were independent determinants of liver telomere attrition. HCC that developed in livers with long telomeres frequently had wild-type TERT with progenitor features and BAP1 mutations. In contrast, HCC that developed on livers with short telomeres were enriched in the non-proliferative HCC class and frequently had somatic TERT promoter mutations. In HCCs, telomere length is stabilized in a narrow biological range around 5.7 kb, similar to non-tumor livers, by various mechanisms that activate TERT expression. Long telomeres are characteristic of very aggressive HCCs, associated with the G3 transcriptomic subclass, TP53 alterations and poor prognosis. In HCC cell lines, TERT silencing with ASO was efficient in highly proliferative and poorly differentiated cells. Treatment for 3 to 16 weeks induced cell proliferation arrest in 12 cell lines through telomere shortening, DNA damage and activation of apoptosis. The therapeutic effect was also obtained in a xenograft mouse model.Telomere maintenance in HCC carcinogenesis is diverse, and is associated with tumor progression and aggressiveness. The efficacy of anti-TERT ASO treatment in cell lines revealed the oncogenic addiction to TERT in HCC, providing a preclinical rationale for anti-TERT ASO treatment in HCC clinical trials.Telomeres are repeated DNA sequences that protect chromosomes and naturally shorten in most adult cells because of the inactivation of the TERT gene, coding for the telomerase enzyme. Here we show that telomere attrition in the liver, modulated by aging, sex, fibrosis and alcohol, associates with specific clinical and molecular features of hepatocellular carcinoma, the most frequent primary liver cancer. We also show that liver cancer is dependent on TERT reactivation and telomere maintenance, which could be targeted through a novel therapeutic approach called antisense oligonucleotides.

Los cambios registrados en el clima, los patrones demográficos y el acceso a los recursos implican que en los próximos veinte años se producirá una transformación en la naturaleza de los riesgos y que éstos serán más graves que los del pasado. Las respuestas que se aportan en este contexto en evolución, muestran la pertinencia constante y el papel vital y decisivo que el Derecho del trabajo tiene, a través de la creación de oportunidades de empleo decente y la generación de ingresos, en los contextos de crisis en pos de la prevención, recuperación y desarrollo de la resiliencia.

CTNNB1 mutations activating s-catenin are frequent somatic events in hepatocellular carcinoma (HCC) and adenoma (HCA), particularly associated with a risk of malignant transformation. We aimed to understand the relationship between CTNNB1 mutation types, tumor phenotype, and level of s-catenin activation in the malignant transformation. To this purpose, CTNNB1 mutation spectrum was analyzed in 220 HCAs, 373 HCCs, and 17 borderline HCA/HCC lesions. s-catenin activation level was assessed in tumors by quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry (IHC), in cellulo by TOP-Flash assay. Overall, s-catenin activity was higher in malignant mutated tumors, compared to adenomas, and this was related to different spectrum of CTNNB1 mutations in HCCs and HCAs. In benign tumors, we defined three levels of s-catenin activation related to specific mutations: (1) S45, K335, and N387 mutations led to weak activation; (2) T41 mutations were related to moderate activity; and (3) highly active mutations included exon 3 deletions and amino acid substitutions within the s-TRCP binding site (D32-S37). Accordingly, in vitro, K335I and N387K mutants showed a lower activity than S33C. Tumors with highly active mutations demonstrated strong/homogeneous glutamine synthase (GS) staining and were associated with malignancy. In contrast, weak mutants demonstrated heterogeneous pattern of GS staining and were more frequent in HCAs except for the S45 mutants identified similarly in 20% of mutated HCAs and HCCs; however, in most of the HCCs, the weak S45 mutant alleles were duplicated, resulting in a final high s-catenin activity. Conclusion: In conclusion, high s-catenin activity driven by specific CTNNB1 mutations and S45 allele duplication is associated with malignant transformation. Consequently, HCAs with S45 and all high/moderate mutants should be identified with precise IHC criteria or mutation screening. (Hepatology 2016)

Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus. Additional risk factors remain to be identified, particularly in patients who develop HCC without cirrhosis. We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC.

SummaryHepatocellular adenomas (HCA) are benign liver tumors predominantly developed in women using oral contraceptives. Here, exome sequencing identified recurrent somatic FRK mutations that induce constitutive kinase activity, STAT3 activation, and cell proliferation sensitive to Src inhibitors. We also found uncommon recurrent mutations activating JAK1, gp130, or β-catenin. Chromosome copy number and methylation profiling revealed patterns that correlated with specific gene mutations and tumor phenotypes. Finally, integrative analysis of HCAs transformed to hepatocellular carcinoma revealed β-catenin mutation as an early alteration and TERT promoter mutations as associated with the last step of the adenoma-carcinoma transition. In conclusion, we identified the genomic diversity in benign hepatocyte proliferation, several therapeutic targets, and the key genomic determinants of the adenoma-carcinoma transformation sequence.

Adeno-associated virus type 2 (AAV2) is a defective DNA virus that was previously considered to be non-pathogenic. We identified somatic AAV2 integration in a subset of 11 hepatocellular carcinomas (HCC) that mainly developed in normal liver without known etiology through recurrent insertional mutagenesis in cancer driver genes such as telomerase reverse transcriptase (TERT), cyclin A2 (CCNA2), cyclin E1 (CCNE1), tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10), and lysine (K)-specific methyltransferase 2B (KMT2B).

Background: Hepatocellular carcinomas (HCC) are related to various etiologies including alcohol intake, obesity, hemochromatosis, hepatitis B and hepatitis C virus infection. Additional risk factors remain to be identified particularly in HCC developed without cirrhosis. We identified a fragment of Adeno-Associated Virus type 2 (AAV2, a defective DNA virus considered as non-pathogenic) inserted in an HCC genome and this result prompted us to screen a large series of HCC samples. Methods: We screened 150 HCC and matched non-tumor liver samples for AAV2 sequences using PCR and next generation sequencing. Expression of the genes targeted by viral integration was quantified and the functional consequences of AAV2 integration within TERT promoter was analyzed in 3 HCC cell lines using luciferase assay Results: Among the 150 tumors, we identified 7 cases (5%) with a clonal AAV2 insertion whereas no clonal insertion was found in the corresponding non-tumor liver tissues. In the first HCC tumor, we identified a 208 base pairs insertion of an AAV2 fragment within the TERT (coding for the telomerase reverse transcriptase) promoter. In HCC cell lines, we demonstrated that this AAV2 insertion resulted in an increased TERT promoter activity. We next identified 6 additional clonal somatic integrations of AAV2, all occurred within known cancer driver genes, CCNA2 (4 cases), CCNE1 or TNFSF10 leading to over-expression of the targeted gene without generating chimeric viral-endogeneous functional fusion transcripts. In CCNA2 (coding for cyclin A2), the four integration sites were identified within intron 2 and AAV2 insertion ranged from 219 to 1,975bp occurring in both orientations. In CCNE1 (coding for cyclin E1), we identified a 368bp AAV2 integration within intron 4. In the remaining case, AAV2 integration occurred in the 3′ UTR of TNSFS10 (coding for the receptor TRAIL), 244 bp after the stop codon. In 6 out of the 7 tumors, the inserted AAV2 included the 3′inverse tandem region. Strikingly, almost all the tumors were developed on non-fibrotic liver (6 out of 7 cases, P Conclusion: Infection by AAV2, that is very frequent the general population (around 30 to 60% of individuals), is involved in the pathogenesis of rare human HCC by recurrent somatic integration in cancer driver genes challenging its safety as a vector for gene therapy. Citation Format: Shalini Datta, Jean-Charles Nault, Andrea Franconi, Sandrine Imbeaud, Maxime Mallet, Gabrielle Couchy, Eric Letouze, Camilla Pilati, Benjamin Verret, Jean-Frédéric Blanc, Charles Balabaud, Julien Calderaro, Alexis Laurent, Mélanie Letexier, Paulette Bioulac-Sage, Fabien Calvo, Jessica Zucman-Rossi. Adeno-associated virus 2 (AAV2) induces recurrent insertional mutagenesis in human hepatocellular carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 919. doi:10.1158/1538-7445.AM2015-919