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<0.05) without known risk factor (5 out of 7, P<0.05), thus suggesting a particular pathogenic role of AAV2 in this subset of patients.
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