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Oncogene activation leads to replication stress and promotes genomic instability. Here we combine optical mapping and whole-genome sequencing (WGS) to explore in depth the nature of structural variants (SV) induced by replication stress in cyclin-activated hepatocellular carcinomas (CCN-HCC). In addition to classical tandem duplications, CCN-HCC displayed frequent intra-chromosomal and interchromosomal templated insertion cycles (TIC), likely resulting from template switching events. Template switching preferentially involves active topologically associated domains that are proximal to one another within the 3D genome. Template sizes depend on the type of cyclin activation and are coordinated within each TIC. Replication stress induced continuous accumulation of SVs during CCN-HCC progression, fostering the acquisition of new driver alterations and large-scale copy-number changes at TIC borders. Together, this analysis sheds light on the mechanisms, dynamics, and consequences of SV accumulation in tumors with oncogene-induced replication stress. Significance: Optical mapping and whole-genome sequencing integration unravels a unique signature of replication stress–induced structural variants that drive genomic evolution and the acquisition of driver events in CCN-HCC.

Mitazalimab is a human CD40 agonistic IgG1antibody being developed as cancer immunotherapy. Targeting CD40 kickstarts the cancer immunity cycle by licensing DCs leading to tumor-specific T cell priming and activation. Furthermore, in PDAC, CD40 agonism activates myeloid cells and promotes the degradation of the desmoplastic tumor stroma, improving the influx of T cells and chemotherapeutic agents into the tumor. Mitazalimab has shown to be safe and well tolerated (at doses up to 1200 μg/kg) with signs of clinical activity in solid tumors in a Phase I study (NCT02829099). Most drug related adverse events (AE) were grade 1 or 2. OPTIMIZE-1 (NCT04888312) is a phase 1b/2, open-label, multicenter study designed to evaluate safety, tolerability, and efficacy of mitazalimab in combination with mFOLFIRINOX in adults diagnosed with previously untreated mPDAC. The objective of the first (phase 1b) part of the study was to determine the RP2D of mitazalimab + mFOLFIRINOX. Mitazalimab was escalated from 450 µg/kg to 900 µg/kg following a Bayesian optimal interval design with at least 3 patients enrolled per dose level. In the first 21-day treatment cycle (Dose Limiting Toxicity assessment period), mitazalimab is administered intravenously on day 1 and 10 and mFOLFIRINOX starts on day 8. In the second and subsequent cycles, treatment follows a 14-day cycle schedule where mitazalimab is administered 2 days after mFOLFIRINOX. In part 2 of the study (phase 2), mitazalimab at the RP2D will be administered in combination with mFOLFIRINOX. Primary endpoint is RECIST-defined overall response rate. Progression-free survival and overall survival will be assessed as secondary endpoints. We report data from phase 1b (dose escalation) part of this study. As of March 9, 2022, 11 patients were treated with mitazalimab: 5 at 450 µg/kg and 6 at 900 µg/kg mitazalimab doses. One patient in the 900 µg/kg dose cohort withdrew from the trial for administrative reasons after the first mitazalimab infusion, prior to receiving mFOLFIRINOX and was not included in RP2D determination. Key baseline characteristics included: 7 female, 4 male; median age 63 (range 57-70); ECOG 0-1; median time since mPDAC diagnosis, 25 days. Mitazalimab related AEs were reported in 9/11 patients. Treatment related AEs occurring in >1 patient were fever (60%), muscle pain (50%) and fatigue (20%). At the 450 µg/kg dose, all mitazalimab related AEs were grade 1-2. At the 900 µg/kg dose, 4 patients (67%) experienced grade 1-2 mitazalimab related AEs. One patient in the 900 µg/kg dose experienced mitazalimab related grade 3 fatigue and grade 3 headache that led to treatment discontinuation after the first cycle. There were no mitazalimab related grade 4 or 5 AEs. 1/10 patients required mFOLFIRINOX dose reduction and, at the cutoff date, the range of treatment length was 1-14 weeks. Mitazalimab combined with mFOLFIRINOX is safe and well tolerated. The 900 µg/kg dose of mitazalimab was selected as the RP2D. Phase 2 of the OPTIMIZE-1 trial is currently enrolling patients. Citation Format: Jean-Luc Van Laethem, Ivan Borbath, Hans Prenen, Aurélien Lambert, Karen Geboes, Jean-Frédéric Blanc, Yago Pico de Coaña, Karin Enell-Smith, Lena Schultz, Karin Nordbladh, Peter Ellmark, Sumeet Ambarkhane, Malin Carlsson, Philippe Cassier. Mitazalimab (CD40 agonist) in combination with mFOLFIRINOX in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC): Safety data and recommended dose for phase 2 (RP2D) from OPTIMIZE-1, a phase 1b/2 study [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A018.

Oncogene activation leads to replication stress and promotes genomic instability. Here we combine optical mapping and whole genome sequencing to explore in-depth the nature of structural variants (SVs) induced by replication stress in cyclin-activated hepatocellular carcinomas (CCN-HCC). In addition to classical tandem duplications, CCN-HCC display frequent intra- and inter-chromosomal templated insertion cycles (TIC) likely resulting from template switching events. Template switching preferentially involves active topologically associated domains that are close in the 3D genome organization. Template sizes depend on the type of cyclin activation and are coordinated within each TIC. Replication stress induces continuous accumulation of SVs during CCN-HCC progression, fostering the acquisition of new driver alterations and large-scale copy-number changes at TIC borders. Together, this analysis sheds light on the mechanisms, dynamics and consequences of SV accumulation in tumors with oncogene-induced replication stress. Citation Format: Quentin Bayard, Pierre Cordier, Camille Péneau, Sandrine Imbeaud, Theo Z. Hirsch, Victor Renault, Jean-Charles Nault, Jean-Frédéric Blanc, Julien Calderaro, Chantal Desdouets, Jessica Zucman-Rossi, Eric Letouzé. Structure, dynamics and consequences of replication stress-induced structural variants in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB545.

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

Background and Aims: Hepatocellular carcinoma (HCC) is a heterogeneous disease mainly developing in chronic liver disease and caused be a variety of etiologies. Hepatocarcinogenesis is a multi-step process in which pre-cancerous lesions can ultimately transform into advanced liver cancer. Genomic analyses promise to unravel the mutagenic processes in order to identify molecular profiles and to optimize personalized patient care. Methods: We performed exome sequences of 243 liver tumors from European patients (France, Italy and Spain), associated with cirrhosis (F4, n = 118), fibrosis (F2+F3, n = 46), or non-fibrotic livers (F0+F1, n = 79). Cirrhosis-associated tumors presented different stages along HCC progression: 7 dysplastic macronodules, 7 early, 17 small and progressed, 58 classic, and 29 poor-prognosis HCC. Tumors were categorized by varying risk factors: Significant alcohol intake (41%), HCV (26%), non-alcoholic steatohepatitis (18%), HBV (14%), hemochromatosis (7%), no known etiology (11%). Results: We identified 8 mutational signatures of which 6 (Signatures 1A, 1B, 4, 5, 6, and 16) were previously validated in a pan-cancer analysis, while 2 (Signatures 23 and 24) are novel. Hierarchical clustering, based on mutational signatures, revealed 6 groups (MSig1 to 6) and 4 singletons which were significantly associated with specific risk factors, mainly combined alcohol/tobacco consumption, and aflatoxin B1. Next, we identified 161 putative driver genes associated with 11 recurrent pathways. Associations of mutations defined 3 groups of genes centered on CTNNB1 (alcohol), TP53 (HBV), and AXIN1. Analyses according to tumor stage progression revealed TERT promoter mutation as an early event whereas FGF/CCND1 amplification, TP53 and CDKN2A alterations, appeared at more advanced stages in aggressive tumors. Altogether, 28% of patients harbored at least one damaging alteration potentially targetable by an FDA-approved drug and 86% by a drug which has been studied in phase I to phase III clinical trials. Conclusions: In conclusion, our study identified relationships between environmental exposures and mutational patterns in HCC as well as the landscape of driver genes and pathways altered in different clinical stages and etiological backgrounds. For patient care, genomic alterations identified in targetable genes will be useful to determine HCC patients that could potentially benefit from targeted treatment in future clinical trials. Citation Format: Kornelius Schulze, Sandrine Imbeaud, Eric Letouzé, Ludmil B. Alexandrov, Julien Calderaro, Sandra Rebouissou, Gabrielle Couchy, Clément Meiller, Jayendra Shinde, Shalini Datta, Frederic Soysouvanh, Anna-Line Calatayud, Roser Pinyol, Laura Pelletier, Charles Balabaud, Alexis Laurent, Jean-Frederic Blanc, Viccenzo Mazzaferro, Fabien Calvo, Augusto Villanueva, Jean-Charles Nault, Paulette Bioulac-Sage, Michael R. Stratton, Josep M. Llovet, Jessica Zucman-Rossi. Exome sequencing of 243 liver tumors identifies new mutational signatures and potential therapeutic targets. [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 2973. doi:10.1158/1538-7445.AM2015-2973