medRxiv preprint doi: https://doi.org/10.1101/2020.11.25.20238477; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 1 HPV16 and HPV18 type-specific APOBEC3 and integration profiles in different 2 diagnostic categories of cervical samples 3 4 Sonja Lagström1,2,3, Alexander Hesselberg Løvestad4, Sinan Uğur Umu2, Ole Herman 5 Ambur4, Mari Nygård2, Trine B. Rounge2,6,#,*, Irene Kraus Christiansen1,7,#,*, 6 7 Author affiliations: 8 1 Department of Microbiology and Infection Control, Akershus University Hospital, 9 Lørenskog, Norway 10 2 Department of Research, Cancer Registry of Norway, Oslo, Norway 11 3 Institute of Clinical Medicine, University of Oslo, Oslo, Norway 12 4 Faculty of Health Sciences, OsloMet - Oslo Metropolitan University, Oslo, Norway 13 5 Norwegian Cervical Cancer Screening Programme, Cancer Registry of Norway, Oslo, 14 Norway 15 6 Department of Informatics, University of Oslo, Oslo, Norway 16 7 Department of Clinical Molecular Biology (EpiGen), Division of Medicine, Akershus 17 University Hospital and University of Oslo, Lørenskog, Norway 18 19 20 #Equal contribution 21 *Corresponding authors 22 E-mail addresses: [email protected] (TBR), 23 [email protected] (IKC) 24 NOTE: This preprint reports new research that has not been certified1 by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.11.25.20238477; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 25 Abstract 26 27 Human papillomavirus 16 and 18 are the most predominant types in cervical 28 cancer. Only a small fraction of HPV infections progress to cancer, indicating that genomic 29 factors, such as minor nucleotide variation caused by APOBEC3 and chromosomal 30 integration, contribute to the carcinogenesis. 31 We analysed minor nucleotide variants (MNVs) and integration in HPV16 and 32 HPV18 positive cervical samples with different morphology. Samples were sequenced 33 using an HPV whole genome sequencing protocol TaME-seq. A total of 80 HPV16 and 34 51 HPV18 positive cervical cell samples passed the sequencing depth criteria of 300× 35 reads, showing the following distribution: non-progressive disease (HPV16 n=21, HPV18 36 n=12); cervical intraepithelial neoplasia (CIN) grade 2 (HPV16 n=27, HPV18 n=9); 37 CIN3/adenocarcinoma in situ (AIS) (HPV16 n=27, HPV18 n=30); cervical cancer (HPV16 38 n=5). 39 Similar rates of MNVs in HPV16 and HPV18 samples were observed for most viral 40 genes but for HPV16, the non-coding region (NCR) showed a trend towards increasing 41 variation with increasing lesion severity. APOBEC3 signatures were observed in HPV16 42 lesions, while similar mutation patterns were not detected for HPV18. The proportion of 43 samples with integration was 13% for HPV16 and 59% for HPV18 positive samples, with 44 a noticeable portion located within or close to cancer-related genes. 45 46 2 medRxiv preprint doi: https://doi.org/10.1101/2020.11.25.20238477; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 47 Keywords 48 Human papillomavirus, minor nucleotide variation, APOBEC3, chromosomal integration, 49 viral genomic deletion 50 51 Abbreviations 52 AID activation-induced cytidine deaminase 53 AIS adenocarcinoma in situ 54 ASC-US atypical squamous cells of undetermined significance 55 CIN cervical intraepithelial neoplasia 56 dN/dS ratio of non-synonymous to synonymous substitutions 57 HPV human papillomavirus 58 LSIL low-grade squamous intraepithelial lesion 59 MNV minor nucleotide variant 60 NCR non-coding region 61 ncRNA non-coding RNA 62 NGS next-generation sequencing 63 SCC squamous cell carcinoma 64 URR upstream regulatory region 65 UTR untranslated region 66 3 medRxiv preprint doi: https://doi.org/10.1101/2020.11.25.20238477; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 67 1. Introduction 68 69 A persistent infection with one of the carcinogenic HPV genotypes is accepted as a 70 necessary cause of cervical cancer development [1]. Of the 12 carcinogenic types [2], 71 HPV16 and HPV18 are associated with about 70% of all cervical cancers [3]. HPV16 is 72 predominantly associated with squamous cell carcinomas (SCC), while HPV18 is more 73 often detected in adenocarcinomas [3], suggesting that these HPV types differ in their 74 target cell specificity [4]. Nevertheless, only a small fraction of HPV infections will persist 75 and progress to cancer [5], indicating that additional factors and genomic events are 76 necessary for the HPV-induced carcinogenic process. 77 78 The 7.9 kb double stranded HPV DNA genome consists of early region (E1, E2, E4-7) 79 genes, late region (L1, L2) genes and an upstream regulatory region (URR) [6]. To date, 80 more than 200 HPV genotypes have been identified, based on at least 10% difference 81 within the conserved L1 gene sequence [7]. HPV types harbouring minor genetic variation 82 are grouped into lineages (1–10% whole genome nucleotide difference) and sublineages 83 (0.5-1.0% difference) [8]. HPV evolve slowly partly since the HPV genome replication is 84 dependent on host cell high-fidelity polymerases [9]. However, recent studies have 85 revealed variability below the level of HPV sublineages, which may indicate intra-host viral 86 diversification and evolution [10-12]. 87 88 Viral genetic instability is caused by various mutagenic processes [13]. One of the 89 mechanisms suggested to cause minor nucleotide variants (MNVs) and impact HPV 90 mutational drift involves the anti-viral host-defence enzyme apolipoprotein B mRNA- 91 editing enzyme, catalytic polypeptide-like 3 (APOBEC3) protein [14]. APOBEC3 proteins 92 are cytidine deaminases causing deoxycytidine (C) to deoxythymidine (T) mutations 93 during viral genome synthesis. The mutations can lead to defects in viral genome 94 replication necessary for the viral life cycle [15]. APOBEC3 mutational signatures have 95 been found in HPV genomes in cervical pre-cancerous and cancer samples [10, 16, 17], 96 and has recently been associated with viral clearance [18]. Findings of hypovariability of 4 medRxiv preprint doi: https://doi.org/10.1101/2020.11.25.20238477; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 97 the E7 gene suggest negative selection opposite of APOBEC3-related editing and an 98 essential gene conservation for progression to cancer [19, 20]. 99 100 HPV integration into the host genome is regarded as a driving event in cervical 101 carcinogenesis and is observed in >80% of HPV-induced cancers [21]. Integrations 102 causing disruption or complete deletion of the E1 or E2 gene result in constitutive 103 expression of the viral E6 and E7 oncogenes [22], leading to inactivation of cell cycle 104 checkpoints and genomic instability [23]. Integration may also lead to disruption of host 105 genes, such as oncogenes or tumour-suppressor genes, modified expression of adjacent 106 genes, as well as other genomic alterations, which may promote HPV-induced 107 carcinogenesis [24-26]. In high-grade lesions and cancers, integrations in certain 108 chromosomal loci, including loci 3q28, 8q24.21 and 13q22.1, have been reported more 109 often than in other loci [27], suggesting selective growth advantages for cells with site- 110 specific integrations in e.g. important regulatory genes. Increasing integration frequencies 111 have been reported upon comparison of cervical precancerous and cancer lesions [28, 112 29]. 113 114 Recently, we developed a novel next-generation sequencing (NGS) strategy TaME-seq 115 for simultaneous analysis of HPV genomic variability and chromosomal integration [30]. 116 Employing the TaME-seq method, we have explored HPV16 and HPV18 genomic 117 variability and integration in HPV positive cervical samples with different morphologies. 118 Differences in HPV variability between the diagnostic categories may shed light on intra- 119 host viral genome dynamics and evolution processes in cervical carcinogenesis. In 120 addition, integration analysis will contribute to a better understanding of this event during 121 HPV-induced carcinogenesis. 122 5 medRxiv preprint doi: https://doi.org/10.1101/2020.11.25.20238477; this version posted November 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 123 2. Material and methods 124 125 2.1. Sample selection