Almost all cervical cancers are caused by human papillomaviruses (HPVs). In most cases, HPV DNA is integrated into the human genome. We found that tumor-specific, HPV-human DNA junctions are detectable in serum cell-free DNA of a fraction of cervical cancer patients at the time of initial treatment and/or at 6 months following treatment. Retrospective analysis revealed these junctions were more frequently detectable in women in whom the cancer later recurred. We also found that cervical cancers caused by HPV types outside of phylogenetic clade α9 had a higher recurrence frequency than those caused by α9 types in both our study and The Cancer Genome Atlas cervical cancer database, despite the higher prevalence ofα9 types, including HPV16, in cervical cancer. Thus, HPV-human DNA junction detection in serum cell-free DNA and HPV type determination in tumor tissue may help predict recurrence risk. Screening serum cell-free DNA for junctions may also offer an unambiguous non-invasive means to monitor absence of recurrence following treatment.

Viruses are a leading cause of human morbidity and mortality. Certain viruses, including human papillomaviruses (HPVs), play a significant role in the etiology of cancer. Detection of viral DNA insertions in the human genome from next generation sequencing data defines viral associations with cancer and other diseases, identifies impacted organs and tissues, provides insights into disease mechanisms and has the potential to enhance clinical evaluations. In this study, we developed VirusIntegrationFinder (CTAT-VIF), a tool for surveying human genome insertions of various human viruses using both DNA and RNA sequencing data. We applied CTAT-VIF to analyze a dataset of over 30,000 tumor and normal samples, as well as more than 1,000 cancer cell lines. This effort resulted in the compilation of a catalog of over 30,0000 virus-human DNA or RNA junctions at more than 20,000 insertion loci and reassessed viral cancer-insertion hotspots across the human genome. Furthermore, we characterized the functional impacts of insertions with respect to human copy number alterations, effects on the expression of flanking human genes, and the identification of potentially oncogenic chimeric human and human/virus fusion transcripts at insertion loci. In addition to confirming known viral associations with specific tumor types, our study revealed both shared and virus-specific insertion hotspots in addition to variable functional impacts based on virus type. Besides some rare events of interest, we also found evidence for sequencing contamination, which underscores the need for vigilance when studying viral content or genome integrations. ### Competing Interest Statement The authors have declared no competing interest.

Most human papillomavirus (HPV)-associated cancers harbor viral DNA integrated into the human genome as extrachromosomal circles, intrachromosomal segments, or both. Distinguishing intrachromosomal from identical-sequence extrachromosomal DNA (ecDNA) by sequencing alone is challenging, and the architecture of large-scale HPV-human DNA structures remains incompletely understood. To address this, we applied complementary genomic tools, spanning single-nucleotide to megabase resolution, to the HPV16-positive oropharyngeal cancer-cell line UM-SCC-47. These revealed that an initial integration event formed a 23 kb extrachromosomal heterocatemer circle comprising 7.5 kb of HPV16 DNA and 16 kb of the human TP63 gene. Subsequent genomic rearrangements generated heterocatemer tandem arrays extending to 0.6 megabases, plus additional large-scale rearrangements involving the HPV- TP63 structures, as revealed by long-read DNA sequencing and optical genome mapping. Fluorescent in situ Hybridization (FISH) showed that the heterocatemers were intrachromosomally localized at chromosome 3 at the TP63 locus in 100% of the cells. Long-read RNA sequencing further showed that these intrachromosomal templates produced spliced, polyadenylated transcripts. A subset of cells also harbored HPV16 ecDNA derived from the intrachromosomal HPV- TP63 DNAs. These findings define previously unrecognized higher-order architecture of integrated HPV DNA and highlight the power of FISH for distinguishing intrachromosomal from extrachromosomal DNA structures. GRAPHICAL ABSTRACT:

HPV infections are associated with a fraction of vulvar cancers. Through hybridization capture and DNA sequencing, HPV DNA was detected in five of thirteen vulvar cancers. HPV16 DNA was integrated into human DNA in three of the five. The insertions were in introns of human NCKAP1, C5orf67, and LRP1B. Integrations in NCKAP1 and C5orf67 were flanked by short direct repeats in the human DNA, consistent with HPV DNA insertions at sites of abortive, staggered, endonucleolytic incisions. The insertion in C5orf67 was present as a 36 kbp, human-HPV-hetero-catemeric DNA as either an extrachromosomal circle or a tandem repeat within the human genome. The human circularization/repeat junction was defined at single nucleotide resolution. The integrated viral DNA segments all retained an intact upstream regulatory region and the adjacent viral E6 and E7 oncogenes. RNA sequencing revealed that the only HPV genes consistently transcribed from the integrated viral DNAs were E7 and E6*I. The other two HPV DNA+ tumors had coinfections, but no evidence for integration. HPV-positive and HPV-negative vulvar cancers exhibited contrasting human, global gene expression patterns partially overlapping with previously observed differences between HPV-positive and HPV-negative cervical and oropharyngeal cancers. A substantial fraction of the differentially expressed genes involved immune system function. Thus, transcription and HPV DNA integration in vulvar cancers resemble those in other HPV-positive cancers. This study emphasizes the power of hybridization capture coupled with DNA and RNA sequencing to identify a broad spectrum of HPV types, determine human genome integration status of viral DNAs, and elucidate their structures.

Supplementary Table 3 from Insertional Mutagenesis in Mice Deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 Reveals Cancer Gene Interactions and Correlations with Tumor Phenotypes

Supplementary Figure Legends 1-3 from Insertional Mutagenesis in Mice Deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 Reveals Cancer Gene Interactions and Correlations with Tumor Phenotypes

Identifying tumor-specific antigens across different types of malignancies and HLA haplotypes remains challenging. Recently, the expression of tumor-specific Human Endogenous Retroelements emerged as a potential new class of cytotoxic T-cell response mediators (1). HLA-F, a non-classic HLA-I molecule, has been hypothesized to present peptides to T-cells and to regulate immunity through interactions with distinct NK-cell receptors (2). HLA-F low genetic diversity and limited peptide repertoire represent an unexplored avenue in cancer immunotherapy. HLA-F-bound peptides were eluted from five patient-derived T-cell lymphoma cell lines and total T-cells purified from ten healthy donor PBMCs; Retroelement peptides were identified in the HLA-F ligandome. To quantify and validate their expression, RNA-sequencing data from the same samples were screened using ERVmap database (3). Lastly, to characterize their expression, we incorporate genome-wide characterization of DNA methylation patterns. We identified a set of retroelement peptides bound to HLA-F shared by patient-derived T-cell lymphoma cell lines but not total T-cells isolated from healthy donor PBMCs, suggesting a potential role of HLA-F in cancer-immune responses. Among those we characterized four tumor-specific retroelement peptides, validated by the presence of their RNA transcripts as well as by DNA integration and chromosomal location. Tumor-specific HLA-F-bound retroelement peptides hold great promise for the discovery of previously unknown tumor-specific markers and therapeutic targets. More importantly, the characterization of their immunogenicity may yield insights broadly applicable to a greater number of patients due to HLA-F monomorphism.

Supplementary Figure Legends 1-3 from Insertional Mutagenesis in Mice Deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 Reveals Cancer Gene Interactions and Correlations with Tumor Phenotypes

Developmental etiologies causing complex congenital aortic root abnormalities are unknown. Here we show that deletion of Sox17 in aortic root endothelium in mice causes underdeveloped aortic root leading to a bicuspid aortic valve due to the absence of non-coronary leaflet and mispositioned left coronary ostium. The respective defects are associated with reduced proliferation of non-coronary leaflet mesenchyme and aortic root smooth muscle derived from the second heart field cardiomyocytes. Mechanistically, SOX17 occupies a Pdgfb transcriptional enhancer to promote its transcription and Sox17 deletion inhibits the endothelial Pdgfb transcription and PDGFB growth signaling to the non-coronary leaflet mesenchyme. Restoration of PDGFB in aortic root endothelium rescues the non-coronary leaflet and left coronary ostium defects in Sox17 nulls. These data support a SOX17-PDGFB axis underlying aortic root development that is critical for aortic valve and coronary ostium patterning, thereby informing a potential shared disease mechanism for concurrent anomalous aortic valve and coronary arteries.

Cervical carcinogenesis, the second leading cause of cancer death in women worldwide, is caused by multiple types of human papillomaviruses (HPVs). To investigate a possible role for HPV in a cervical carcinoma that was HPV-negative by PCR testing, we performed HPV DNA hybridization capture plus massively parallel sequencing. This detected a subgenomic, URR-E6-E7-E1 segment of HPV70 DNA, a type not generally associated with cervical cancer, inserted in an intron of the B-cell lymphoma/leukemia 11B (BCL11B) gene in the human genome. Long range DNA sequencing confirmed the virus and flanking BCL11B DNA structures including both insertion junctions. Global transcriptomic analysis detected multiple, alternatively spliced, HPV70-BCL11B, fusion transcripts with fused open reading frames. The insertion and fusion transcripts were present in an intraepithelial precursor phase of tumorigenesis. These results suggest oncogenicity of HPV70, identify novel BCL11B variants with potential oncogenic implications, and underscore the advantages of thorough genomic analyses to elucidate insights into HPV-associated tumorigenesis.

Abstract Purpose/Objectives: Integration of human papillomavirus (HPV) DNA into the human genome occurs in a fraction of viral infections and is a near ubiquitous factor in cervical tumorigenesis. While use of next-generation sequencing (NGS) methods has significantly advanced our understanding of HPV DNA integration and its consequences, short read approaches may yield false positives or misinterpretation of associated genomic structural variants. With the goal of resolving genomic rearrangements, we applied both HPV16 hybridization capture-NGS and long read Nanopore technology to the well-studied CaSki cervical carcinoma cell line that has many sites of HPV16 DNA integration. Methods: CaSki genomic DNA was sheared, ligated to Illumina adaptors, hybridized to HPV16 capture probes (Roche Nimblegen SeqCap EZ System), and sequenced by Illumina HiSeq 2500, paired end, 300 bp mode. Reads were aligned to a custom human (GRCh37/hg19) plus HPV16 reference genome using BWA mem, and junction fragments were computationally identified using Delly and SplazerS. For long read analysis, isolated CaSki DNA was sheared to 20 kb. Genomic libraries were prepared and sequenced on an Oxford Nanopore MinION Mk1b device (ONT) using standard 48 hour scripts. Base calling and fastq file extraction were performed with Albacore v2.0. Reads were aligned using Ngmlr, and structural variants called using Sniffles. Results: For Illumina analysis, 34 million demultiplexed read pairs were obtained with average, HPV genome coverage depth of 2.8*106. 47 previously identified, HPV-human junctions were verified along with 193 novel junctions. Use of PCR has validated 4 of 6 novel junctions tested to date. Multiple junctions were noted to be clustered at some chromosomal locations, with 30 (13.5%) called less than 1kb apart on the human genomic side. For long read analysis, ~150K reads were obtained. Mean read length was 16K with genome and HPV16 coverage of 0.60X and 220X, respectively. Integrations were detected on chromosomes 2, 5, 7, 10, 19, 20, and X, and all consisted of >2 tandem viral genomes. Discussion: Our molecular approaches generated unprecedented long-range analyses of HPV DNA integration sites in the CaSki cervical carcinoma cell line. HPV DNA was often present in tandem arrays. We interpret the clusters of nearby HPV-human junctions as potential evidence of frequent, ongoing, subclonal, genomic rearrangements at these sites. Combining deep-sequencing and long-range methodologies allowed important insights, and we are currently analyzing clinical cancer samples using them. Citation Format: Anne Van Arsdale, Jack Lenz, Nicole Patterson, Elaine Maggi, Dennis Y. Kuo, Cristina Montagna. Elucidation of integrated HPV DNA structure in a cervical carcinoma cell line by combined high resolution and long range sequencing analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-334.

Objectives: Integration of human papillomavirus (HPV) DNA into the human genome is a secondary consequence of persistent infection that is thought to be a ubiquitous factor in cervical tumorigenesis. The objectives of this study are identification of HPV DNA integration events using next-generation sequencing (NGS) techniques early in the disease process and assessment of the dynamics on temporal progression of cervical dysplasia.

Retroviruses comprise over 8% of the human genome (1, 2). Human endogenous retroviruses (HERVs) exist as DNA remnants of infections that occurred in germ lineage cells of our ancestors. Most of this viral DNA is mutated, often including various large disruptions, but some components are intact or otherwise functional. What viral components exist in human genomes, and what gene products do they encode that might interact with the nonviral parts of us? When did they arrive in the genomes of our ancestors, and are they still active today? Does an intact, infectious, retroviral provirus lurk in the genomes of some of us? Wildschutte et al. (3) shed new light on these issues by characterizing the most recently acquired proviruses in human genomes, a subset of the virus HERV-K called HML-2 (for human mouse mammary tumor virus like-2), which are present at various allele frequencies <1 in the human population, i.e., not in everyone.

Aimed at refining the safety profile of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications we investigated the impact of chromatin insulators (CI) on vector-mediated genotoxicity. Specifically, we studied four recently identified CI whose function is mediated by CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates, and cloned these CI in the LTRs of a SIN. LV with a strong enhancer/promoter in internal position (CI. SIN. LV). We took advantage of two sensitive in vivo genotoxicity assays based on the systemic injection of LVs in newborn tumor-prone Cdkn2a−/− and Cdkn2a+/− mice, that allow to measure vector-induced genotoxicity as accelerated tumor onset proportional to the genotoxic potential of the tested LV. CI. SIN. LVs displayed slightly not statistically significant improvement in the median survival time vs. the uninsulated SIN. LV counterpart (ranging from 193.5 to 214 days vs. 186 days, respectively) in Cdkn2a−/− mice. In Cdkn2a+/− mice, two insulated vectors significantly improved the median survival time, which resulted non-statistically different from Mock mice (450 and 511 vs. 505.5 days respectively), while the other two CI. SIN. LVs studied resulted to be still slightly genotoxic (median survival time: 412 and 429.5 days). To gain more insights on the safety profile of these LVs we retrieved and analyzed the vector integration sites (IS) (n>14000 IS) and identified common integration sites (CIS) in the murine tumors generated in our experimental framework and in both murine models. In Cdkn2a−/− mice, uninsulated SIN. LV-induced tumors harbored activating integrations targeting Map3k8 oncogene, while tumors obtained in mice treated with two out of four different insulated LVs significantly reduced the frequency of tumors with Map3k8-activating insertions. The reduced targeting frequency of Map3k8 was accompanied by a skewing of integrations inactivating Pten, Rasa1 or other tumor-suppressors, an escape genotoxicity mechanism on which insulators cannot act. In Cdkn2a−/+ mice we identified different predominant CIS genes targeted by the different insulated vectors. These data show that heterozygous Cdkn2a−/+ mice allow discriminating between more subtle shades of genotoxicity of the different vector designs and are therefore instrumental to understand the different molecular mechanisms of insertional mutagenesis and ways to avoid them. Interestingly by comparing the results from both in vivo assays we observed that one CI displayed superior safety profile in terms of significant improvement in the median survival time and/or in terms of reduced oncogenic CIS identified. In summary we validated new human-origin insulator elements able to block SIN. LV genotoxicity in vivo. Overall, these data highlight the importance of stringent in vivo genotoxicity testing of improved vector versions and support the use of CI for future gene therapy applications.

Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications. By taking advantage of an in vivo genotoxicity assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten. Here by using this stringent in vivo genotoxicity assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates. Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape genotoxicity mechanism on which CI cannot act. These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo. The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.