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Oncogenic viruses: Lessons learned using next-generation sequencing technologies
European Journal of Cancer 61 (2016) 61e68
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.ejcancer.com
Review
Oncogenic viruses: Lessons learned using next-generation sequencing technologies Ronan Flippot a, Gabriel G. Malouf a,*, Xiaoping Su b, David Khayat a, Jean-Philippe Spano a a
University Hospital Pitie´ Salpeˆtrie`re, Department of Medical Oncology, University Pierre and Marie Curie, Paris, France Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, U.S.A b
Received 29 November 2015; received in revised form 25 March 2016; accepted 30 March 2016
KEYWORDS Cancer; Integration; Next-generation sequencing; Virus; NGS
Abstract Fifteen percent of cancers are driven by oncogenic human viruses. Four of those viruses, hepatitis B virus, human papillomavirus, Merkel cell polyomavirus, and human T-cell lymphotropic virus, integrate the host genome. Viral oncogenesis is the result of epigenetic and genetic alterations that happen during viral integration. So far, little data have been available regarding integration mechanisms and modifications in the host genome. However, the emergence of high-throughput sequencing and bioinformatic tools enables researchers to establish the landscape of genomic alterations and predict the events that follow viral integration. Cooperative working groups are currently investigating these factors in large data sets. Herein, we provide novel insights into the initiating events of cancer onset during infection with integrative viruses. Although much remains to be discovered, many improvements are expected from the clinical point of view, from better prognosis classifications to better therapeutic strategies. ª 2016 Elsevier Ltd. All rights reserved.
1. Introduction
* Corresponding author: Hoˆpital Salpeˆtrie`re, de´partement d’oncologie me´dicale, 47-83 Boulevard de l’hoˆpital, 75013, Paris, France. E-mail address: [email protected] (G.G. Malouf). http://dx.doi.org/10.1016/j.ejca.2016.03.086 0959-8049/ª 2016 Elsevier Ltd. All rights reserved.
Cancers are the leading cause of mortality in developed western countries. In a variety of situations, pathogens like bacteria, viruses and parasites are suspected to be the key event for cancer development. Cancer related bacteria are mostly represented by Helicobacter pylori in gastric carcinoma [1], or Fusobacterium in colorectal
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cancer [2]. Parasites such as Fasciola hepatica or Schistosoma haematobium are responsible for cholangiocarcinomas [3] and bladder cancer [4], respectively. Seven viruses have been reported to be associated with human cancers [5]. Of those, hepatitis C virus (HCV), Epstein-Barr virus (EBV) and human herpes virus 8 (HHV8) are non-integrative viruses, while hepatitis B virus (HBV), human papillomavirus (HPV), Merkel cell polyomavirus (MCPV), and human T-cell lymphotropic virus (HTLV) integrate the host genome. The causal association between viruses and cancers has been established in about 10e15% of malignant tumours worldwide [6]. Mechanistically, most virusassociated tumours are thought to follow a multistep carcinogenesis model, with a continuum between dysplasia and invasive cancer [7]. Cell transformation mechanisms are diverse [8], ranging from induced expression of viral and cellular oncogenes [5], to epigenetic modifications [9], miRNA regulations [9] and protumoural inflammation [10]. The incidence of virus-associated tumours has long been underestimated, as next-generation sequencing (NGS) of large tumour data sets was not available. Furthermore, the oncogenic mechanisms have been poorly understood, but we have observed that tumours caused by integrative viruses remain unique in their natural history, prognosis, and response to treatment [6]. Thus, exploring viral integration patterns and hostepathogen interactions is crucial for the understanding and management of virus-associated tumours. The development of high-throughput DNA and RNA sequencing, in conjunction with new bioinformatic tools, allows for deep, whole-genome sequencing of the host and pathogen. Recent improvements in genomics and transcriptomics have made it possible to precisely identify viral sequences in tumour genomes, which has led to the discovery of new tumourassociated viruses [11]. The identification of redundant integration sites and recurrent changes in gene expression is essential to the comprehension of this type of oncogenesis. Further extending these techniques to large-scale studies can establish the genomic landscapes of virus and tumour associations with great precision. This article aims to provide an updated perspective on NGS techniques and software for the study of integrative tumour-associated viruses. We describe their contribution to the understanding of the oncogenic mechanisms that occur during viral integration and the clinical relevance of such developments. 2. Impact of NGS and bioinformatics In the last decade, new techniques have led to a drastic improvement in the comprehension of genetics and epigenetics, all of which can be applied to tumourassociated viruses. Foremost, NGS has provided a giant
leap towards whole-genome sequencing and the comprehensive characterization of modifications in gene expression.
2.1. Charting genomic and epigenomic architecture The use of RNA microarrays to study gene expression became possible in the 1990s [12,13]. Probe-based methods use fluorescent markers of RNA hybridization to homologous cDNA to compare the differences in gene expression between a study genome and a reference genome, in which the relative changes in gene expression are determined by the change in fluorescence. This method was the first to directly compare the genomes of the host and pathogen in vitro and in vivo. However, it requires the physical separation of both host and pathogen prior to processing, which can alter the evidence of hostepathogen interactions in gene expression changes. In addition, it does not cover non-coding RNAs nor study mRNA splicing variants [14]. NGS is a powerful tool for studying the changes in gene expression between cell lineages from different tissues or in different environments. Indeed, RNA sequencing allows for the study of the whole transcriptome through standardized techniques. Nucleic acids are extracted from fresh frozen or formalin-fixed paraffin-embedded tissues, and nucleic acid sequences are fragmented into about 200-nucleotide inserts for sequencing. The most common among the diverse sequencing methods is synthesis sequencing, in which deoxynucleotide triphosphates labelled with fluorescent markers are incorporated into the nucleic acid chain [15]. Other base detection techniques have appeared in the last decade, such as pH-based methods or transmembrane channels that transport sequences with specific templates [16,17]. The gene expression level is correlated with the number of reads per exon, normalized by the length of said exon. Thanks to deep and parallel sequencing, these NGS techniques can cover virtually every transcription variant. Such advancements have made it easier and more affordable to sequence virus and cellular lineages of interest such as tumour samples. These advances in sequencing also benefit the study of epigenetics and chromatin remodelling processes. ChiP-sequencing combines chromatin immunoprecipitation and NGS to define the state of the chromatin and the level of gene expression: fragmented DNA associated with a transcription factor or a specific histone variant is immunoprecipitated and sequenced [18]. Using antibodies directed against markers of repression, such as H3K27, or active transcription, such as H3K4me3, it is possible to identify repressed genes or transcriptionally active genes in a tumour sample [19].
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2.2. Viral detection and integration sites Before NGS, viral integration was detected through in situ hybridization, using known viral sequences to identify integration sites in the human genome. Those sites were amplified by polymerase chain reaction (PCR) and then sequenced by Sanger sequencing. This method lacks sensitivity, as only parts of the genome are sequenced. In addition, Sanger sequencing lacks depth and resolution: transcript borders and insertion sites cannot be defined precisely, and each DNA fragment can only be sequenced once [14]. Consequently, it is impossible to analyse splice variants and differences in gene expression. In contrast, NGS can perform hostepathogen sequencing concurrently. Bioinformatic tools developed since 2010 are able to identify viral integration, the sites of integration, and the link between gene expression changes and viral integration. These bioinformatic tools share common algorithms (Fig. 1). First, the genome of an infected cell is fragmented, then mapped onto a reference human genome. The reads that do not match the human genome are extracted, and compared with a database of viral genomes. The presence of viral sequences indicates that viral integration occurred. Finally, sequencing chimeric reads, containing both human and pathogen DNA, determines the integration site on both the viral and host levels [20,21,22,23]. This method has led to the discovery of a new virus involved in Merkel cell carcinoma, the Merkel cell virus [11], which indicates that these bioinformatic tools might help in the discovery of new oncogenic pathogens. 2.3. Cooperative working groups Open-access databases of multiple tissue samples and viruses have been developed. Among them is The Cancer Genome Atlas (TCGA; http://cancergenome.nih. gov/), which was started in 2006 to establish the
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Table 1 Oncogenic alterations unveiled by next-generation sequencing in integrative virus-associated tumours. Virus
Cancer types
Gene alterations
Mechanism
HBV
HCC
TERT MLL4
HPV 16 HPV 18
HNSCC and anogenital SCC
MCV
MCC
HTLV
T-cell leukaemia
ERBB2 NOTCH1 RAD51B MCV Large T antigen Interleukin Interleukin receptors STAT pathway
Viral integration in gene or promoter Viral integration in gene Viral oncogene expression Viral integration in gene
genetic profiling of a large variety of tumours, and the Genome Information Broker for Viruses, which provides more than 18,000 genome sequences of known viruses. These databases are used for research purposes and by cooperative organizations, such as the International Cancer Genome Consortium, created in 2008 to achieve a comprehensive description of cancer genomes and epigenomes, or the TCGA pathogen working group, which relies on the comprehensive characterization of hostepathogen interactions in pathogenassociated tumours. 3. New insights in tumour-associated viruses: large-scale studies In parallel with the progress of genomic and bioinformatic tools, large-scale studies have been undertaken to study the landscape of virus-associated tumours. More than 8000 tumours from TCGA have been sequenced in two major studies, which assessed the impact of genomic alterations following viral insertion events [5,24]. With more studies ongoing, we intend to
Fig. 1. Principles of dual RNA sequencing of host and pathogen. 1. The whole genome is fragmented into reads prior to sequencing. 2. Identification of the integrative virus by performing next-generation sequencing (NGS) on non-human reads. 3. Identification of 5ʹ and 3ʹ insertion sites by performing NGS on chimeric reads.
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learn more about the oncogenic mechanisms of integrative viruses (Table 1).
impact of the integration events on oncogenic proteins hint at a direct link between viral insertion and malignant transformation.
3.1. Integrative DNA viruses Five tumour-associated DNA viruses have been described so far: HBV, HPV, MCV, EBV, and HHV8. Although EBV and HHV8 do not typically integrate the host genome, HBV, HPV and MCPV are integrative viruses and are involved in different pathways of carcinogenesis according to the region of viral integration. 3.1.1. Hepatitis B virus HBV infection has been implicated in 20% of hepatocellular carcinomas (HCCs) in western countries, and in up to 60% of HCCs in Southeast Asia, where frequent co-infection with HCV is observed [25]. HBV is a double-stranded DNA virus organized in a covalently closed circular DNA within the cell nucleus, and has the ability to integrate the host genome. It, as well as HCV, drives oncogenesis through chronic inflammation and molecular mechanisms, such as the activation of signalling pathways (NFKB, MAPK, AKT) that are mediated by the oncoprotein HBx. HBV integration is known to occur in actively transcribed regions [26]. Large-scale studies using NGS technologies [27,28] have demonstrated the preferential insertion of HBV within MLL4 and TERT genes or inside their promoters in HCC [5,24,29]. Recent studies have found inactivation of the MLL gene family and the reactivation of TERT in 20% [30] and 60% [31] of HCCs. Indeed, MLL4 is an epigenetic regulator acting as a transcriptional activator [32], and its selective inactivation by viral insertion might account for the downregulation of several anti-oncogenes. TERT is a gene that codes for human telomerase reverse transcriptase, which plays a key role in cell immortalization. Viral integration in TERT might induce activating mutations, while viral integration inside the TERT promoter can induce the overexpression of human telomerase reverse transcriptase. However, to date we do not know whether integration events are selective or the recurrent sites of integration only result from a selective bias. Of note, alterations of TERT and MLL have been reported in non-viral-driven HCCs [30,31], which indicates that viral integration is not always necessary for specific oncogenic alterations. This underlines the need for further assessments regarding the consequences of integration events in oncogenic pathways. Other cancers might also show the integration of HBV in the genome. Indeed, integration events have been confirmed in two cases of renal cell carcinoma [5,24]. Although the incidence was very low (<0.01%), these events involve alterations in HBS, a suspected viral oncogene, and insertion sites within the cellular SHH gene, which is involved in a pro-oncogenic pathway. The
3.1.2. Human papillomavirus HPV is a double-stranded DNA virus of which the most frequent oncogenic subtypes are HPV16 and HPV18. Oncogenic HPV infection is responsible for cervical, anogenital and head and neck squamous cell carcinoma, and the patterns of oncogenesis are similar for all locations [33,34]. It is suggested that HPV-driven carcinomas follow a multistep carcinogenesis, with viral integration being a key event in tumour progression. HPV-induced squamous cell carcinomas have been reported to have better outcomes and are usually more sensitive to radiation and chemotherapy. It has been speculated that this might be explained by the liberation of viral antigens during therapy, leading to a more efficient immune response [35]. Integration sites within the genome have been more precisely defined by NGS. They are thought to be located in clusters near DNA copy number breakpoints, involving actively transcribed gene regions and miRNA regions [36]. These insertion events might induce genomic instability and changes in gene expression next to the integration sites, but also in more distant regions of the genome. In particular, recurrent integration of the viral genome in upstream regions of the proto-oncogene c-Myc is associated with its upregulation, which favours cellular immortalization and proliferation [37,38]. Other recurrent insertion sites are located in oncogenes such as NOTCH1 and ERBB2, involved in cellular proliferation and survival, or tumour suppressor genes such as RAD51B, involved in homologous recombination. However, their precise roles in HPV-driven oncogenesis are not fully understood [24,39]. Some data tend to provide a new role for HPV integration in other epithelial cancers. Indeed, the HPV genome has been found repeatedly in squamous cell lung carcinomas, endometrial cancer and bladder urothelial carcinomas [5,24]. Viral insertion sites involve alterations of putative oncogenes, which might indicate that viral infection is a driver event in those cancers. In addition, the rare presence of HPV has been reported in cancers of the kidney, liver and colon, without enough significance to implicate the virus in the initiation of those cancers. Studies in progress should explore those putative associations. 3.1.3. Merkel cell polyomavirus MCV is an integrative double-stranded DNA virus belonging to the polyomavirus family. It was discovered in 2008 and has been reported to be the cause of 80% of Merkel cell carcinomas (MCCs), an aggressive subtype of skin cancer affecting elderly and immunosuppressed patients [40]. Viral transcripts of MCV are present in
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physiological conditions of human skin along with transcripts of other polyomaviruses, but viral integration is only found in malignant tissues. MCV was the first virus to be discovered using NGS techniques. Indeed, deep sequencing led to the discovery of unknown sequences in 80% of MCCs. Those sequences have been identified as a polyomavirus genome [11]. In that study, integration site analysis did not show any preferential insertion sites within the human genome. However, the sequence of a large T antigen has been found, which might account for its oncogenic potential. Indeed, the large T antigen is known to bind and inactivate tumour suppressor genes Rb and p53 in the polyomavirus family. Oncogenic potential is suspected in other tumour types. In fact, integration of the viral genome and expression of T antigens with the same molecular patterns as those found in MCC have been found in nonsmall-cell lung carcinomas [41]. This finding could indicate a wider role for polyomaviruses in human cancers. 3.2. Integrative RNA viruses Two oncogenic RNA viruses have been described in virus-associated tumours, HCV and HTLV1. Only HTLV1 has the ability to integrate the host genome. 3.2.1. Human T-cell lymphotropic virus 1 HTLV1 is a single-stranded RNA virus. After infection of human T-lymphocytes by HTLV1, 10% of infected individuals might develop acute trichocellular leukaemia (ATL), a lymphoid malignancy that accounts for 2% of all leukaemias [42]. TAX and HBZ are the two main viral oncogenes. TAX promotes tumour survival through the upregulation of NFKB and mTOR pathways. HBZ helps infected cells escape the immune system and promotes cell cycle progression [43,44]. NGS has identified the integration of HTLV1 in immune regulatory genes, such as interleukin, the interleukin receptor, and downstream STAT proteins [45,46]. These data suggest that impairment of the interleukin signalling pathway might be one of the oncogenic events triggered by the integration of HTLV into leukocytes. Furthermore, NGS techniques have been able to identify the virus subtypes and the clone size of ATLs, which might be useful for therapeutic management [47]. 3.3. Other putative integrative tumour-associated viruses Other viruses are marginally reported in a variety of cancers, with diverse prevalence depending on the sample and detection method. Rare associations include HHV1 in head and neck squamous cell carcinoma, enteroviruses in colorectal cancers [24], and HPV16 and
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the BK polyomavirus in urinary tract cancers [48]. It is unclear whether these associations are fortuitous or indicate direct participation in the onset of cancer. EBV and Kaposi’s sarcoma-associated herpes virus are generally non-integrative viruses; however, deep sequencing of numerous lymphoma cell lines has found integration events involving fragments of viral genomes. It is unclear whether those integration events are the consequence of genomic instability or are proper oncogenic events [45]. The human immunodeficiency virus (HIV) has not been proven to be a tumour-associated virus, despite AIDS involving a predisposition to the development of solid tumours and lymphomas [49]. However, there is recent evidence of recurrent HIV integration into oncogenes, which may contribute to persistent infection in proliferative cells [50]. These new findings indicate that HIV has the potential to activate oncogenes. Further studies are needed to explore the direct role of HIV integration in HIV-related malignancies. 4. Therapeutic challenges The identification of novel oncogenic events linked with viral integration might improve the management of those cancers. Indeed, molecular profiling of virusassociated tumours might improve prognostic classifications and identify targetable alterations at a patient’s level. It will also be of major interest to determine whether viral integration influences the efficacy of targeted therapies in tumours with targetable alterations. Furthermore, findings of new integration events might be especially important for cancer control. Indeed, it is known that antiviral therapies help reduce the risk of cancer onset and recurrence in solid and haematologic tumours [51], and remain an effective cancer treatment in lymphomas induced by oncoviruses [52]. Thus, better cancer prevention and control might be achieved through broader and enlightened use of antiviral therapies. These findings are also meaningful to explore oncoviruses tropism. Indeed, some viruses are able to switch cell types, such as EBV, which can infect epithelial gastric cells from lymphocytes [53], while other viruses are identified in usually non-pathogen-associated tumours, such as HBV in renal cell carcinoma. The exploration of the molecular mechanisms that allow such events might drive new therapeutic developments. Finally, the promising field of immunotherapy might be heavily influenced by the characterization of virusassociated tumours. Indeed, the expression of viral epitopes in those tumours might confer a strong immunogenicity [54]. Molecular tools are able to assess the proportion of neo- or viral epitopes, which might be a predictive factor of response to immune checkpoints inhibitors such as anti-CTLA4 or anti-PD1 therapies
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[55]. Moreover, the identification of viral antigens expressed on tumour cells should be useful for engineered T-cell therapies such as chimeric-antigen receptor lymphocytes [56]. 5. Discussion NGS has paved the way for a greater understanding of virus-associated tumours, thanks to the study of the genetic and epigenetic architecture of tumour genomes. The discovery of new associations between viruses and cancers will define new subsets of tumours with specific oncogenic alterations and specific outcomes. Molecular screening might be useful for patients with virusassociated tumours, as the identification of targetable alterations will provide more opportunities for treatment and clinical trial recruitment. From a technical standpoint, there is still room for improvement. Third-generation sequencing principles are designed to overcome the inadequacies of secondgeneration sequencing. The accuracy of sequencing methods can be improved by avoiding common issues: PCR-based techniques that generate inequalities of amplification in processed samples, and cDNA conversion responsible for single-point conversion errors in RNA sequencing [14]. Suppressing sample fragmentation and achieving single molecule sequencing might help to achieve de novo assembly of tumour genomes, without the need for mapping and averaging techniques [57]. These molecular tools for the characterization of virus-associated tumours also bring new questions and perspectives. Epigenetic alterations are reported to be widely involved in cancer progression [58], and the chromatin state directly influences the mutation rate in cancer cells [59]. Thus, the consequences of viral integration in tumour epigenetics should be of key interest for further studies. Regarding therapeutics, many improvements are possible, from better cancer prevention to better drug targeting and new immunotherapeutic developments. Together, this indicates that the exploration of oncogenic viruses with modern tools will provide new opportunities for better patient care. Conflict of interest statement None declared.
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Review
Oncogenic viruses: Lessons learned using next-generation sequencing technologies Ronan Flippot a, Gabriel G. Malouf a,*, Xiaoping Su b, David Khayat a, Jean-Philippe Spano a a
University Hospital Pitie´ Salpeˆtrie`re, Department of Medical Oncology, University Pierre and Marie Curie, Paris, France Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, U.S.A b
Received 29 November 2015; received in revised form 25 March 2016; accepted 30 March 2016
KEYWORDS Cancer; Integration; Next-generation sequencing; Virus; NGS
Abstract Fifteen percent of cancers are driven by oncogenic human viruses. Four of those viruses, hepatitis B virus, human papillomavirus, Merkel cell polyomavirus, and human T-cell lymphotropic virus, integrate the host genome. Viral oncogenesis is the result of epigenetic and genetic alterations that happen during viral integration. So far, little data have been available regarding integration mechanisms and modifications in the host genome. However, the emergence of high-throughput sequencing and bioinformatic tools enables researchers to establish the landscape of genomic alterations and predict the events that follow viral integration. Cooperative working groups are currently investigating these factors in large data sets. Herein, we provide novel insights into the initiating events of cancer onset during infection with integrative viruses. Although much remains to be discovered, many improvements are expected from the clinical point of view, from better prognosis classifications to better therapeutic strategies. ª 2016 Elsevier Ltd. All rights reserved.
1. Introduction
* Corresponding author: Hoˆpital Salpeˆtrie`re, de´partement d’oncologie me´dicale, 47-83 Boulevard de l’hoˆpital, 75013, Paris, France. E-mail address: [email protected] (G.G. Malouf). http://dx.doi.org/10.1016/j.ejca.2016.03.086 0959-8049/ª 2016 Elsevier Ltd. All rights reserved.
Cancers are the leading cause of mortality in developed western countries. In a variety of situations, pathogens like bacteria, viruses and parasites are suspected to be the key event for cancer development. Cancer related bacteria are mostly represented by Helicobacter pylori in gastric carcinoma [1], or Fusobacterium in colorectal
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cancer [2]. Parasites such as Fasciola hepatica or Schistosoma haematobium are responsible for cholangiocarcinomas [3] and bladder cancer [4], respectively. Seven viruses have been reported to be associated with human cancers [5]. Of those, hepatitis C virus (HCV), Epstein-Barr virus (EBV) and human herpes virus 8 (HHV8) are non-integrative viruses, while hepatitis B virus (HBV), human papillomavirus (HPV), Merkel cell polyomavirus (MCPV), and human T-cell lymphotropic virus (HTLV) integrate the host genome. The causal association between viruses and cancers has been established in about 10e15% of malignant tumours worldwide [6]. Mechanistically, most virusassociated tumours are thought to follow a multistep carcinogenesis model, with a continuum between dysplasia and invasive cancer [7]. Cell transformation mechanisms are diverse [8], ranging from induced expression of viral and cellular oncogenes [5], to epigenetic modifications [9], miRNA regulations [9] and protumoural inflammation [10]. The incidence of virus-associated tumours has long been underestimated, as next-generation sequencing (NGS) of large tumour data sets was not available. Furthermore, the oncogenic mechanisms have been poorly understood, but we have observed that tumours caused by integrative viruses remain unique in their natural history, prognosis, and response to treatment [6]. Thus, exploring viral integration patterns and hostepathogen interactions is crucial for the understanding and management of virus-associated tumours. The development of high-throughput DNA and RNA sequencing, in conjunction with new bioinformatic tools, allows for deep, whole-genome sequencing of the host and pathogen. Recent improvements in genomics and transcriptomics have made it possible to precisely identify viral sequences in tumour genomes, which has led to the discovery of new tumourassociated viruses [11]. The identification of redundant integration sites and recurrent changes in gene expression is essential to the comprehension of this type of oncogenesis. Further extending these techniques to large-scale studies can establish the genomic landscapes of virus and tumour associations with great precision. This article aims to provide an updated perspective on NGS techniques and software for the study of integrative tumour-associated viruses. We describe their contribution to the understanding of the oncogenic mechanisms that occur during viral integration and the clinical relevance of such developments. 2. Impact of NGS and bioinformatics In the last decade, new techniques have led to a drastic improvement in the comprehension of genetics and epigenetics, all of which can be applied to tumourassociated viruses. Foremost, NGS has provided a giant
leap towards whole-genome sequencing and the comprehensive characterization of modifications in gene expression.
2.1. Charting genomic and epigenomic architecture The use of RNA microarrays to study gene expression became possible in the 1990s [12,13]. Probe-based methods use fluorescent markers of RNA hybridization to homologous cDNA to compare the differences in gene expression between a study genome and a reference genome, in which the relative changes in gene expression are determined by the change in fluorescence. This method was the first to directly compare the genomes of the host and pathogen in vitro and in vivo. However, it requires the physical separation of both host and pathogen prior to processing, which can alter the evidence of hostepathogen interactions in gene expression changes. In addition, it does not cover non-coding RNAs nor study mRNA splicing variants [14]. NGS is a powerful tool for studying the changes in gene expression between cell lineages from different tissues or in different environments. Indeed, RNA sequencing allows for the study of the whole transcriptome through standardized techniques. Nucleic acids are extracted from fresh frozen or formalin-fixed paraffin-embedded tissues, and nucleic acid sequences are fragmented into about 200-nucleotide inserts for sequencing. The most common among the diverse sequencing methods is synthesis sequencing, in which deoxynucleotide triphosphates labelled with fluorescent markers are incorporated into the nucleic acid chain [15]. Other base detection techniques have appeared in the last decade, such as pH-based methods or transmembrane channels that transport sequences with specific templates [16,17]. The gene expression level is correlated with the number of reads per exon, normalized by the length of said exon. Thanks to deep and parallel sequencing, these NGS techniques can cover virtually every transcription variant. Such advancements have made it easier and more affordable to sequence virus and cellular lineages of interest such as tumour samples. These advances in sequencing also benefit the study of epigenetics and chromatin remodelling processes. ChiP-sequencing combines chromatin immunoprecipitation and NGS to define the state of the chromatin and the level of gene expression: fragmented DNA associated with a transcription factor or a specific histone variant is immunoprecipitated and sequenced [18]. Using antibodies directed against markers of repression, such as H3K27, or active transcription, such as H3K4me3, it is possible to identify repressed genes or transcriptionally active genes in a tumour sample [19].
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2.2. Viral detection and integration sites Before NGS, viral integration was detected through in situ hybridization, using known viral sequences to identify integration sites in the human genome. Those sites were amplified by polymerase chain reaction (PCR) and then sequenced by Sanger sequencing. This method lacks sensitivity, as only parts of the genome are sequenced. In addition, Sanger sequencing lacks depth and resolution: transcript borders and insertion sites cannot be defined precisely, and each DNA fragment can only be sequenced once [14]. Consequently, it is impossible to analyse splice variants and differences in gene expression. In contrast, NGS can perform hostepathogen sequencing concurrently. Bioinformatic tools developed since 2010 are able to identify viral integration, the sites of integration, and the link between gene expression changes and viral integration. These bioinformatic tools share common algorithms (Fig. 1). First, the genome of an infected cell is fragmented, then mapped onto a reference human genome. The reads that do not match the human genome are extracted, and compared with a database of viral genomes. The presence of viral sequences indicates that viral integration occurred. Finally, sequencing chimeric reads, containing both human and pathogen DNA, determines the integration site on both the viral and host levels [20,21,22,23]. This method has led to the discovery of a new virus involved in Merkel cell carcinoma, the Merkel cell virus [11], which indicates that these bioinformatic tools might help in the discovery of new oncogenic pathogens. 2.3. Cooperative working groups Open-access databases of multiple tissue samples and viruses have been developed. Among them is The Cancer Genome Atlas (TCGA; http://cancergenome.nih. gov/), which was started in 2006 to establish the
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Table 1 Oncogenic alterations unveiled by next-generation sequencing in integrative virus-associated tumours. Virus
Cancer types
Gene alterations
Mechanism
HBV
HCC
TERT MLL4
HPV 16 HPV 18
HNSCC and anogenital SCC
MCV
MCC
HTLV
T-cell leukaemia
ERBB2 NOTCH1 RAD51B MCV Large T antigen Interleukin Interleukin receptors STAT pathway
Viral integration in gene or promoter Viral integration in gene Viral oncogene expression Viral integration in gene
genetic profiling of a large variety of tumours, and the Genome Information Broker for Viruses, which provides more than 18,000 genome sequences of known viruses. These databases are used for research purposes and by cooperative organizations, such as the International Cancer Genome Consortium, created in 2008 to achieve a comprehensive description of cancer genomes and epigenomes, or the TCGA pathogen working group, which relies on the comprehensive characterization of hostepathogen interactions in pathogenassociated tumours. 3. New insights in tumour-associated viruses: large-scale studies In parallel with the progress of genomic and bioinformatic tools, large-scale studies have been undertaken to study the landscape of virus-associated tumours. More than 8000 tumours from TCGA have been sequenced in two major studies, which assessed the impact of genomic alterations following viral insertion events [5,24]. With more studies ongoing, we intend to
Fig. 1. Principles of dual RNA sequencing of host and pathogen. 1. The whole genome is fragmented into reads prior to sequencing. 2. Identification of the integrative virus by performing next-generation sequencing (NGS) on non-human reads. 3. Identification of 5ʹ and 3ʹ insertion sites by performing NGS on chimeric reads.
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learn more about the oncogenic mechanisms of integrative viruses (Table 1).
impact of the integration events on oncogenic proteins hint at a direct link between viral insertion and malignant transformation.
3.1. Integrative DNA viruses Five tumour-associated DNA viruses have been described so far: HBV, HPV, MCV, EBV, and HHV8. Although EBV and HHV8 do not typically integrate the host genome, HBV, HPV and MCPV are integrative viruses and are involved in different pathways of carcinogenesis according to the region of viral integration. 3.1.1. Hepatitis B virus HBV infection has been implicated in 20% of hepatocellular carcinomas (HCCs) in western countries, and in up to 60% of HCCs in Southeast Asia, where frequent co-infection with HCV is observed [25]. HBV is a double-stranded DNA virus organized in a covalently closed circular DNA within the cell nucleus, and has the ability to integrate the host genome. It, as well as HCV, drives oncogenesis through chronic inflammation and molecular mechanisms, such as the activation of signalling pathways (NFKB, MAPK, AKT) that are mediated by the oncoprotein HBx. HBV integration is known to occur in actively transcribed regions [26]. Large-scale studies using NGS technologies [27,28] have demonstrated the preferential insertion of HBV within MLL4 and TERT genes or inside their promoters in HCC [5,24,29]. Recent studies have found inactivation of the MLL gene family and the reactivation of TERT in 20% [30] and 60% [31] of HCCs. Indeed, MLL4 is an epigenetic regulator acting as a transcriptional activator [32], and its selective inactivation by viral insertion might account for the downregulation of several anti-oncogenes. TERT is a gene that codes for human telomerase reverse transcriptase, which plays a key role in cell immortalization. Viral integration in TERT might induce activating mutations, while viral integration inside the TERT promoter can induce the overexpression of human telomerase reverse transcriptase. However, to date we do not know whether integration events are selective or the recurrent sites of integration only result from a selective bias. Of note, alterations of TERT and MLL have been reported in non-viral-driven HCCs [30,31], which indicates that viral integration is not always necessary for specific oncogenic alterations. This underlines the need for further assessments regarding the consequences of integration events in oncogenic pathways. Other cancers might also show the integration of HBV in the genome. Indeed, integration events have been confirmed in two cases of renal cell carcinoma [5,24]. Although the incidence was very low (<0.01%), these events involve alterations in HBS, a suspected viral oncogene, and insertion sites within the cellular SHH gene, which is involved in a pro-oncogenic pathway. The
3.1.2. Human papillomavirus HPV is a double-stranded DNA virus of which the most frequent oncogenic subtypes are HPV16 and HPV18. Oncogenic HPV infection is responsible for cervical, anogenital and head and neck squamous cell carcinoma, and the patterns of oncogenesis are similar for all locations [33,34]. It is suggested that HPV-driven carcinomas follow a multistep carcinogenesis, with viral integration being a key event in tumour progression. HPV-induced squamous cell carcinomas have been reported to have better outcomes and are usually more sensitive to radiation and chemotherapy. It has been speculated that this might be explained by the liberation of viral antigens during therapy, leading to a more efficient immune response [35]. Integration sites within the genome have been more precisely defined by NGS. They are thought to be located in clusters near DNA copy number breakpoints, involving actively transcribed gene regions and miRNA regions [36]. These insertion events might induce genomic instability and changes in gene expression next to the integration sites, but also in more distant regions of the genome. In particular, recurrent integration of the viral genome in upstream regions of the proto-oncogene c-Myc is associated with its upregulation, which favours cellular immortalization and proliferation [37,38]. Other recurrent insertion sites are located in oncogenes such as NOTCH1 and ERBB2, involved in cellular proliferation and survival, or tumour suppressor genes such as RAD51B, involved in homologous recombination. However, their precise roles in HPV-driven oncogenesis are not fully understood [24,39]. Some data tend to provide a new role for HPV integration in other epithelial cancers. Indeed, the HPV genome has been found repeatedly in squamous cell lung carcinomas, endometrial cancer and bladder urothelial carcinomas [5,24]. Viral insertion sites involve alterations of putative oncogenes, which might indicate that viral infection is a driver event in those cancers. In addition, the rare presence of HPV has been reported in cancers of the kidney, liver and colon, without enough significance to implicate the virus in the initiation of those cancers. Studies in progress should explore those putative associations. 3.1.3. Merkel cell polyomavirus MCV is an integrative double-stranded DNA virus belonging to the polyomavirus family. It was discovered in 2008 and has been reported to be the cause of 80% of Merkel cell carcinomas (MCCs), an aggressive subtype of skin cancer affecting elderly and immunosuppressed patients [40]. Viral transcripts of MCV are present in
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physiological conditions of human skin along with transcripts of other polyomaviruses, but viral integration is only found in malignant tissues. MCV was the first virus to be discovered using NGS techniques. Indeed, deep sequencing led to the discovery of unknown sequences in 80% of MCCs. Those sequences have been identified as a polyomavirus genome [11]. In that study, integration site analysis did not show any preferential insertion sites within the human genome. However, the sequence of a large T antigen has been found, which might account for its oncogenic potential. Indeed, the large T antigen is known to bind and inactivate tumour suppressor genes Rb and p53 in the polyomavirus family. Oncogenic potential is suspected in other tumour types. In fact, integration of the viral genome and expression of T antigens with the same molecular patterns as those found in MCC have been found in nonsmall-cell lung carcinomas [41]. This finding could indicate a wider role for polyomaviruses in human cancers. 3.2. Integrative RNA viruses Two oncogenic RNA viruses have been described in virus-associated tumours, HCV and HTLV1. Only HTLV1 has the ability to integrate the host genome. 3.2.1. Human T-cell lymphotropic virus 1 HTLV1 is a single-stranded RNA virus. After infection of human T-lymphocytes by HTLV1, 10% of infected individuals might develop acute trichocellular leukaemia (ATL), a lymphoid malignancy that accounts for 2% of all leukaemias [42]. TAX and HBZ are the two main viral oncogenes. TAX promotes tumour survival through the upregulation of NFKB and mTOR pathways. HBZ helps infected cells escape the immune system and promotes cell cycle progression [43,44]. NGS has identified the integration of HTLV1 in immune regulatory genes, such as interleukin, the interleukin receptor, and downstream STAT proteins [45,46]. These data suggest that impairment of the interleukin signalling pathway might be one of the oncogenic events triggered by the integration of HTLV into leukocytes. Furthermore, NGS techniques have been able to identify the virus subtypes and the clone size of ATLs, which might be useful for therapeutic management [47]. 3.3. Other putative integrative tumour-associated viruses Other viruses are marginally reported in a variety of cancers, with diverse prevalence depending on the sample and detection method. Rare associations include HHV1 in head and neck squamous cell carcinoma, enteroviruses in colorectal cancers [24], and HPV16 and
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the BK polyomavirus in urinary tract cancers [48]. It is unclear whether these associations are fortuitous or indicate direct participation in the onset of cancer. EBV and Kaposi’s sarcoma-associated herpes virus are generally non-integrative viruses; however, deep sequencing of numerous lymphoma cell lines has found integration events involving fragments of viral genomes. It is unclear whether those integration events are the consequence of genomic instability or are proper oncogenic events [45]. The human immunodeficiency virus (HIV) has not been proven to be a tumour-associated virus, despite AIDS involving a predisposition to the development of solid tumours and lymphomas [49]. However, there is recent evidence of recurrent HIV integration into oncogenes, which may contribute to persistent infection in proliferative cells [50]. These new findings indicate that HIV has the potential to activate oncogenes. Further studies are needed to explore the direct role of HIV integration in HIV-related malignancies. 4. Therapeutic challenges The identification of novel oncogenic events linked with viral integration might improve the management of those cancers. Indeed, molecular profiling of virusassociated tumours might improve prognostic classifications and identify targetable alterations at a patient’s level. It will also be of major interest to determine whether viral integration influences the efficacy of targeted therapies in tumours with targetable alterations. Furthermore, findings of new integration events might be especially important for cancer control. Indeed, it is known that antiviral therapies help reduce the risk of cancer onset and recurrence in solid and haematologic tumours [51], and remain an effective cancer treatment in lymphomas induced by oncoviruses [52]. Thus, better cancer prevention and control might be achieved through broader and enlightened use of antiviral therapies. These findings are also meaningful to explore oncoviruses tropism. Indeed, some viruses are able to switch cell types, such as EBV, which can infect epithelial gastric cells from lymphocytes [53], while other viruses are identified in usually non-pathogen-associated tumours, such as HBV in renal cell carcinoma. The exploration of the molecular mechanisms that allow such events might drive new therapeutic developments. Finally, the promising field of immunotherapy might be heavily influenced by the characterization of virusassociated tumours. Indeed, the expression of viral epitopes in those tumours might confer a strong immunogenicity [54]. Molecular tools are able to assess the proportion of neo- or viral epitopes, which might be a predictive factor of response to immune checkpoints inhibitors such as anti-CTLA4 or anti-PD1 therapies
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[55]. Moreover, the identification of viral antigens expressed on tumour cells should be useful for engineered T-cell therapies such as chimeric-antigen receptor lymphocytes [56]. 5. Discussion NGS has paved the way for a greater understanding of virus-associated tumours, thanks to the study of the genetic and epigenetic architecture of tumour genomes. The discovery of new associations between viruses and cancers will define new subsets of tumours with specific oncogenic alterations and specific outcomes. Molecular screening might be useful for patients with virusassociated tumours, as the identification of targetable alterations will provide more opportunities for treatment and clinical trial recruitment. From a technical standpoint, there is still room for improvement. Third-generation sequencing principles are designed to overcome the inadequacies of secondgeneration sequencing. The accuracy of sequencing methods can be improved by avoiding common issues: PCR-based techniques that generate inequalities of amplification in processed samples, and cDNA conversion responsible for single-point conversion errors in RNA sequencing [14]. Suppressing sample fragmentation and achieving single molecule sequencing might help to achieve de novo assembly of tumour genomes, without the need for mapping and averaging techniques [57]. These molecular tools for the characterization of virus-associated tumours also bring new questions and perspectives. Epigenetic alterations are reported to be widely involved in cancer progression [58], and the chromatin state directly influences the mutation rate in cancer cells [59]. Thus, the consequences of viral integration in tumour epigenetics should be of key interest for further studies. Regarding therapeutics, many improvements are possible, from better cancer prevention to better drug targeting and new immunotherapeutic developments. Together, this indicates that the exploration of oncogenic viruses with modern tools will provide new opportunities for better patient care. Conflict of interest statement None declared.
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