Human papilloma virus integration sites and genomic signatures in head and neck squamous cell carcinoma

A prevalence of around 26% of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has been previously reported. HPV induced oncogenesis mainly involving E6 and E7 viral oncoproteins. In some cases, HPV viral DNA has been detected to integrate with the host genome and possibly contributes to carcinogenesis by affecting the gene expression. We retrospectively assessed HPV integration sites and signatures in 80 HPV positive patients with HNSCC, by using a double capture‐HPV method followed by next‐generation Sequencing. We detected HPV16 in 90% of the analyzed cohort and confirmed five previously described mechanistic signatures of HPV integration [episomal (EPI), integrated in a truncated form revealing two HPV‐chromosomal junctions colinear (2J‐COL) or nonlinear (2J‐NL), multiple hybrid junctions clustering in a single chromosomal region (MJ‐CL) or scattered over different chromosomal regions (MJ‐SC) of the human genome]. Our results suggested that HPV remained episomal in 38.8% of the cases or was integrated/mixed in the remaining 61.2% of patients with HNSCC. We showed a lack of association of HPV genomic signatures to tumour and patient characteristics, as well as patient survival. Similar to other HPV associated cancers, low HPV copy number was associated with worse prognosis. We identified 267 HPV‐human junctions scattered on most chromosomes. Remarkably, we observed four recurrent integration regions: PDL1/PDL2/PLGRKT (8.2%), MYC/PVT1 (6.1%), MACROD2 (4.1%) and KLF5/KLF12 regions (4.1%). We detected the overexpression of PDL1 and MYC upon integration by gene expression analysis. In conclusion, we identified recurrent targeting of several cancer genes such as PDL1 and MYC upon HPV integration, suggesting a role of altered gene expression by HPV integration during HNSCC carcinogenesis.     

Multi-omics mapping of human papillomavirus integration sites illuminates novel cervical cancer target genes

Background Integration of human papillomavirus (HPV) into the host genome is a dominant feature of invasive cervical cancer (ICC), yet the tumorigenicity of cis genomic changes at integration sites remains largely understudied.Methods Combining multi-omics data from The Cancer Genome Atlas with patient-matched long-read sequencing of HPV integration sites, we developed a strategy for using HPV integration events to identify and prioritise novel candidate ICC target genes (integration-detected genes (IDGs)). Four IDGs were then chosen for in vitro functional studies employing small interfering RNA-mediated knockdown in cell migration, proliferation and colony formation assays.Results PacBio data revealed 267 unique human–HPV breakpoints comprising 87 total integration events in eight tumours. Candidate IDGs were filtered based on the following criteria: (1) proximity to integration site, (2) clonal representation of integration event, (3) tumour-specific expression (Z-score) and (4) association with ICC survival. Four candidates prioritised based on their unknown function in ICC (BNC1, RSBN1, USP36 and TAOK3) exhibited oncogenic properties in cervical cancer cell lines. Further, annotation of integration events provided clues regarding potential mechanisms underlying altered IDG expression in both integrated and non-integrated ICC tumours.Conclusions HPV integration events can guide the identification of novel IDGs for further study in cervical carcinogenesis and as putative therapeutic targets.Subject terms: Cervical cancer, Cancer genomics, Targeted resequencing, Data mining, Tumour virus infections     

Frequent detection of human papilloma viruses in cervical dysplasia by PCR single-strand DNA-conformational polymorphism analysis

BACKGROUND: To clarify the pathogenicity of multiple human papilloma virus (HPV) infection, we applied SSCP (single-strand DNA conformation polymorphism) analysis for cervical neoplastic lesions. MATERIALS AND METHODS: Two hundred and sixty-six cervical swab specimens from normal cervix (n=64), cervical dysplasia (n=95), carcinoma in situ (n=79) and cervical cancer (n=28), were studied by nested PCR-SSCP analysis using L1 consensus primers. RESULTS: In 95 samples of cervical dysplasia, HPV infection was detected in 98.9% (94 out of 95), multiple HPV infection was detected in 38.3% (36 out of 94). In 19 squamous cell carcinomas (SCC) and 9 adenocarcinomas, the detection rate of HPV infection was 84.2% (16 out of 19) and 55.6% (5 out of 9), respectively, and all HPV-positive cases showed infection of a single HPV, among which HPV 16 occupied 68.6% (11 out of 16) in SCC and HPV 18 occupied 100% (5 out of 5) in adenocarcinoma. CONCLUSION: Multiple HPV infections may be concerned with pathogenicity in cervical dysplasia; however, the single infection with only a few HPV types, such as type 16 in SCC and type 18 in adenocarcinoma, may play a role in cervical carcinogenesis.     

利用FISH分析HPV16在Hela细胞中的整合位点

 目的 探讨HPV16病毒感染导致子宫颈癌发生的染色体畸变。方法 利用荧光原位杂交（FISH）技术检测HPV16病毒感染导致的Hela细胞中染色体核型的变化。结果 Hela细胞系中存在9号染色体的三体畸变，但3号染色体表现为正常的二倍体核型；Hela细胞中没有发现HPV16的整合位点，但当外源性的HPV16感染Hela细胞后，HPV16病毒DNA整合在3号染色体上；9号和3号染色体变异在子宫颈癌的发生发展中起着重要的作用，而HPV16的感染主要引起3号染色体的变异。结论 HPV16可以引起Hela细胞的3号染色体的畸变，从而导致子宫颈癌的发生。     

Human papilloma viruses and p53 mutations in normal,pre-malignant and malignant oral epithelia

HPV infections have been previously observed in oral cancers, and inactivation of the p53 gene has been shown to be one of the most common genetic alterations in human tumors. We examined 179 oral specimens from 70 individuals with histologic findings of either normal mucosa (n = 6) or oral disease that ranged from mild dysplasia to invasive squamous-cell carcinoma (n = 64) to determine the occurrence of both HPV infection and p53 mutations and their relationship with several clinical factors. HPV infection was detected by PCR amplification of viral DNA, and the presence of p53 mutations was assayed using the single-strand conformation polymorphism (SSCP)-PCR technique. HPV infection was found in 31% of individuals with oral disease and was not seen in healthy individuals. Mutations in exons 5, 6, 7 or 8 of the p53 gene were detected in 37.5% of patients with oral lesions and in a biopsy from 1 healthy individual who was a heavy smoker. Approximately one-third of lesions classified as pre-malignant (dysplasia and carcinoma in situ) and 42% of invasive carcinomas contained p53 mutations. The majority of these mutations were G:T transversions located within exons 7 and 8. Tumor tissues from 6 patients with oral lesions were found both to be HPV-16-positive and to contain p53 mutations; of these, 4 were poorly differentiated carcinomas that were diagnosed as late-stage disease. In this study, p53 mutations were detected in the early stages of cancer development. © 1996 Wiley-Liss, Inc.     

Human papilloma virus status and chromosomal imbalances in primary cervical carcinomas and tumour cell lines

Human papilloma virus (HPV) infection is the crucial step in the initiation of cervical carcinomas. In addition, HPV18 has been implicated in tumour progression and adverse clinical outcome. We determined the HPV types in 12 primary cervical carcinomas and 12 cell lines and compared the findings with the comparative genetic hybridisation (CGH) pattern of chromosomal alterations. The most frequent alteration was the deletion at 3p14 followed by the loss of 2q34-q36 along with 3q gain. High risk HPV types were detected in all samples except one primary tumour. In contrast to the normal distribution, HPV18 was present in 75% of cases including all cell lines. The cell lines carried a higher number of genetic alterations and a different CGH pattern for several chromosomes than the primary tumours, despite microdissection. Purely HPV18 positive cases indicated a high incidence of imbalances at specific loci with peaks of the histogram coinciding with known HPV integration sites. The study suggests that HPV infection is associated with a recurrent pattern of chromosomal changes in cervical carcinomas and that the development and progression of these alterations is triggered by integration into the host genome.     

Human papilloma virus (HPV) integration signature in Cervical Cancer: identification of MACROD2 gene as HPV hot spot integration site

Background Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality with infection by human papilloma virus (HPV) being the most important risk factor. We analysed the association between different viral integration signatures, clinical parameters and outcome in pre-treated CCs.Methods Different integration signatures were identified using HPV double capture followed by next-generation sequencing (NGS) in 272 CC patients from the BioRAIDs study [{"type":"clinical-trial","attrs":{"text":"NCT02428842","term_id":"NCT02428842"}}NCT02428842]. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed.Results Episomal HPV was much less frequent in CC as compared to anal carcinoma (p < 0.0001). We identified >300 different HPV-chromosomal junctions (inter- or intra-genic). The most frequent integration site in CC was in MACROD2 gene followed by MIPOL1/TTC6 and TP63. HPV integration signatures were not associated with histological subtype, FIGO staging, treatment or PFS. HPVs were more frequently episomal in PIK3CA mutated tumours (p = 0.023). Viral integration type was dependent on HPV genotype (p < 0.0001); HPV18 and HPV45 being always integrated. High HPV copy number was associated with longer PFS (p = 0.011).Conclusions This is to our knowledge the first study assessing the prognostic value of HPV integration in a prospectively annotated CC cohort, which detects a hotspot of HPV integration at MACROD2; involved in impaired PARP1 activity and chromosome instability.Subject terms: Oncology, Molecular medicine, Biomarkers, Molecular biology     

Human papilloma virus (HPV) DNA associated with prognosis of cervical cancer after radiotherapy

The importance of human papilloma virus (HPV) infection in the outcome of cervical cancer after radiotherapy remains unknown. Our study explored whether the HPV status of tumors is associated with the outcome of radiotherapy in patients with cervical cancer.

A total of 84 patients with cervical cancer (6 Stage I, 10 Stage II, 49 Stage III, and 19 Stage IV) who underwent definitive radiotherapy between January 1995 and June 2000 were included in this study. Tumor samples were obtained from all patients by punch biopsy before radiotherapy. The presence of HPV and its type were analyzed by polymerase chain reaction (PCR) based assay using the consensus primers for E6 and L1 regions. Actuarial methods were used to calculate overall survival and disease-free survival.

A total of 42 patients (50%) had cancer recurrence after radiotherapy. HPV-positive tumors were found in 76.2% (64 cases) of patients. HPV-negative patients survived for significantly shorter time periods compared to the HPV-positive patients in the overall survival (p = 0.007) and the disease-free survival (p = 0.005). According to multivariate analysis, HPV status is a significant predictor of both overall (p = 0.02) and disease-free survival time (p = 0.005).

The results of this study suggest that HPV-negative patients with cervical carcinoma have a significantly poorer prognosis after radiotherapy, and HPV status may be used as a marker to optimize the treatment of patients with this type of cancer.     

Identification of potential human oncogenes by mapping the common viral integration sites in avian nephroblastoma

Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.     

人乳头瘤病毒水平与宫颈局部免疫功能对高危型人乳头瘤病毒感染宫颈癌患者的影响

目的 探讨人乳头瘤病毒(HPV)水平与宫颈局部免疫功能对高危型HPV感染宫颈癌患者的影响.方法 收集76例宫颈癌患者(宫颈癌组)、40例慢性宫颈炎患者(慢性宫颈炎组)和40例宫颈上皮内瘤变患者(宫颈上皮内瘤变组)临床资料.检测并比较3组患者HPV水平和免疫功能指标[CD4+、CD8+、CD4+/CD8+、CD56+和调...     

Interference of papilloma viruses with p53-dependent and -independent apoptotic pathways

Papillomavirus infection interferes with four distinct and dominant levels of intra- and intercellular control of oncogenesis, three of which are based on the induction of apoptosis. Papillomaviruses cause abrogation of the cellular senescence program and interfere with p53-dependent DNA repair or apoptosis triggered by DNA damage. As a result, spontaneous or induced mutations are not eradicated and the transformed state may be established through oncogene activation and tumor suppressor gene inactivation. The interference of papillomaviruses with a recently described p53-independent intercellular control step, in which nontransformed cells induce apoptosis specifically in their transformed neighbouring cells, allows survival of papillomavirus-expressing transformed cells. Finally, p53-dependent hypoxia-triggered apoptosis in microtumors may be overcome by papillomaviruses and thus lead to efficient and rapid tumor progression.     

His-1 and His-2: identification and chromosomal mapping of two commonly rearranged sites of viral integration in a myeloid leukemia.

To identify genes that contribute to myeloid leukemogenesis we have cloned viral integration sites from a CasBrM-MuLV-induced interleukin 3-independent myeloid leukemia cell line. Genomic probes derived from cellular sequences flanking two integrated proviruses were used to screen restriction digests of DNAs from a panel of 52 hematopoietic cell lines, 30 of which were established from CasBrM-MuLV- or MoMuLV-induced mouse leukemias. Probes from one integration site (His-1) defined a region that was rearranged in 3/52 cell lines, and probes from a second integration site (His-2) identified a rearrangement in 2/52 cell lines. Both cases of His-2 rearrangements occurred in concert with viral insertions in the His-1 locus. Genetic mapping of these loci using interspecific backcross analysis assigned the His-1 locus to mouse chromosome 2 and the His-2 locus to mouse chromosome 19. In situ hybridization with a probe from the human homologous region mapped the His-1 locus to human chromosome 2q14-q21. No recombinants were observed between His-2 and Gin-1, a common site of provirus integration in Gross passage A MuLV-induced T-cell leukemias, in 131 backcross animals, suggesting that these loci are tightly linked. The His-1 locus maps to mouse chromosome 2 distinct from any known oncogene or common site of integration but near the proximal breakpoint for a deletion that is observed in over 90% of radiation-induced leukemias.     

Part II: spinal-cord neoplasms--primary tumours of the bony spine and adjacent soft tissues

Primary tumours of the bony spine and adjacent soft tissues most frequently present with pain although neurological deficits and spinal deformity can be present too. Knowledge of the spectrum of lesions that can affect the bony spine and the surrounding soft tissues is crucial in directing appropriate investigation and treatment. Patients need individualised approaches and treatment plans in view of the variations in tumour aggressiveness, spinal level, location within the vertebral body or posterior elements, involvement of soft tissues and structures surrounding the vertebral column, neurological deficits, and spinal instability.