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Vulvar squamous cell carcinoma (VSCC) and precancerous high-grade vulvar intraepithelial neoplasia (VIN) can develop through human papillomavirus (HPV)-dependent and -independent pathways, indicating a heterogeneous disease. Only a minority of VIN progress, but current clinical and histological classifications are insufficient to predict the cancer risk. Here we analyzed copy number alterations (CNA) to assess the molecular heterogeneity of vulvar lesions in relation to HPV and cancer risk. HPV-status and CNA by means of whole-genome next-generation shallow-sequencing were assessed in 24 VSCC and 41 VIN lesions. The latter included VIN of women with associated VSCC (VINVSCC) and women who did not develop VSCC during >10 year follow-up (VINnoVSCC). HPV-testing resulted in 41 HPV-positive lesions (16 VINVSCC, 14 VINnoVSCC and 11 VSCC) and 24 HPV-negative lesions (11 VINVSCC and 13 VSCC). HPV-positive and -negative VSCC showed a partially overlapping pattern of recurrent CNA, including frequent gains of 3q and 8q. In contrast, amplification of 11q13/cyclinD1 was exclusively found in HPV-negative lesions. HPV-negative VINVSCC had less CNA than VSCC (P = 0.009), mainly 8q gains and 8p losses. In HPV-positive lesions, CNA frequencies were lower in VINnoVSCC than in VINVSCC (P = 0.022) and VSCC. Interestingly, a 1pq gain was detected in 81% of VINVSCC and only 29% of VINnoVSCC. In conclusion, HPV-dependent and -independent vulvar carcinogenesis is characterized by distinct CNA patterns at the VIN stage, while more comparable patterns are present at the cancer stage. Progression risk in VIN is reflected by the extent of CNA, in particularly chromosome 1 gain. Citation Format: Maaike CG Bleeker, Dorian RA Swarts, Quirinus JM Voorham, Saskia M. Wilting, Marc van Beurden, Renske DM Steenbergen. Molecular heterogeneity in HPV-dependent and -independent vulvar carcinogenesis: Chromosome 1 gain reflects the cancer risk in vulvar intraepithelial neoplasia [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 5050.

Although persistent infection with high-risk human papillomavirus (HPV) is generally acknowledged as necessary cause for cervical cancer, additional molecular changes are required for the development of cancer. Those changes include chromosomal aberrations that result in deregulated expression of coding and non-coding RNAs. In this study we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines, to identify the sequential order and relevance of these molecular alterations for HPV-induced transformation. Genome-wide chromosomal, mRNA and miRNA expression profiles were generated from 4 HPV-transformed keratinocyte cell lines at 8 different passages representing progressive stages of transformation. Unsupervised hierarchical clustering of DNA copy number, mRNA and miRNA profiles revealed that in all 4 cell lines most pronounced changes were associated with the acquisition of anchorage independence, a hallmark of transformation. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to DNA copy number alterations. Focal adhesion, TGF-β signalling and mTOR signalling pathways were enriched among these genes. In addition, longitudinal analysis identified more than 600 potential miRNA-mRNA interactions that were negatively correlated during passaging. Four interactions were also predicted by 3 independent prediction algorithms, and have in part already been functionally validated, thereby demonstrating the validity of our approach. In conclusion, integrated longitudinal analysis of our HPV-induced transformation model enabled us to pinpoint relevant interconnected molecular changes and affected signalling pathways. Increased understanding of the interplay between different molecular alterations and affected pathways will be of importance for the clinical management of patients diagnosed with HPV-induced (pre)cancerous lesions. Citation Format: Iris Babion, Viktorian Miok, Annelieke Jaspers, Wessel N. van Wieringen, Renske D. Steenbergen, Saskia M. Wilting. Comprehensive molecular profiling of HPV-induced transformation over time [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 5059.

Cervical cancer develops from precancerous high-grade cervical intraepithelial neoplasia (CIN) harboring a transforming infection with high-risk human papillomavirus, which is characterized by p16INK4a overexpression. Once such a lesion has developed, progression toward an invasive squamous cell carcinoma (SCC) may take one or more decades, underlining the heterogeneity of these lesions in terms of duration of existence and progression risk. We performed array-based comparative genomic hybridization (array CGH) on 46 p16INK4a immunopositive CIN2/3 lesions to determine whether this heterogeneity is reflected in their chromosomal profiles. Chromosomal profiles of CIN2/3 lesions were related to those of invasive cervical SCC and promoter methylation of CADM1, a tumor suppressor gene known to be functionally involved in the tumorigenic phenotype of cervical cancer cells. Frequent alterations found in CIN2/3 lesions included gains located at chromosome 1, 3, 7, and 20 and losses located at 4, 11, 16, 17, and 19. Unsupervised hierarchical clustering identified two subsets of CIN2/3 lesions, chromosomal profiles of one of which closely resembled invasive SCCs. Gains of 1, 3q, and 20 were characteristic for CIN2/3 lesions with chromosomal signatures resembling carcinomas. In addition, dense promoter methylation of the CADM1 gene was significantly more frequent in these CIN2/3 lesions (P = 0.004). No chromosomal alterations were detected in six CIN1 lesions, five of which were completely p16INK4a immunonegative. These findings suggest that biomarkers associated with gains at chromosomes 1, 3q, and 20 are potential hallmarks of advanced p16INK4a-positive CIN2/3 lesions with a high short-term risk of progression. [Cancer Res 2009;69(2):647–55]

Deregulated expression of microRNAs (miRNAs) is common and biologically relevant in cervical carcinogenesis and appears only partly related to chromosomal changes. We recently identified 34 miRNAs showing decreased expression in cervical high-grade precursor lesions and carcinomas not associated with a chromosomal loss, 6 of which were located within a CpG island. This study aimed to investigate to what extent these miRNAs are subject to DNA methylation-mediated silencing in cervical carcinogenesis. Methylation-specific PCR (MSP) analysis on a cell line panel representing different stages of human papillomavirus (HPV) induced transformation revealed an increase in methylation of hsa-miR-149, -203, and -375 with progression to malignancy, whereas expression of these miRNAs was restored upon treatment with a demethylating agent. All three miRNAs showed significantly increased levels of methylation in cervical carcinomas, whereas methylation levels of hsa-miR-203 and -375 were also significantly increased in high-grade cervical intraepithelial neoplasia (CIN). A pilot analysis showed that increased hsa-miR-203 methylation was also detectable in HPV-positive cervical scrapes of women with high-grade CIN compared to controls. Similar to recent findings on hsa-miR-375, ectopic expression of hsa-miR-203 in cervical cancer cells decreased both the proliferation rate and anchorage independent growth. We found evidence for methylation-mediated silencing of hsa-miR-149, -203 and -375 in cervical cancer. Methylation of the latter two was already apparent in precancerous lesions and represent functionally relevant events in HPV-mediated transformation. In addition, as demonstrated for hsa-miR-203, these methylated miRNAs may provide valuable markers for assessment of the presence of (pre)cancerous cervical lesions in HPV-positive women. Citation Format: Saskia Marije Wilting, Wina Verlaat, Annelieke Jaspers, Nour A. Makazaji, Reuven Agami, Chris JLM Meijer, Peter JF Snijders, Renske DM Steenbergen. Methylation-mediated silencing of microRNAs during cervical carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2971. doi:10.1158/1538-7445.AM2013-2971

Persistent infection with high-risk human papillomavirus (hrHPV) types is causally related to the development of cervical cancer and a subset of other anogenital and head and neck cancers. HrHPV-induced malignant transformation of epithelial cells is associated with (epi)genetic aberrations in host cell genes, including alterations in DNA methylation often affecting tumor suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is characterized by deregulated expression of the viral oncogenes E6 and E7 in dividing epithelial cells. Hereto, primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation karyotype with genome-wide expression profiles of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV-containing cell lines was confirmed using a real-time methylation specific PCR (MSP) detection platform. Except from NPTX-1, methylation of none of these genes has to the best of our knowledge been linked to hrHPV-induced cancers before. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens revealed an increase in methylation of all six genes with disease progression. All genes were commonly methylated in cervical carcinomas, with highest frequencies of up to 100% detected for FAM19A4, PHACTR3 and PRDM14. NKX2-8, PHACTR3 and PRDM14 were already methylated in approximately one third of precancerous lesions, indicating that their alterations represent relatively early events in cervical carcinogenesis. In conclusion, MSDK analysis of HPV16 transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel methylation events in cervical carcinogenesis, including methylation of FAM19A4, LHX1, NKX2-8, PHACTR3 and PRDM14 gene promoters. These genes may provide promising triage markers for assessment of the presence of (pre)cancerous cervical lesions in hrHPV-positive women. Citation Format: Renske D M Steenbergen, Maté Ongenaert, Suzanne Snellenberg, Chris JLM Meijer, Kornelia Polyak, Noga Bloushtain-Qimron, Peter JF Snijders, Wim van Criekinge. Methylation specific digital karyotyping of human papillomavirus type 16 E6E7 expressing keratinocytes identifies novel methylation targets in cervical carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4248. doi:10.1158/1538-7445.AM2013-4248

High-grade cervical intraepithelial neoplastic (CIN2/3) lesions represent a heterogeneous disease both with respect to clinical behaviour and chromosomal aberrations detected. We hypothesized that the extent of chromosomal aberrations reflects the duration of their existence. To test this hypothesis, chromosomal profiles were determined of CIN3 lesions of women with a known 5 year history of high-risk human papillomavirus virus (hrHPV) infection, in which duration of prior hrHPV infection was considered a proxy for duration of CIN3 lesion existence. Eight women had a Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2011-2718

At present little is known about the involvement of alterations in microRNA (miRNA) expression patterns during the development of cervical cancer. In this study we investigated genome-wide miRNA expression profiles in normal cervical squamous epithelium, high-grade precancerous lesions (CIN2/3), squamous cell carcinomas (SCC) and adenocarcinomas (AdCAs), all of which were high-risk HPV positive. In addition, miRNA expression profiles were integrated with previously generated genome-wide chromosomal profiles of the same samples. Unsupervised hierarchical clustering showed that miRNA expression profiling enables the distinction of normal cervical squamous epithelium, CIN2/3 lesions, SCCs and AdCAs. Differential miRNA expression analysis comparing normals, CIN2/3 lesions and SCCs identified early (temporal), late, and continuously differentially expressed miRNAs in cervical carcinogenesis (n=106). Differential expression of selected miRNAs could be validated by qRT-PCR in the same samples as well as in cervical scrapes of women with abnormal cytology and underlying high-grade CIN disease compared to control scrapes. In general, miRNAs located in the same cluster showed similar expression profiles, suggesting common regulatory mechanisms. For 5 of the 106 differentially expressed miRNAs, altered expression could be directly linked to underlying chromosomal alterations. Finally, cell viability assays indicated that ectopic expression of 2 selected miRNAs influenced cell proliferation in vitro, indicating functional relevance of differential miRNA expression during cervical carcinogenesis. In conclusion, deregulation of miRNA expression patterns occurs during cervical carcinogenesis and has functional relevance for cervical cancer development. Since deregulated miRNA expression in cervical (pre)malignant disease is reflected and detectable in cervical scrapes, miRNAs represent a highly promising class of disease markers for further improvement of the triage of high-risk HPV positive women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4959. doi:10.1158/1538-7445.AM2011-4959

BACKGROUND Exosomes are 40-100 nm membrane vesicles that are released by cancer and normal cells, after the fusion of multivesicular bodies with the plasma membrane. Multiple functions have been attributed to exosomes including antigen presentation and intercellular communication. Importantly, exosomes carry tumor-specific antigens and have been identified in various biofluids. Therefore, exosomes may provide an attractive platform for biomarker discovery. Mass spectrometry-based proteomics has contributed significantly to our understanding of the molecular composition of exosomes. Yet a comprehensive quantitative analysis of exosomes and corresponding cell lysates derived from a panel of different cancer cell lines and primary cells is missing. AIM Identification of exosome core proteins and cancer-type specific proteins to obtain insight into aberrant exosomal functions in cancer and for candidate biomarker discovery APPROACH Quantitative proteomics of exosomes released by a panel of 9 cancer cell lines and 2 normal cell types and their corresponding total cell lysates. METHODS Exosomes were harvested by differential centrifugation from the following human cancer cell lines: HT29, HCT116, SW480, SW1398, CaCo2; H460; MCF-7, PC3 and LNCaP. The primary human cells included are endothelial cells and keratinocytes. The proteomics workflow is based on protein fractionation by 1DGE, in-gel digestion, nano-liquid chromatography coupled to tandem mass spectrometry on an LTQ-FTMS. Spectral counting was used for protein quantitation. Dedicated statistics and bioinformatics tools were used for significance analysis of altered protein abundances and coupling to biological functions as described (Pham et al., Bioinformatics 2010; Albrethsen et al., Mol.Cell.Prot. 2010). RESULTS Exosome isolation by differential centrifugation was reproducible as deduced from replicate analyses. The purity of the exosome preparation was verified using analysis of specific exosome markers in our dataset, electron microscopy and western blot and was shown to be good. The total dataset of exosomes and cell lysates contains ∼4500 proteins with 1300-1400 exosome proteins per cell line. The core exosome proteome shared by all cells analyzed was highly enriched for the terms protein synthesis, RNA binding and lipid raft proteins among others. We are currently comparing the exosome protein profile of each cell type with the protein profile of the cell lysate, as well as with the exosomes from other cell types and coupling the data to gene ontology and pathway analysis tools. CONCLUSIONS & OUTLOOK Exosomes may be considered a relevant new platform for biomarker discovery. In cancer exosomes, we identified established tumor type-specific antigens and proteins belonging to oncogenic pathways. For the validation of potential biomarkers in biofluids, we will employ targeted mass spectrometry and antibodies against the candidate biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5115. doi:10.1158/1538-7445.AM2011-5115