The AACR Project GENIE is an international data-sharing consortium focused on generating an evidence base for precision cancer medicine by integrating clinical-grade cancer genomic data with clinical outcome data for tens of thousands of cancer patients treated at multiple institutions worldwide. In conjunction with the first public data release from approximately 19,000 samples, we describe the goals, structure, and data standards of the consortium and report conclusions from high-level analysis of the initial phase of genomic data. We also provide examples of the clinical utility of GENIE data, such as an estimate of clinical actionability across multiple cancer types (>30%) and prediction of accrual rates to the NCI-MATCH trial that accurately reflect recently reported actual match rates. The GENIE database is expected to grow to >100,000 samples within 5 years and should serve as a powerful tool for precision cancer medicine. Significance The AACR Project GENIE aims to catalyze sharing of integrated genomic and clinical datasets across multiple institutions worldwide, and thereby enable precision cancer medicine research, including the identification of novel therapeutic targets, design of biomarker-driven clinical trials, and identification of genomic determinants of response to therapy.

Combined detection of cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation-associated protein (MAL) promoter methylation in cervical scrapes is a promising triage strategy for high-risk human papillomavirus (hrHPV)-positive women. Here, CADM1 and MAL DNA methylation levels were analysed in cervical scrapes of hrHPV-positive women with no underlying high-grade disease, high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. CADM1 and MAL methylation levels in scrapes were first related to CIN-grade of the corresponding biopsy and second to CIN-grade stratified by the presence of 'normal' or 'abnormal' cytology as present in the accompanying scrape preceding the cervical biopsy. The scrapes included 167 women with CIN1, 54 with CIN2/3 and 44 with carcinoma. In a separate series of hrHPV-positive scrapes of women with CIN2/3 (n 5 48), methylation levels were related to duration of preceding hrHPV infection (PHI; <5 and 5 years). Methylation levels were determined by quantitative methylation-specific PCR and normal cytology scrapes of hrHPV-positive women with histologically CIN1 served as reference. CADM1 and MAL methylation levels increased proportional to severity of the underlying lesion, showing an increase of 5.3-and 6.2-fold in CIN2/3, respectively, and 143.5-and 454.9-fold in carcinomas, respectively, compared to the reference. Methylation levels were also elevated in CIN2/3 with a longer duration of PHI (i.e. 11.5-and 13.6-fold, respectively). Moreover, per histological category, methylation levels were higher in accompanying scrapes with abnormal cytology than in scrapes with normal cytology. Concluding, CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes are related to the degree and duration of underlying cervical disease and markedly increased in cervical cancer.

We recently identified MAL (T-lymphocyte maturation associated protein) as the most down-regulated gene in cervical oncogenesis. Here, we examined the mechanism underlying MAL silencing, its functional role in cervical carcinogenesis, and the relevance of detecting MAL alterations for risk assessment of hrHPV-positive women. MAL mRNA expression and promoter methylation were analysed in primary keratinocytes, hrHPV-immortalized keratinocytes, cervical cancer cell lines, biopsies, and scrapings by quantitative (methylation-specific) PCR. SiHa cells were transfected with MAL cDNA and assayed for proliferation, migration, and anchorage-independent growth. MAL mRNA was (nearly) undetectable in all HPV-immortalized and cervical cancer cells, but could be up-regulated upon methylation inhibition. MAL promoter methylation at two promoter regions (M1 and M2) was detected in all HPV-immortalized cells and cancer cells. Ectopic expression of MAL in SiHa cells suppressed proliferation, migration, and anchorage-independent growth. None (0/22) of normal cervical biopsies, 9% (6/66) of CIN1 lesions, 53% (34/64) of CIN3 lesions, 90% (85/94) of cervical squamous cell carcinomas (SCCs), and 93% (26/28) of cervical adenocarcinomas (AdCAs) demonstrated MAL promoter methylation at both promoter regions. Moreover, detection of MAL promoter methylation in cervical scrapings was predictive for underlying high-grade lesions. Both in biopsies and in scrapings, MAL promoter methylation was significantly correlated with reduced mRNA expression. MAL gene silencing by promoter methylation is a frequent and biologically essential event in HPV-induced cervical carcinogenesis. Hence, MAL promoter methylation and/or mRNA expression analysis on cervical scrapings may provide a valuable diagnostic tool to improve the detection of CIN3, SCC, and AdCA.

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Cervical cancer results from persistent infection with high-risk human papillomavirus (hrHPV). Common genetic aberrations in cervical (pre)cancers encompass large genomic regions with numerous genes, hampering identification of driver genes. This study aimed to identify genes functionally involved in HPV-mediated transformation by analysis of focal aberrations (<3 Mb) in high-grade cervical intraepithelial neoplasia (hgCIN). Focal chromosomal aberrations were determined in high-resolution array comparative genomic hybridization data of 60 hgCIN. Genes located within focal aberrations were validated using 2 external gene expression datasets or qRT-PCR. Functional roles of candidate genes EYA2 (20q13) and hsa-miR-375 (2q35) were studied by siRNA-mediated knock-down and overexpression, respectively, in hrHPV-containing cell lines. We identified 74 focal aberrations encoding 305 genes. Concurrent altered expression in hgCIN and/or cervical carcinomas compared with normal cervical samples was shown for ATP13A3, HES1, OPA1, HRASLS, EYA2, ZMYND8, APOBEC2, and NCR2. Gene silencing of EYA2 significantly reduced viability, migratory capacity, and anchorage-independent growth of HPV16-transformed keratinocytes. For hsa-miR-375, a direct correlation between a (focal) loss and significantly reduced expression was found. Downregulation of hsa-miR-375 expression was confirmed in an independent series of cervical tissues. Ectopic expression of hsa-miR-375 in 2 cervical carcinoma cell lines reduced cellular viability. Our data provide a proof of concept that chromosomal aberrations are actively contributing to HPV-induced carcinogenesis and identify EYA2 and hsa-miR-375 as oncogene and tumor suppressor gene, respectively.