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Cervical Cancer Study in Ugandan Women Finds HPV-Specific Tumor DNA Methylation, Gene Expression

NEW YORK – An analysis of cervical cancer in Ugandan patients has mapped epigenome and transcriptome landscapes that are specific to the human papillomavirus clade from which that particular cancer arose.

In a study published on Monday in Nature Genetics, an international team of researchers wrote that cervical cancer is the most common cancer affecting sub-Saharan African women, and is particularly prevalent among HIV-positive individuals. HPV-16 (clade A9) is common in both squamous cell carcinomas and adenocarcinomas, while HPV-18 (clade A7) is associated with adenocarcinomas and inferior survival. Although cervical cancer prevention strategies include vaccination and screening for HPV, vaccine use remains low in low- and middle-income countries where HIV is prevalent, they said.

In order to comprehensively profile cancer genomes, transcriptomes, and epigenomes in this population, the researchers characterized 118 tumors from Ugandan patients, 72 of whom were HIV-positive, and performed extended mutation analysis on an additional 89 tumors.

They found HPV-clade-specific differences in tumor DNA methylation, promoter- and enhancer-associated histone marks, gene expression, and pathway dysregulation, and further noted changes in histone modification at HPV integration events that were correlated with upregulation of nearby genes and endogenous retroviruses.

When they performed whole-genome sequencing on the samples from their discovery cohort, the investigators identified an average of 22,942 somatic mutations per sample, including 311 coding mutations. They detected APOBEC mutation signatures 2 and 13, confirming previous reports and in line with a mutational process driven by a cellular response to viral infections. Tumors with a high proportion of mutations with APOBEC signatures exhibited significantly more coding mutations than those with lower proportions. The researchers also found that 15 samples exhibited moderate to high homologous recombination deficiency (HRD) scores, indicative of a dysfunctional HR repair pathway, but found no differences in mutation burden, mutation signatures, or HRD score between HIV-positive and HIV-negative samples.

Of the 12 significantly mutated genes in the cohort, PIK3CA was the most recurrent. They found that a higher proportion of HIV-negative tumors had PIK3CA mutations, and PIK3CA expression was 1.3 times higher in HIV-negative samples. Notably, the researchers added, 87 percent of the cohort had at least one mutation in an annotated chromatin modifier gene.

When they performed targeted sequencing of 2,735 selected genes in their extension cohort, the investigators confirmed mutations in 11 of the 12 significantly mutated genes they found in the discovery cohort, and observed similar mutation frequencies between the discovery and extension cohorts.

Further, an analysis of copy number landscapes showed that broad copy number alterations were comparable between HIV-positive and HIV-negative samples.

They next used whole-genome sequencing to identify seven high-confidence noncoding hotspots in 11 percent of the discovery cohort samples. All reported hotspots were present in HIV-positive and HIV-negative samples, and the samples with mutations exhibited a moderate proportion of mutations with APOBEC signatures, the researchers said.

Whole-genome sequencing also detected 17 HPV types and their associated clades in the cohort. High-risk HPV-16 (clade A9), HPV-18, and HPV-45 (clade A7) were the most abundant types. When the researchers compared their data to TCGA data, they found that clade A7 was more prevalent in their cohort than in the TCGA cohort, particularly among the squamous cell carcinomas. They also found no difference in HPV types between HIV-positive and HIV-negative tumors.

They went on to characterize the tumors' expression and DNA methylation landscapes, and correlated them with tumor features. They identified three gene expression clusters, enriched for adenocarcinomas (cluster 1), keratinizing squamous cell carcinomas (cluster 2), or non-keratinizing squamous cell carcinomas (cluster 3). Cluster 1 was enriched for samples with clade A7 HPV and cluster 3 was enriched for PIK3CA-mutated samples, the investigators noted. Cluster 2 was also enriched in samples with higher tumor grade.

They also studied the genomic impacts of 1,010 unique HPV integration sites in 109 of the 118 tumors. The researchers identified 257 integration events, and found that clade A7 integration events contained more integration sites per event than clade A9 events. Of the genes near integration events, 61 displayed significantly higher expression in samples with integration, and events containing a higher number of integration sites were associated with increased fold change in expression.

Further, the researchers said, clade A7 integration events appeared to have a more pronounced effect on expression than clade A9 events, which may result from the higher number of integration sites per event in this clade. Of the 16 genes identified in multiple samples near integration events, eight were significantly upregulated in integrated samples, including the oncogenes ERBB2 and TP63.

"Clade A7-infected samples exhibited distinct gene expression patterns converging on pathways linked to the extracellular matrix and to cell adhesion and migration, indicating a more aggressive phenotype," the authors wrote. "While inferior prognosis associated with clade A7 has previously been reported in invasive cervical cancer, our study provides insight into the cellular pathways that may promote the aggressive phenotype in these tumors. Genes upregulated in samples with clade A7 HPV, such as PXDN, are upregulated in cancers that have more potential to progress through the epithelial-mesenchymal transition."