Next-Generation Sequencing for Simultaneous Determination of Human Papillomavirus Load, Subtype, and Associated Genomic Copy Number Changes in Tumors

Abstract

Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a powerful predictive and prognostic biomarker. We describe how the use of next-generation sequencing can provide a novel method for the detection of HPV in DNA isolated from formalin-fixed paraffin-embedded tissues. Using this methodology in a cohort of 44 head and neck tumors, we identified the samples that contained HPV sequences, the viral subtype involved, and a direct readout of viral load. Specificity of HPV detection by sequencing compared to traditional detection methods using either PCR or p16 immunohistochemistry was 100%. Sensitivity was 50% when either compared to PCR [confidence interval (CI) = 29% to 71%] or 75% when compared to p16 (CI = 47% to 91%). In addition, we demonstrate the ability of next-generation sequencing to detect other HPV subtypes that would not have been detected by traditional methods, and we demonstrated the ability to apply this method to any tumor and any virus in a panel of eight human cancer cell lines. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of next-generation sequencing for the detection of HPV provides a multiplicity of data with clinical significance in a single test.

Is next-generation sequencing an important tool in HPV subtype diagnosis?

“…A third method uses low-coverage whole-genome sequencing to measure viral load and subtype, as well as providing a map of large-scale chromosomal damage [18]. No viral-specific capture or PCR is required.…”

Survival of Head and Neck Cancer Cells Relies upon LZK Kinase-Mediated Stabilization of Mutant p53

“…Analysis was run according to previously published procedures (35). No viral genomes were detected in any sample, so cell lines were determined to be HPV-negative with 95% confidence (36).…”

Role of Next-Generation Sequencing in Understanding the Interactions between Human Papillomavirus and the Cervicovaginal Microbiome

“…NGS is a promising method for HPV typing because of its high sensitivity in multiple infection [1] and its potential ability to detect a broad spectrum of HPV types, subtypes and variants [13,14], as well as unknown/uncharacterized HPV types [15]. NGS is also able to detect other HPV subtypes that would not have been detected by traditional methods [16]. This has important implications on HPV incidence and genotype distribution data, obtained through commercial HPV detection kits, which may not detect all HPV types, especially in HIV-positive women, which are often infected with multiple, rare and undetermined HPV types [14].…”

“…NGS bypasses or reduces many of the problems associated with PCR-based HPV detection methods, including the requirement for prior sequence information, false-positives due to cross-reactivity between types, false-negatives associated with low viral loads, and biased amplification that makes identifying all the types present in a multiple infection difficult [14]. Although the significance of these types in the context of their association to cervical disease remains to be investigated, the ability to detect all HPV subtypes using NGS could potentially lead to a more accurate prevalence of this virus and better understanding of its role in the etiology of epithelial cancers [16]. Furthermore, NGS seems to perform well in settings where very low genome quantities are available, such as formalin-fixed paraffin-embedded tissue [1,16].…”

“…Although the significance of these types in the context of their association to cervical disease remains to be investigated, the ability to detect all HPV subtypes using NGS could potentially lead to a more accurate prevalence of this virus and better understanding of its role in the etiology of epithelial cancers [16]. Furthermore, NGS seems to perform well in settings where very low genome quantities are available, such as formalin-fixed paraffin-embedded tissue [1,16]. …”