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.
Subject(s)
Gene Dosage , Head and Neck Neoplasms/diagnosis , Papillomaviridae/classification , Papillomaviridae/genetics , Papillomavirus Infections/diagnosis , Tumor Virus Infections/diagnosis , Viral Load/genetics , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/virology , Cyclin-Dependent Kinase Inhibitor p16 , DNA, Viral/genetics , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/virology , High-Throughput Nucleotide Sequencing , Humans , Immunoenzyme Techniques , Middle Aged , Neoplasm Proteins/metabolism , Papillomavirus Infections/genetics , Papillomavirus Infections/virology , Polymerase Chain Reaction , Sequence Analysis, DNA , Tumor Virus Infections/genetics , Tumor Virus Infections/virologyABSTRACT
Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal abnormalities are detected in individual tumours. We have used next generation sequencing at low coverage to produce high resolution copy number karyograms of a series of 89 non-small cell lung tumours specifically of the squamous cell subtype. Because this methodology is able to create karyograms from formalin-fixed paraffin-embedded material, we were able to use archival stored samples for which survival data were available and correlate frequently occurring copy number changes with disease outcome. No single region of genomic change showed significant correlation with survival. However, adopting a whole-genome approach, we devised an algorithm that relates to total genomic damage, specifically the relative ratios of copy number states across the genome. This algorithm generated a novel index, which is an independent prognostic indicator in early stage squamous cell carcinoma of the lung.
