Comparison of next generation sequencing, droplet digital PCR, and quantitative real-time PCR for the earlier detection and quantification of HPV in HPV-positive oropharyngeal cancer

Oral oncology

2022 Mar 22

Mattox, AK;D'Souza, G;Khan, Z;Allen, H;Henson, S;Siewert, TY;Koch, W;Pardoll, DM;Fakhry, C;
PMID: 35334415 | DOI: 10.1016/j.oraloncology.2022.105805

Human papillomavirus (HPV) causes nearly 80% of oropharynx cancers diagnosed in the United States, with incidence increasing each year. Analysis of cfDNA in plasma and oral rinse has the potential to detect these cases earlier than their typical presentation, but their utility and the best method to detect HPV in plasma and oral rinse samples is unknown.We directly compared next generation sequencing (NGS), droplet digital PCR (ddPCR), and quantitative real-time PCR (qPCR) for their ability to detect HPV16 DNA in plasma and oral rinse from 66 patients diagnosed with HPV16-positive oropharyngeal cancer (HPV16-OPC).HPV DNA detection by NGS and ddPCR in plasma samples both had good sensitivity (70%) for HPV16-OPC compared to 20.6% sensitivity by qPCR (p < 0.001). In oral rinse, NGS demonstrated a superior sensitivity of 75.0% as compared to both ddPCR (8.3%, p < 0.001) and qPCR (2.1%, p < 0.001). In a limited cohort of follow up patients, HPV levels detected in plasma by NGS but not ddPCR or qPCR reflected disease remission or progression.These results suggest that NGS has the best sensitivity for detecting HPV in both plasma and oral rinse and may play a role in monitoring patients for disease recurrence. Additional studies are needed to define the specificity of NGS for similar patient cohorts.

Single cell transcriptomic analysis of HPV16-infected epithelium identifies a keratinocyte subpopulation implicated in cancer

Nature communications

2023 Apr 08

Bedard, MC;Chihanga, T;Carlile, A;Jackson, R;Brusadelli, MG;Lee, D;VonHandorf, A;Rochman, M;Dexheimer, PJ;Chalmers, J;Nuovo, G;Lehn, M;Williams, DEJ;Kulkarni, A;Carey, M;Jackson, A;Billingsley, C;Tang, A;Zender, C;Patil, Y;Wise-Draper, TM;Herzog, TJ;Ferris, RL;Kendler, A;Aronow, BJ;Kofron, M;Rothenberg, ME;Weirauch, MT;Van Doorslaer, K;Wikenheiser-Brokamp, KA;Lambert, PF;Adam, M;Steven Potter, S;Wells, SI;
PMID: 37031202 | DOI: 10.1038/s41467-023-37377-0

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

Priorities of human papillomavirus-associated oropharyngeal cancer patients at diagnosis and after treatment

Oral Oncol

2019 Jun 04

Windon MJ, Fakhry C, Faraji F, Troy T, Gourin CG, Kiess AP, Koch W, Eisele DW, D'Souza G.
PMID: 31345377 | DOI: 10.1016/j.oraloncology.2019.05.016

INTRODUCTION:

Little is known regarding how human papillomavirus-positive oropharyngeal cancer (HPV-OPC) patient goals change with treatment. This study evaluates whether patient ranking of non-oncologic priorities relative to cure and survival shift after treatment as compared to priorities at diagnosis.

MATERIALS AND METHODS:

This is a prospective study of HPV-OPC patient survey responses at diagnosis and after treatment. The relative importance of 12 treatment-related priorities was ranked on an ordinal scale (1 as highest). Median rank (MR) was compared using Wilcoxon matched-pairs signed-rank tests. Prevalence of high concern for 11 treatment-related issues was compared using paired t-test. The effect of patient characteristics on change in priority rank and concern was evaluated using linear regression.

RESULTS:

Among 37 patients, patient priorities were generally unchanged after treatment compared with at diagnosis, with cure and survival persistently ranked top priority. Having a moist mouth uniquely rose in importance after treatment. Patient characteristics largely did not affect change in priority rank. Concerns decreased after treatment, except concern regarding recurrence.

DISCUSSION:

Treatment-related priorities are largely similar at diagnosis and after treatment regardless of patient characteristics. The treatment experience does not result in a shift of priorities from cure and survival to non-oncologic domains over cure and survival. The rise in importance of moist mouth implies that xerostomia may have been underappreciated as a sequelae of treatment. A decrease in most treatment-related concerns is encouraging, whereas the persistence of specific areas of concern may inform patient counseling

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

Search form

Probes for HPV

Content for comparison

Gene

Product

Research area

Category

INTRODUCTION:

MATERIALS AND METHODS:

RESULTS:

DISCUSSION:

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China