A comparative study of extracellular vesicle-associated and cell-free DNA and RNA for HPV detection in oropharyngeal squamous cell carcinoma

Abstract

Purpose: This study compares the detection sensitivity of two separate liquid biopsy sources, cell-free (cf) DNA/RNA and extracellular vesicle (EV)-associated DNA/RNA (EV-DNA/RNA), to identify circulating Human Papilloma Virus (HPV) DNA/RNA in plasma obtained from patients with oropharyngeal squamous cell carcinoma (OPCSCC). We also report on the longitudinal changes observed in HPV-DNA levels in response to treatment. Experimental design: A prospective study was conducted that included 22 patients with locally advanced disease and six patients with metastatic OPCSCC. Twenty-three patients had HPV-related OPCSCC defined by p16 immunohistochemistry. Levels of circulating HPV-DNA and HPV-RNA from plasma-derived cf-DNA/RNA and EV-DNA/RNA were quantified using digital droplet PCR. Results: Circulating HPV-DNA was detected with higher sensitivity in cf-DNA compared to EV-DNA at 91% vs. 42% (p = <0.001). Similarly, circulating tumoral HPV-RNA was detected at a higher sensitivity in cf-RNA compared to EV-RNA, at 83% vs. 50% (p = 0.0019). In the locally advanced cohort, 100% (n = 16) of HPV-OPCSCC patients demonstrated a reduction in circulating HPV-DNA levels in cf-DNA following curative treatment, with 81% of patients demonstrating complete clearance to undetectable levels. However, in metastatic HPV-OPCSCC patients (n = 4), HPV-DNA levels did not correlate with treatment response. Conclusion: Our study demonstrates that although HPV-DNA/RNA can be detected in EV associated DNA/RNA, cf-DNA/RNA is the more sensitive liquid biopsy medium. As circulating HPV-DNA levels were found to only correlate with treatment response in the locally advanced but not metastatic setting in our small cohort of patients, the use of HPV-DNA as a dynamic biomarker to monitor treatment response requires further evaluation.

Cell-Free HPV-DNA as a Biomarker for Oropharyngeal Squamous Cell Carcinoma—A Step Towards Personalized Medicine?

“…OPSCC patients with UICC (Union for International Cancer Control) stage of 0–IV who received surgery, radio(chemo)therapy, and/or immunotherapy were incorporated (for more information, see Table S1 ). According to collection of data/samples, five studies had a prospective design [ 24 , 27 , 28 , 30 , 37 ], whereas four studies were purely retrospective [ 23 , 25 , 29 , 33 ]. Seven studies did not report how data/samples were collected [ 22 , 26 , 31 , 32 , 34 , 35 , 36 ] ( Table 1 , Table S1 ).…”

“…One study [ 27 ] used “HPV16-detect” and compared sensitivity and specificity via real-time PCR. Additionally, eight studies [ 25 , 28 , 29 , 30 , 33 , 35 , 36 , 37 ] used droplet digital PCR (ddPCR). Damerla et al [ 29 ] and Jeannot et al [ 33 ] compared ddPCR with qPCR and they reported more sensitive values by using ddPCR at lower cfHPV-DNA concentrations.…”

“…Probes and primers used in the 16 studies included in the review are displayed in Table S3 . Thirteen studies extracted cfHPV-DNA from plasma [ 22 , 23 , 25 , 27 , 28 , 29 , 30 , 31 , 32 , 34 , 35 , 36 , 37 ], whereas two other studies extracted DNA from serum [ 24 , 26 ] and Jeannot et al used both plasma and serum [ 33 ].…”

“…The risk of bias was rated high, low, or unclear according to QUADAS-2. Nine studies presented with high quality [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 31 ], whereas one study increased the risk of bias due to an isolated focus on patients with tumor stage III-IV [ 30 ].…”

Decline in circulating viral and human tumor markers after resection of head and neck carcinoma

“…Prior studies of HNSCC have evaluated tumor markers using polymerase chain reaction (PCR) or sequencing technologies to detect human gene variants or to quantify HPV load in plasma 17,24‐28,30,31,37‐41 . The ability of sequencing panels to track multiple tumor markers at once should generate more precise measurements of tumor clones and emerging aggressive subclones.…”