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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: Oral Oncol. 2022 Mar 22;128:105805. doi: 10.1016/j.oraloncology.2022.105805

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

Austin K Mattox 1, Gypsyamber D’Souza 2,*, Zubair Khan 3, Hailey Allen 3, Stephanie Henson 3, Tanguy Y Siewert 1, Wayne Koch 3, Drew M Pardoll 4, Carole Fakhry 3,4,*
PMCID: PMC9058207  NIHMSID: NIHMS1791903  PMID: 35334415

Abstract

Background

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.

Materials and Methods

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).

Results

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.

Conclusions

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.

Keywords: Liquid biopsy, head and neck cancer, oropharyngeal cancer, HPV, next generation sequencing, ddPCR, quantitative real-time PCR

INTRODUCTION

Head and neck squamous cell carcinomas are the seventh most common cancer worldwide [1, 2], and their incidence is increasing due to human papillomavirus (HPV)-positive tumors of the oropharynx (HPV-OPC) [3–7]. Despite good overall prognosis for HPV-positive patients, disease progression affects up to 30% [8–11]. Presently, identification of patients at increased risk for developing recurrence after definitive treatment to no evidence of disease (NED) relies upon clinical characteristics including recursive partitioning analyses and nomograms, each of which have their limitations. Proposed biomarkers, such as mutant allele tumor heterogeneity (MATH), TRAF3/CYLD mutations, HPV type, estrogen receptor α expression, presence of tumor infiltrating lymphocytes, and PD-1/PD-L1 expression have yet to be integrated into the clinical risk stratification or as a means to identify evidence of impending recurrence [12–16]. Instead, recurrence is identified clinically.

Recent studies have shown that oral and plasma HPV DNA are detectable prior to recurrence in some patients [17–20], suggesting that HPV DNA may represent microscopic HPV-OPC. Because viral DNA is shed from tumor cells into blood and/or saliva, oral and plasma HPV DNA may serve as biomarkers for disease surveillance [18, 21, 22]. This approach would help identify patients at low risk of recurrence, who could potentially undergo reduced clinical surveillance, or high risk of recurrence who may need closer surveillance to detect recurrence earlier when tumor burden is low and surgically salvageable, an independent marker of prognosis [23]. Additionally, detection of patients at high risk for recurrence might identify patients who could benefit from systemic adjuvant therapy where viral DNA levels could then be used to monitor response to therapy.

Studies of oral and plasma HPV detection primarily use multiplex PCR followed by line blot hybridization [24–27], quantitative real-time PCR (qPCR) [17, 27–32], droplet digital PCR (ddPCR) [21, 33], or next generation sequencing (NGS) [18]. Among those diagnosed with HPV-OPC, HPV detection in oral rinse or plasma has a broad range of sensitivities, from 19% to 89% compared to tumor biopsy confirmed disease. While these technologies have individually been used to quantify HPV copy number in the plasma and oral rinses of patients with HPV-OPC, no study to date has directly compared qPCR, ddPCR, and NGS on the same set of samples to determine which is most sensitive.

We sought to determine whether qPCR, ddPCR, or NGS was the most sensitive for detecting HPV16 DNA in the plasma and oral rinses of patients with biopsy-confirmed HPV16-OPC (Figure 1). We also sought to determine which technology was most sensitive for predicting disease recurrence after initial treatment.

FIGURE 1.

FIGURE 1.

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HPV DNA is shed from HPV-OPC cancers into the saliva and plasma of patients and may be detected by quantitative real-time PCR, droplet digital PCR, and/or next generation sequencing.

MATERIALS & METHODS

Study Participants

This retrospective study used oral rinse and plasma samples prospectively collected from patients who had provided informed consent to an institutional review-board approved biospecimen collection protocol at Johns Hopkins (IRB No. NA_00037133). Samples were collected at diagnosis and during clinical follow-up at varying intervals based on when a patient returned for additional treatments and/or routine care. Eligibility for follow-up analysis included clinical diagnosis of HPV16-OPC and availability of oral and/or plasma samples at more than one visit. All participants consented to oral rinses and collection of blood samples as previously described [17]. DNA was extracted using a QIAamp circulating nucleic acid kit (Qiagen, Cat. No. 55114) per the manufacturer’s instructions. In order to assess the quality of the DNA used in the three assays, we evaluated the positivity of the housekeeping gene β-globin in each sample by qPCR. The median CT value using 10 ng of input DNA was 23.2 ± 3.3, with no sample having a CT > 30.

358 plasma and oral rinse samples were collected across all time points for 66 patients. At the time of diagnosis, there were 45 patients with both plasma and oral rinse samples, 18 with only plasma, and 3 with only oral rinse. 47 patients had longitudinal samples collected after diagnosis and treatment, including 98 oral rinse and 149 plasma samples (follow-up range 5 days to 39.6 months after diagnosis).

HPV Status

Tumor HPV testing was performed at Johns Hopkins for clinical purposes and consisted of p16 immunohistochemistry (MTM Laboratories, Heidelberg, Germany) and HPV16 E6/E7 RNA in situ hybridization (ISH, RNA-scope, Advanced Cell Diagnostics, Hayward, CA) [34]. p16 expression was considered positive if ≥ 70% strong and diffuse nuclear and cytoplasmic staining pattern was observed [35, 36]. Tumors were considered HPV16 positive if both HPV16 ISH and p16 staining were positive.

Next Generation Sequencing (NGS)

The presence of HPV16 DNA was assessed using primers for E6 and included a random 14 nucleotide unique identifier sequence (UID) at the 5’ end of the forward primer. The forward primer sequence was 5’-NNNNNNNNNNNNNNCAGGACACAGTGGCTTTTGA-3’ and the reverse primer sequence was 5’-ACAGCAATACAACAAACCGTTG-3’ (IDT, Coralville, IA), which resulted in a 71bp amplicon. 10 ng of DNA was amplified as previously described [18]. High-quality sequence reads generated by a MiSeq (Illumina, San Diego, CA) were selected on the basis of Q > 30 [37]. The template-specific portion of the reads was then matched to the E6 reference sequence. Reads from a common template molecule were grouped by UID. Only molecules that matched the reference sequence were included in downstream analyses. Plasma and/or oral rinse samples with a UID count ≥ 1 were scored as positive for HPV DNA. Using serial dilution of HPV16 DNA, analytical sensitivity and precision was determined to be 1 ± 0.5 UIDs per reaction. Sequences described in this study have been deposited with the European Genome-phenome Archive (EGAS00001005062).

Droplet Digital PCR (ddPCR)

The presence of HPV16 DNA was assessed in 10 ng of input DNA using the same primers as for NGS, except without the random 14 nucleotide UID. Reactions were partitioned into a median of 18,268 droplets per well using the QX200 droplet generator (Bio-Rad, Hercules, CA). Emulsified reactions were amplified on a 96-well thermal cycler with SYBR Green (Bio-Rad). Plates were read and analyzed using QuantaSoft software (Bio-Rad) to assess the number of droplets positive for HPV16. Each sample was run in technical duplicate, and the assay threshold sensitivity was set at three standard deviations above the background observed in no template (nuclease-free water) controls. Using serial dilution of HPV16 DNA, analytical sensitivity and precision was determined to be 2 ± 1.1 copies per reaction.

Quantitative Real-Time PCR (qPCR)

The presence of HPV16 DNA was assessed in 10 ng of input DNA using the same primers as for ddPCR. The assay was performed using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, Catalog no. A25780) with a 20 μL volume on a QuantStudio3 (Applied Biosystems) per the manufacturer’s instructions. Standard curves were generated using serial dilutions of HPV16 plasmid DNA (ATCC, Manassas, VA, Catalog no. 45113D). PCR consisted of a denaturation step of 98°C for 3 minutes, followed by 40 cycles of 98°C for 10 seconds, 56°C for 2 minutes, and 72°C for 2 minutes, followed by melt curve analysis. Each sample was run in technical duplicate, and the assay threshold sensitivity was set at three standard deviations above the background observed in no template (nuclease-free water) controls. Using serial dilution of HPV16 DNA, analytical sensitivity and precision was determined to be 8 ± 3.4 copies per reaction.

Statistical Analysis

The proportion of HPV16-OPC cases that had HPV16 DNA detected in oral rinse and/or plasma by NGS, ddPCR, and qPCR were compared using a Chi-Squared test. Comparisons of the quantification of UIDs and/or HPV copy number by tumor stage was tested by one-way ANOVA. p values ≤ 0.05 were considered statistically significant.

47 of 66 patients had follow-up appointments where plasma and/or oral rinse samples were collected. All 47 patients had plasma samples and 35 also had oral rinse samples from their second study visit (median of 193 days after diagnosis, IQR 131–262 days) that were tested for HPV DNA to assess the positive predictive value (PPV) and negative predictive value (NPV) of each assay to predict recurrence. The results were correlated with clinical status as determined by MRI or CT scans.

RESULTS

Patient and Sample Characteristics at Presentation

Clinicopathological characteristics of the 66 patients are shown in Table 1. At initial presentation, the median age was 59 years old, 84.8% were male, and 89.4% were Caucasian. The majority of patients had AJCC 7th edition advanced stage disease (20% Stage III; 70% Stage IV), and 84.9% underwent primary surgery.

TABLE 1.

Summary of clinical characteristics of patients with HPV16-OPC included in the study

Characteristics of HPV16-OPC Study Population All Patients
Median age at diagnosis (Q1–Q3) 59 (51.5–65.0)
Sex, no. (%) N = 66
 Male 56 (84.8)
 Female 10 (15.2)
Race, no. (%) N = 66
 White (Caucasian) 59 (89.4)
 Black (African American) 3 (4.5)
 Other 4 (6.1)
AJCC 7th edition stage of disease*, no. (%) N = 60
 I 3 (5.0)
 II 3 (5.0)
 III 12 (20.0)
 IV 42 (70.0)
TNM Staging, no. (%)
 Primary Tumor* N = 64
  T1 35 (54.7)
  T2 19 (29.7)
  T3 8 (12.5)
  T4 2 (3.1)
 Regional Lymph Nodes* N = 64
  N0 7 (10.9)
  N1 13 (20.3)
  N2 38 (59.4)
  N3 5 (7.8)
 Distant Metastasis N = 66
  Mx 9 (13.6)
  M0 56 (84.8)
  M1 5 (7.8)
Treatment modality, no. (%) N = 66
 Surgery only 25 (37.9)
 Radiation with or without chemotherapy 7 (10.6)
 Surgery and radiation 16 (24.2)
 Surgery, radiation, and chemotherapy 15 (22.7)
 Observation only 3 (4.5)
Length in months of follow up, median (range) 50.2 (0.5–170.5)
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*

There were 6 people with unknown/missing information on AJCC stage. There were 2 people with unknown/missing information on primary tumor and lymph node staging.

Sensitivities of HPV16 DNA by NGS, ddPCR, and qPCR In Samples at Diagnosis

We first compared HPV16 detection in plasma by each technology to the known HPV tumor status at diagnosis (Figures 2 and S1). NGS and ddPCR detected about two-thirds of cases with sensitivities of 68.3% and 69.8%, respectively. By contrast, sensitivity by qPCR (20.6%) was significantly lower than both NGS and ddPCR (p < 0.001).

FIGURE 2. Sensitivity at diagnosis for detection of HPV in plasma and oral rinse is greatest by next generation sequencing.

FIGURE 2.

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** p < 0.01, *** p < 0.001.

We next evaluated HPV16 detection in oral rinses at diagnosis. NGS had a sensitivity of 75.0%, which was significantly higher than ddPCR (8.3%, p < 0.001) and qPCR (2.1%, p < 0.001). In plasma samples, NGS and ddPCR had a concordance of 54.0% for calling samples positive and a correlation coefficient of 0.20 for HPV copy number (Figure 3). qPCR and NGS had a concordance of 20.1% (R2 = 0.38), and qPCR and ddPCR had a concordance of 17.5% (R2 = 0.30). Concordance of detecting HPV DNA was less than 9% between any two technologies in oral rinse samples (qPCR and NGS (R2 = 0.002), ddPCR and NGS (R2 = 0.09), ddPCR and qPCR (R2 = 0.12), Figure 3).

FIGURE 3. NGS and ddPCR demonstrate the best positive concordance in both plasma and oral rinse.

FIGURE 3.

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The dashed red line indicates the threshold for sensitivity for each assay, and the red shaded area indicates samples that are positive by both technologies in each plot.

Sensitivity of HPV DNA detection in plasma versus oral rinse was also compared (Figure 4). When tested by NGS, plasma (68%) and oral rinse (75%) had good sensitivity (p = 0.44) and demonstrated a robust dynamic range of detecting 1 to 212 HPV DNA molecules. However, plasma was superior to oral rinse for detecting HPV DNA by both ddPCR (70% vs 8%, p < 0.001) and qPCR (21% vs 2% p = 0.004). We did not detect discernable differences in HPV DNA by stage (Figure S2).

FIGURE 4. NGS in plasma and oral rinse and ddPCR in plasma show good sensitivity for HPV DNA detection at the time of diagnosis.

FIGURE 4.

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The dashed red line indicates the threshold for sensitivity for each assay. ** p < 0.01, *** p < 0.001.

Utility of HPV16 DNA detection by NGS, ddPCR, and qPCR in Follow-Up Samples

47 patients had one or more follow-up sample(s) collected after treatment, during which 7 patients were observed to experience a clinical recurrence.

We calculated the PPV and NPV of NGS (Table 2) and ddPCR (Table 3) for recurrence at the time of the second study visit. Given the poor sensitivity of qPCR at diagnosis, PPV and NPV were not calculated for qPCR. In plasma, PPV was moderate for NGS (57%) and ddPCR (71%, p = 0.67), and NPV was low (38% for NGS and 23% for ddPCR, p = 0.13). In oral rinse, PPV was high by NGS (100%), although the sample size was limited with 6 positive samples, and was non-significantly higher than ddPCR (33%, p = 0.14). However, NPV was low by NGS and was significantly lower by NGS than ddPCR (38% vs 93%, p < 0.001).

TABLE 2.

Longitudinal follow-up for disease recurrence by NGS in 47 patients with plasma samples available and 35 patients with oral rinse samples available at the second follow-up visit.

Disease By Imaging and Clinical Impression
Yes
(Plasma N = 7)
(Oral Rinse N = 6)		 No
(Plasma N = 40)
(Oral Rinse N = 29)
Plasma
(N = 47)		 Positive 57.1% (PPV)
(N = 4)		 62.5%
(N = 25)
Negative 42.9%
(N = 3)		 37.5% (NPV)
(N = 15)
Oral Rinse
(N = 35)		 Positive 100.0% (PPV)
(N = 6)		 62.1%
(N = 18)
Negative 0.0%
(N = 0)		 37.9% (NPV)
(N = 11)
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TABLE 3.

Longitudinal follow up for disease recurrence by ddPCR in 47 patients with plasma samples available and 35 patients with oral rinse samples available at the second follow-up visit.

Disease By Imaging and Clinical Impression
Yes
(Plasma N = 7)
(Oral Rinse N = 6)		 No
(Plasma N = 40)
(Oral Rinse N = 29)
Plasma
(N = 47)		 Positive 71.4% (PPV)
(N = 5)		 77.5%
(N = 31)
Negative 28.6%
(N = 2)		 22.5% (NPV)
(N = 9)
Oral Rinse
(N = 35)		 Positive 33.3% (PPV)
(N = 2)		 6.9%
(N = 2)
Negative 66.7%
(N = 4)		 93.1% (NPV)
(N = 27)
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One patient had seven plasma samples collected during follow-up. Longitudinal testing by NGS, ddPCR, and qPCR were explored in this patient as a case example and are shown in Figure 5. Only NGS was positive at diagnosis when the patient had HPV-OPC in the right tongue base and positive cervical lymph node. After resection of the primary tumor and lymph nodes and adjuvant chemoradiotherapy, HPV DNA levels were undetectable by NGS, ddPCR, and qPCR at the second time point (169 days after surgery and 106 days after completion of chemoradiotherapy), mirroring MRI scans that indicated NED. The patient had two interval MRIs at 265 days and 366 days post-surgery that demonstrated NED. All three plasma assays performed on those same days did not detect HPV DNA. 489 days after surgery, an MRI detected a sub-plural nodule in the left lower lung lobe, which was biopsy-confirmed as metastatic from the primary HPV-OPC. Only NGS at day 489 was positive for HPV DNA. Following 20 Gy of radiation and chemotherapy with carboplatin/cetuximab, the patient’s sub-plural nodule resolved, and HPV DNA was again undetectable by NGS on day 727. At the last follow-up appointment 912 days after surgery, the patient presented with metastatic masses in the left lower lobe and right lower lobe of the lungs. Only NGS showed detectable levels of HPV in the patient’s plasma at this final visit.

FIGURE 5. HPV levels detected in plasma by NGS but not ddPCR or qPCR mirror the clinical course of HPV-OPC.

FIGURE 5.

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The green dot represents an assay performed at each follow up visit. Red arrows designate tumors on MRI or CT scans.

DISCUSSION

Detection of HPV16 DNA after treatment may herald clinical recurrence [19, 20, 38]. Given the improved survival of surgical salvage [39], early identification of recurrent disease is paramount. Therefore, there is increased interest in identifying the most sensitive technology for HPV detection and strengths and weaknesses of plasma and oral rinse. To our knowledge, this is the first study to compare the three most common HPV detection technologies in use. In this retrospective cohort of 66 patients with HPV16-OPC, NGS in either plasma or oral rinse, and ddPCR in plasma but not oral rinse demonstrated sensitivities of around 70% for detecting HPV DNA.

The primary mechanism of HPV detection to date in the literature has been qPCR. In this analysis, sensitivity of qPCR was lower than expected in both plasma and oral rinses. While a study by Capone et al. using qPCR for HPV E7 achieved a similar sensitivity of 23.5% in plasma compared to 20.6% in the present study [27], more recent studies achieve three-fold higher sensitivities. Cao et al. simultaneously detected HPV E6 and E7 in plasma and achieved a sensitivity of 65% among the 40 HPV16-OPC samples studied [28]. Dahlstrom et al. simultaneously detected HPV E6 and E7 in serum and achieved a sensitivity of 60.5% among the 114 HPV16-OPC samples studied. Ahn et al. expanded their analysis to also include oral rinses. Using a similar assay design, their study achieved a sensitivity of 67.3% in plasma and 52.8% in saliva among 93 paired blood-oral rinse samples taken at the time of diagnosis [17].

Several differences may have contributed to the low sensitivity of qPCR observed in the present study. First, our study used banked DNA previously isolated from plasma and oral rinses, instead of freshly isolated DNA. It is possible that DNA samples may have degraded over time. Second, each study that achieved a sensitivity greater than 60% in plasma or serum used amplification of both E6 and E7 genes compared to only E6 used in the present study. Third, each study used primer/probe chemistry that combines the sensitivity of PCR with the specificity afforded by the probe. Our qPCR design used SYBR Green as the fluorophore to detect HPV DNA. While SYBR Green-based assays have higher background than primer/probe-based assays, they are significantly less costly and can be run on nearly all qPCR instruments. Low HPV copy number samples may only be detected at high PCR cycle numbers that are also characteristic of background SYBR Green intercalation into primer/dimer artifacts. Thus, it is often impossible to differentiate between false positive and true positive samples with low HPV copy number using non primer/probe-based qPCR. In scenarios where HPV copy number is expected to be high, including amplification of DNA isolated from primary tumor tissue and in metastatic disease, qPCR would likely be sufficient. In screening or surveillance scenarios, however, where the intent of the assay is to identify disease when HPV copy number is low, other technologies or assay designs may be more fruitful.

ddPCR partitions samples into approximately 20,000 oil-water emulsion droplets whose contents are individually amplified [40]. A key benefit of ddPCR is the ability to absolutely quantify the number of DNA copies per sample without a standard curve. In addition, high-copy templates and background are diluted across the droplets, effectively enriching template concentration in HPV-positive partitions, allowing for increased sensitivity over that of qPCR at low copy number (69.8% for ddPCR vs 20.6% for qPCR in the present study). For samples that were positive by both qPCR and ddPCR, quantitative analysis of HPV16 copy number demonstrated robust linearity (R2 = 0.88, p < 0.001).

Our ddPCR sensitivity is comparable to previous studies. Hanna et al. utilized primer/probe-based quintuplex ddPCR for HPV 16, 18, 31, 33, and 45 and achieved sensitivities of 66.7% in plasma and 86.7% in saliva in 15 patients with locoregional HPV-OPC [20]. Wang et al. used ddPCR to achieve sensitivities of 85.7% in plasma and 37.9% in saliva in 21 and 29 patients with locoregional disease, respectively, with HPV16-OPC [18]. More recently, Chera et al. also used primer/probe-based quintuplex ddPCR for HPV 16, 18, 31, 33, and 35 in plasma to detect 89% of HPV-OPC [19, 38].

Notably, our ddPCR assay had a sensitivity of 8.3% in oral rinse, which was significantly lower than that reported in Hanna et al. [20] and Wang et al. [18]. In both prior studies, nearly all patients presented with advanced disease with locoregional or metastatic spread. In Hanna et al., all 15 patients had locoregional disease, and in Wang et al., 93.1% of patients had regional lymph node or metastatic disease involvement. Our patient population had similar characteristics at the time of diagnosis and oral rinse collection, suggesting patient cohort differences do not explain our low sensitivity. As noted in Hanna et al., the sensitivity of HPV DNA detection in oral rinses is impacted by the acidic pH of the mouth, adequacy of rinsing/gargling, behaviors prior to collection such as eating, drinking, or smoking, and contributions of prior radiotherapy and other treatments to xerostomia and hyposalivation. These factors are also unlikely to explain the low sensitivity of ddPCR in our study at the time of diagnosis, as NGS robustly detected 75% of HPV16-OPC cases. If ddPCR is used for HPV DNA quantification, further studies are needed to better understand the factors that affect sensitivity in oral rinse samples. In addition, use of oral rinse for longitudinal monitoring is likely limited to detecting only intraoral recurrences that saliva is likely to sample.

NGS maintains the sensitivity of ddPCR but adds the ability to simultaneously detect multiple targets in the sample, such as other genotypes of HPV that cause HPV-OPC, including 18, 31, 33, and 35. NGS also has the ability to detect mutations in circulating tumor DNA, which would be important for surveillance of HPV-negative OPC or detection of mutations in PIK3CA or NOTCH1 common to HPV-OPC [18]. The ability to sequence allows for simultaneous HPV and DNA mutation detection from the same sample. In addition, the sequencing read alignment step limits false positives, increasing the signal-to-noise ratio at low HPV copy number. In practice, it may be possible to multiplex up to 30 amplicons per well [41], providing a wealth of information from HPV copy number to the presence of clinically actionable mutations.

In our study, NGS and ddPCR were similarly sensitive for detecting HPV DNA in plasma. The choice of which technology to use in a given clinical or research setting is a function of available technology, desired number and variety of alterations to detect, and cost. At institutions with massively parallel sequencing cores, NGS may quantitate many alterations including mutations, aneuploidy, methylation changes, and viral copy number in the same sample but at the expense of cost and more complicated and time-intensive sample preparation. ddPCR retains sensitivity but has a limited ability to multiplex and requires an additional droplet generation step. However, ddPCR is more cost effective than NGS when quantification of only a few targets is desired. qPCR is the simplest and most cost-effective technology used to detect HPV DNA. Depending on assay design, the sensitivity of qPCR is limited by background fluorescent dye intercalation and the generation of a standard curve to quantify HPV copy number. The background is most limiting at low HPV copy number, as the signal-to-noise ratio is not great enough to distinguish single or a few copies of HPV DNA from false positives.

For studies interested in only quantifying HPV copy number, ddPCR would suffice at all ranges of HPV copy number and qPCR when HPV copy number was likely to be high. For studies interested in HPV quantification at all ranges of HPV copy number and in the presence of other alterations such as mutations, NGS is the more appropriate choice. If NGS were the modality of choice for a clinical trial, oral rinse may be a suitable alternative to plasma testing at the time of diagnosis because it is less invasive yet provides similar results for oropharyngeal disease and potentially improved sensitivity for oral disease.

Chera et al. recently conducted a longitudinal study using ddPCR to detect HPV 16, 18, 31, 33, and 35 in the plasma of 115 patients diagnosed with HPV-OPC [19]. At the time of the first abnormal blood test after initial treatment, their assay had a PPV of 54% and NPV of 100%, which rose to a PPV of 94% with two consecutively positive blood tests. In our study, NGS and ddPCR had a PPV of 57% and 71%, respectively, at the time of the first abnormal blood test, although our NPV was much lower at 38% for NGS and 23% for ddPCR. Importantly, the Chera et al. study prospectively collected samples every 6–9 months and performed PET/CT scans at fixed 2–4 month (follow up years 1–2) or 6 month (follow up years 3–5) intervals. Our banked samples were not collected at defined intervals, which may have impacted our PPV and NPV analysis.

Strategies to improve PPV and NPV for recurrence monitoring include collecting plasma and/or oral rinse samples every 3–6 months and requiring that two consecutive tests be positive before imaging with MRI and/or CT. Other composite approaches could include stratifying patients by PD-L1 expression, presence of tumor infiltrating lymphocytes, or concurrent detection of mutations, aneuploidy, or methylation changes in circulating tumor DNA.

Most studies to date have enrolled patients with advanced HPV-OPC with locoregional or metastatic spread. Future studies aimed at assessing HPV DNA detection in the screening setting will need to accrue a significant number of patients with early-stage disease to accurately determine the PPV and NPV of NGS or other technologies. In addition, additional prospective studies should be conducted with blood draws and oral rinse samples assessed at fixed time intervals to complement recent work [19, 20, 38] and determine whether HPV DNA dynamics pre-date radiologic or symptomatic recurrence.

Supplementary Material

1
2
3

  • HPV DNA is often detected by qualitative PCR, real time PCR, and other technologies including NGS and ddPCR are being explored.

  • NGS and ddPCR in plasma and NGS in oral rinse have good sensitivity for HPV16-OPC.

  • HPV levels detected in plasma by NGS may mirror the clinical course of HPV-positive head and neck cancer.

FUNDING:

This work was supported by the National Institutes of Health [R35 DE026631].

CONFLICTS OF INTEREST:

None declared (AKM, GD, ZK, HA, SH, WK, DP, CF). TYS has received honoraria and institutional grants from Merck, Nanobiotix, Regeneron, and AstraZeneca; honoraria from Innate Pharma, eTheRNA, and Nektar; and an institutional grant from Bristol Myers Squibb, all outside the submitted work.

Footnotes

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Supplementary Materials

1
NIHMS1791903-supplement-1.tiff (5.2MB, tiff)
2
NIHMS1791903-supplement-2.tiff (5.2MB, tiff)
3
NIHMS1791903-supplement-3.xlsx (48.3KB, xlsx)

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