A Liquid Biopsy To Detect Transcriptionally Active Human Papillomavirus 16 From Patient Saliva

doi:10.21203/rs.3.rs-3147471/v1

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A Liquid Biopsy To Detect Transcriptionally Active Human Papillomavirus 16 From Patient Saliva

https://doi.org/10.21203/rs.3.rs-3147471/v1

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published 10 Apr, 2024

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Head and neck cancers, particularly oropharyngeal cancers (OPC), have been increasingly associated with human papillomavirus (HPV) infections, specifically HPV16. The current methods for HPV16 detection primarily rely on p16 staining or PCR techniques. However, it is important to note the limitations of conventional PCR, as the presence of viral DNA does not always indicate an ongoing viral infection. Moreover, these tests heavily rely on the availability of tissue samples, which can present challenges in certain situations. In this study, we developed a qPCR multiplex approach to detect HPV16 oncogenes E6 and E7 RNA in saliva samples from OPC patients. Salivary supernatant was used as the liquid biopsy source. We successfully obtained RNA from salivary supernatant, preserving its integrity as indicated by the detection of several housekeeping genes. Our multiplex approach accurately detected E6 and E7 RNA in HPV16-positive cell lines, tissues, and finally in OPC salivary samples. Importantly, the assay specifically targeted HPV16 and not HPV18. This multiplexing technique allowed for reduced sample input without compromising specificity. The multiplex qPCR approach presented in this study demonstrates the potential in accurately detecting viable HPV16 in saliva and tissue samples from OPC patients. Since the assay measures HPV16 RNA, it provides insights into the transcriptional activity of the virus. This could guide clinical decision-making and treatment planning for individuals with HPV-related OPC.

The underlying mechanisms responsible for head and neck cancers (HNC) are multifaceted and encompass both endogenous and exogenous risk factors. Tobacco and alcohol consumption are widely recognised as the primary risk factors for HNC [1]. The synergistic effect of tobacco and alcohol consumption on HNC has been extensively documented. However, human papillomavirus (HPV) infection has been implicated as a key determinant in the development of oropharyngeal cancers (OPC) [2]. It is estimated that nearly 70% of OPC cases are HPV positive, with a substantial proportion of these cases occurring in the tonsils [3]. Additionally, approximately 85% of HPV-positive OPC cases are infected with oncolytic variants, such as HPV 16 or HPV 18 [4]. These HPV-positive OPCs have been found to have distinct clinical characteristics when compared to HPV-negative tumours, particularly with respect to treatment response and overall survival rates [5, 6].

HPV-related OPCs are growing with increasing incidence rates reported in both developed and developing countries. In some regions, the incidence of HPV-positive OPC has now surpassed that of HPV-negative OPC [3]. Studies conducted the United States and Europe have demonstrated a sharp rise in the incidence of HPV-positive OPC, particularly in young adults. In the United States, the incidence of HPV-positive OPC has increased by 225% among young men in the last two decades [7]. In Europe, a similar trend has been observed, with incidence rates of HPV-positive OPC increasing by approximately 60% among young men in the last 10 years [8].

In developing countries, the incidence of HPV-related OPC is rising and is expected to continue to increase [9]. In low- and middle-income countries within both Asia and Africa, the incidence of HPV-positive OPC has been increasing at a faster rate than in developed countries, with HPV 16 and 18 being the most common high-risk types identified [9].

High-risk strains of human papillomaviruses code for several oncogenes, in particular, E6 and E7. Under normal conditions, E6 and E7 are expressed at low levels and are thought to function by creating conditions in the infected oral keratinocytes that favour replication of the virus and prevent apoptosis of host cells [10]. At higher levels, these two oncoproteins have major effects on a variety of cellular functions that may lead to uncontrolled growth [11]. E6 is best known for its ability to bind to and mediate the degradation of the tumour suppressor p53 [12] and other targets involved in cellular apoptotic pathways [13]. As a consequence of these interactions, cells expressing E6 are much less likely to undergo apoptosis. E7 is known for its ability to bind to and inactivate the tumour suppressor Rb protein, disrupting its ability to regulate E2F transcription factors, resulting in disrupted cell cycle regulation [13].

The detection of HPV is a critical component in the diagnosis and management of HPV-related OPC. There are several techniques available for HPV detection, including: polymerase chain reaction (PCR)-based methods; hybrid capture (HC) assays; in situ hybridization (ISH); and p16INK4a (p16) overexpression detection via immunohistochemistry (IHC) [14, 15]. p16 detection is the most used method in the diagnosis of HPV-related OPC [16]. While p16 overexpression is a marker of HPV-associated malignancy, the interpretation of p16 results can be subjective and may result in inter-observer variability [17]. In addition, using the p16 detection method may produce false positive results, as p16 overexpression can occur due to other causes besides HPV infection [18]. Moreover, p16 overexpression is a late event in HPV-associated carcinogenesis, meaning that it may not be present in early-stage cancers [19].

PCR methods have been widely used for the detection of HPV16 [20–26]. However, the major problem with the PCR approach is that the detection of viral DNA does not indicate an active infection. The virus may be dormant, and patients, even though they test positive for HPV, may not go on to develop cancer [26]. Despite the various methods available for HPV detection, there is a lack of RNA-based HPV testing which can indicate an active infection. Furthermore, most of these tests require a tissue biopsy which may limit the scope of testing.

There has been a growing interest in using saliva as a liquid biopsy for diagnosing certain diseases [27–30]. Saliva is an ideal choice as it contains trace amounts of inorganic elements and a diverse population of biological particles, making it a "mirror to the body" that reflects both local and systemic conditions [31]. This makes it plausible that saliva may contain RNA species released by head and neck cancer cells or HPV16 within the oral cavity.

To this end, our study aimed to develop and evaluate a probe-based multiplexing quantitative PCR (qPCR) technique to identify viable HPV16 RNA in the saliva of patients with OPC. Detecting active or replicating virus in the saliva of OPC patients would be a valuable clinical tool that could aid in directing appropriate treatment strategies for these individuals.

Cell lines

The cell lines used in this study included: squamous cell carcinoma from the cervix, SiHa (HPV16 positive); epidermoid carcinoma from the cervix, CaSki (HPV16 positive); adenocarcinoma of the cervix, HeLa (HPV18 positive); and ductal carcinoma of the mammary gland, MCF-7 (HPV negative). All cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) GlutaMAX™ (Thermo Fisher Scientific) with 1% glutamine, 10% foetal calf serum (Thermo Fisher Scientific) in a 37°C incubator with humidified 5% CO2.

Tissue specimens

Tissue specimens were retrieved from patients treated for Squamous Cell Carcinoma (SCC) of the Oropharynx at Royal Prince Alfred Hospital, Sydney, between 2002 and 2006. The study was approved by the Research Ethics Committee at Royal Prince Alfred Hospital, Sydney, Australia (Protocol number X05-0270). Informed consent was obtained for the collection of fresh tissues. Immediately after surgical resection, tissues were snap frozen on dry ice and stored at -70°C. The histology of tissues was assessed by hospital pathologists. For this study, six fresh-frozen tissues samples were selected from p16 positive (n = 4) and p16 negative (n = 2) OPC specimens.

Saliva specimens

All HNC saliva samples were obtained from patients through informed written consent with approval by the ethics board at Royal Prince Alfred Hospital, Sydney, between 2018 and 2022 (Ethics: X19-0195 and 2019/ETH11588). From each patient, 2mL of saliva was collected directly into sterile collection tubes (non-commercial kit) or a commercial kit (DNA/RNA Shield SafeCollect Saliva Collection Kit, Zymo Research). For this proof-of-principle study, we collected saliva specimens from p16 positive (n = 9) and p16 negative (n = 5) HNC patients.

RNA Isolation from Cell Lines

5X10⁶ cultured tissue cells was homogenised by adding 1mL of RNAzol® RT (Molecular Research Center). The homogenate was incubated for five minutes after the addition of 0.4mL RNase-free water (Invitrogen, Thermo Fisher Scientific) for DNA, protein and polysaccharide precipitation, and centrifuged at 12, 000 x g for 10 minutes. The supernatant was then transferred to a fresh tube and 5µL 4-bromoanisole (Molecular Research Center) was added for RNA purification. The sample was incubated for three minutes and centrifuged at 12, 000 x g for 10 minutes. RNA was precipitated out by adding one volume isopropanol (Sigma Aldrich) to the supernatant. The sample was incubated overnight at -20°C and then centrifuged at 12, 000 x g for 10 minutes. The supernatant was then discarded, and the RNA pellet was washed twice with 75% ethanol (Sigma Aldrich) by centrifugation at 12,000 x g for 5 minutes. Lastly, the RNA pellet was resolubilised in 20µL of RNase-free water.

RNA isolation from Tissue

Briefly, 100mg of fresh frozen tissue was diced with a surgical blade, homogenised with a mortar and pestle, and rinsed with 1mL of RNAzol® RT. 0.4mL water was added to the sample and centrifuged at 12,000 x g for 10 minutes to precipitate the DNA and proteins. The sample was purified using BAN and centrifuged again at 12,000 x g for 10 minutes. RNA was precipitated using isopropanol according to the above protocol, and the sample was incubated at -20°C overnight. The RNA was then washed with 75% ethanol twice and the RNA pellet was resuspended in 20 µL of RNase-free water.

RNA isolation from Saliva

Our previously published protocol to isolate total RNA from serum [32] was modified and adapted to isolate total RNA from patient saliva samples. Following the collection of saliva samples from storage (-30°C), the sample was centrifuged at 1,600 x g for 15 minutes at 4°C to separate the cellular debris from the supernatant. The salivary supernatant was then aliquoted into lots of 400µL for processing. Samples were homogenised with 1.5mL Tri-Reagent RT-Liquid Samples (Molecular Research Centre) and 100µL 4-bromoanisole was added for phase separation. Samples were centrifuged at 12,000 x g for 20 minutes at 4°C in 1.5mL phase-lock gel tubes (5PRIME), as per the manufacturer’s protocol.

After the sample was centrifuged, the uppermost clear aqueous phase containing RNA was decanted into a fresh DNA Eppendorf Lo-bind tube. 500µL isopropanol and 5µL Glycogen was added to the sample. The sample was then inverted and stored for incubation overnight at -20°C. Following incubation, the sample was centrifuged at 12,000 x g for 20 minutes at 4°C. Immediately after centrifugation, the supernatant was removed, and the remaining pellet was flash spun (centrifuged) at 16,000 x g for two minutes at 4°C. Any remaining liquid was removed from around the pellet and then the pellet was washed twice with 1mL of 70% ethanol and allowed to air dry prior to re-suspension in 20µL of RNase-free water. For a higher total RNA yield, samples from the same participant were pooled into the same 20µL of RNase-free water.

RNA Quantification and Quality Control

The re-suspended total RNA was quantitated using a Nanodrop™ 1000 3.7.1 UV-Vis Spectrophotometer (Thermo Fisher Scientific). The Nanodrop stage was cleaned with water and 70% ethanol. Following this, the instrument was blanked using 1µL of RNase free water. Using 1µL of sample, the absorbance spectra was measured, and the data obtained from the 260nm peak indicated the concentration of total RNA. Absorbance ratios at 280nm (A₂₆₀/ A₂₈₀) and 230nm (A₂₆₀/A₂₃₀) were noted for purity purposes. Accepted ratios for purity vary with downstream applications, however, typical A₂₆₀/ A₂₈₀ ratios should be between 1.8–2.2, while requirements for A₂₆₀/A₂₃₀ ratios are generally greater than 1.7.

cDNA Synthesis

cDNA synthesis was prepared using the High-Capacity cDNA Reverse Transcription Kit from Thermo Fisher Scientific using standard concentrations. Tubes were then placed in a thermocycler and run using the following conditions: 10 minutes at 25°C, 120 minutes at 37°C, 5 minutes at 85°C and the sample was held at 4°C until collected.

Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR); Multiplexing HPV16 Oncogenes E6/E7

Following cDNA synthesis, samples were diluted 1:4, and RT-qPCR was performed using a 5µL reaction. All qPCR reactions were conducted in triplicate using the StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, USA) with the recommended cycling conditions for the TaqMan Universal PCR Master Mix (Applied Biosystems, Thermo Fischer Scientific, USA). TaqMan assays were designed for the oncogenes E6 and E7, using Primer3Plus (https://primer3plus.com/) based on sequences obtained from NCBI (https://www.ncbi.nlm.nih.gov/refseq/). In order to multiplex these two oncogenes, E6 was labelled with a VIC™ and E7 was labelled with a FAM™ probe.

Data Analysis

qPCR data was imported into GraphPad Prism (Version 8.2.1) and Cq values were plotted against sample using column graphs that compare both singleplex and multiplex qPCR results. PCR efficiencies were determined using LinRegPCR (v 2021.2) and measured on a scale between 1.0 and 2.0, with 2.0 representing 100% efficiency. PCR efficiencies above 1.5 were determined to be acceptable. A two-sided t-test was used to determine whether a significant difference was observed between singleplex and multiplex PCR reactions (Cq values) and their PCR efficiencies (p < 0.05).

HPV16 singleplex and multiplex with HPV16 positive cell lines

The E6 and E7 primer/probe combination were firstly tested by singleplex and multiplex qPCR using the HPV16 positive cell lines, SiHa and Caski, and HPV16 negative cell lines, HeLa and MCF-7. Cq values were measured for two total RNA input amounts, 100ng and 200ng. Both E6 and E7 were detected in the HPV16 positive cell lines only (Fig. 1A & B). Despite a 2-fold increase in RNA input, the expression levels of both probes were lower in SiHa cells, which was determined to be due to this cell line only having two viral insertions of HPV16 [33]. Nevertheless, 100ng of input RNA was found to be adequate to detect both E6 and E7. Furthermore, no significant difference in Cq value was observed between the singleplex and multiplex qPCR methods. PCR efficiencies for HPV16 positive results for both the E6 and E7 were calculated using LinRegPCR. All HPV16 positive samples remained above an efficiency of 1.5, and no significant difference was observed between the mean qPCR efficiency of the singleplex and multiplex of E6 and E7 mRNA in samples at both 100ng and 200ng (p value < 0.05) (Fig. 1C & D). Overall, multiplexed samples fared better than singleplex in terms of PCR efficiency, particularly for the E7 assay.

Minimal RNA required for detection of HPV16

Next, we evaluated the minimum quantity of total RNA necessary to detect E6 (Fig. 2A) and E7 (Fig. 2B). We diluted SiHa and Caski total RNA within a range of 50pg, 100pg, 500pg, 1ng, 2ng, 3ng, and 4ng, using a Cq value of 35 as the designated threshold for a positive outcome. Our findings indicated that at an RNA input of 50pg for both SiHa and Caski in the E6 assay, as well as for SiHa in the E7 assay, the Cq value exceeded 35, resulting in a negative determination. All samples with RNA concentrations above 1ng consistently fell below the Cq threshold. Therefore, the determined minimal amount of RNA required for detection was 100pg. Notably, no significant disparity in Cq values was observed between the singleplex and multiplex methods (p < 0.05). We further computed the PCR efficiency for the E6 and E7 assays in HPV positive samples using LinRegPCR (Fig. 2C and Fig. 2D). While some variations in the mean qPCR efficiencies were noted, the majority of samples remained above the threshold of 1.5. In samples with minimal RNA input, singleplex reactions exhibited superior PCR efficiencies compared to multiplex reactions.

Single and multiplex detection of E6 and E7 using Patient Tissue

Subsequently, we evaluated the expression of E6 and E7 in patient tissues, distinguishing between HPV16 positive and HPV16 negative samples (Fig. 3A, B, C & D). The isolated RNA from these samples was serially diluted, resulting in total inputs of 5ng, 10ng, 50ng, and 100ng for all patient specimens. Both the E6 and E7 assays successfully detected their respective targets in both singleplex and multiplex formats. As anticipated, higher amounts of RNA input led to decreased Cq values. In p16 negative patient tissues, no E6 or E7 mRNA was detected. Based on these findings, an RNA input of 10ng would guarantee a positive result below the designated Cq threshold of 35. Furthermore, PCR efficiencies were determined using LinRegPCR (Fig. 3E & F). While the majority of samples remained above the established threshold of 1.5, we observed variations in average qPCR efficiencies. In samples with minimal RNA input (i.e., less than 10ng), singleplex reactions exhibited superior PCR efficiencies compared to multiplex reactions. Notably, a sample input of 100ng of total RNA demonstrated the highest performance, with PCR efficiencies closest to 2.0.

Detection of viable HPV16 virus in patient saliva

The collection method for saliva is pivotal for any downstream testing. We decided to compare two standard approaches; 1) the collection of whole saliva with no additives, and 2) the collection of saliva using a commercial kit (containing additives). After collection, total RNA was isolated per our laboratory’s published methodology [34]. Figure 4A depicts the RNA concentrations obtained when isolating total RNA from whole saliva. The mean total RNA concentration for the salivary supernatant using the non-commercial kit was 67ng, which was significantly lower in yield compared to the mean total RNA concentration for the salivary supernatant collected using the commercial kit at 1,775 ng. We note that the while the mean total RNA obtained from the commercial kit was much improved from the non-commercial kit, the total RNA concentrations still have a large variation, similar to what we observed in whole saliva total RNA isolations. RNA was isolated from 28 individuals using the non-commercial kit methodology and 18 individuals using the commercial kit.

We then determined if the RNA from these commercial kits would provide usable material for PCR amplification. As shown in Fig. 4B, we were able to detect the following targets, 18S, Beta Actin (ACTB), p53 and Dicer1, with RNA collected from both methods. RNA input into the qPCR was standardised to 100ng. Cq data for the commercial and non-commercial kits were aggregated to understand how the methods compared. For 18S, the mean Cq value for the non-commercial kit was 20.0 and the mean Cq value for the commercial kit was 12.8. Similarly, mean Cq values for ACTB were 29.4 and 25.2 for non-commercial and commercial kits respectively. Cq values for p53 were similar for both kits with a mean Cq value of 30.9 for non-commercial and 31.0 for commercial. Mean Cq values for Dicer1 were 33.7 for non-commercial and 29.5 for commercial. Overall, the commercial kit performed better across the various gene targets.

Multiplex qPCR of E6 and E7 mRNA in Patient Saliva

In the next step, we used isolated RNA from the collected saliva, to determine if a multiplex qPCR could be used to amplify and detect both E6 and E7 targets (Fig. 5B & C). As a proof of principle, 100ng of total RNA was isolated from 1.2mL of saliva from eight HNSCC patient specimens, including three p16 positive and five p16 negative patients. Using a multiplex approach, both E6 and E7 were detected in p16 positive samples. The E6 assay (mean Cq 31.5) had slightly increased levels of expression compared to the E7 assay (mean Cq 34.4). As expected, the E6 and E7 targets were not found in the p16 negative patients. Beta 2 Microglobulin (B2M) was used as a both positive control and reference gene and was amplified and detected in all samples (Fig. 5A).

PCR efficiencies of the HPV16 positive results for both the E6 and E7 assays were calculated using LinRegPCR (Fig. 6A & B). Overall, both E6 and E7 performed with the same mean PCR efficiency of 1.8 across the two assays and methodologies. The E6 assay showed less variability in PCR efficiency than the E7 assay. All p16 positive patient saliva samples remained above the 1.5 efficiency threshold and there was no significant difference in mean qPCR efficiencies between the singleplex and multiplexing of E6 and E7 mRNA (p < 0.05) (Fig. 6C & D).

Patients with HPV-positive OPC commonly exhibit better treatment outcomes and overall survival rates compared to their HPV-negative OPC counterparts [35]. The identification of HPV in OPC can guide treatment selection by identifying patients who may respond more favourably to chemotherapy and radiation therapy. Furthermore, HPV detection in OPC patients serves as a vital diagnostic and monitoring tool [36].

The oncogenes E6 and E7, which are encoded by HPV, play a critical role in the development of HPV-associated cancer by disrupting the normal cell cycle and promoting uncontrolled cell growth. Thus, they are useful biomarkers for the detection of HPV and the identification of high-risk HPV-associated cancer [37, 38] [39].

High-risk HPV infection is also closely linked to the overexpression of p16 protein. Positivity for p16 is now integrated into the recent 8th edition of tumour-node-metastasis (TNM) classification by the Union for International Cancer Control (UICC), and p16-positive OPCs are substantially down-staged compared to p16-negative OPCs [21]. Although p16-IHC is highly sensitive for high-risk HPV infection, it is not an entirely specific surrogate marker [21], which results in false positives due the overexpression of p16 that is not linked to HPV infection. It is estimated that this problem affects approximately 5–10% of all OPCs [40–42].

Consequently, the use of p16-IHC or HPV-specific testing alone as a reliable means of determining HPV status has been called into question, with recent studies identifying a subgroup of patients with discordant p16 and HPV positivity [43]. Specifically, most of the discrepant cases reported to date are p16-positive but HPV DNA-PCR or DNA-ISH negative. In light of these findings, the College of American Pathologists (CAP) recommends additional HPV-specific testing at the discretion of the pathologist and/or treating clinician following p16 testing [44]. These developments underscore the need for greater scrutiny of testing methods and the importance of accurate HPV status determination in guiding clinical decision-making.

It is for this reason that viral RNA expression has been suggested as the gold standard for a viable infection, meaning the virus is transcriptionally active. In this study, we sought to develop and optimise a method to non-invasively detect virally active high-risk HPV16 using a multiplex qRT-PCR in OPC patient saliva.

From the results, we were able to successfully detect transcriptionally active E6 and E7 mRNA transcripts in HPV16 positive cell lines, SiHa and Caski with as low as 100pg of input RNA. Mean PCR efficiencies were approximately 1.8, which suggests optimal PCR amplification. This indicates that the PCR reactions are producing E6 and E7 amplicons with close to 100% efficiency. We then multiplexed the assays in p16 positive OPC patient tissues using various concentrations of input RNA. We detected transcriptionally active E6 and E7 mRNA in the p16 positive patient specimens only and increasing levels of mRNA expression was associated with increased RNA input. While some variability in PCR efficiency for these reactions was observed, particularly for samples with low input RNA (< 10ng), mean PCR efficiencies remained above 1.7.

As expected, when testing the OPC saliva samples, transcriptionally active E6 and E7 mRNA was only found in the p16 patient saliva samples, confirming the specificity of the assays. The E6 assay performed slightly better than the E7 assay with a mean Cq value of 31.5, compared to 34.4 respectively. This reflects other studies [45, 46], where the E6 protein is thought to be more consistently expressed across different types of HPV and different stages of infection, and the expression of E7 is more varied. The mean PCR efficiency was 1.8 across both E6 and E7 assays and the singleplex and multiplex methods, indicating the salivary assays worked with high efficiency.

Saliva testing offers advantages over blood and tissue-based testing due to its non-invasiveness and ease of sample collection, allowing for a time- and cost-effective diagnosis. Multiplexing assays to include both E6 and E7 mRNA targets offers confirmation of active virus (viable infection) and may provide an alternative to the need for DNA-PCR or ISH tests. Furthermore, this methodology is scalable and well-suited for high-throughput screening, making it an attractive option for widespread screening or HPV16 surveillance programs.

Several studies have sought to use saliva for oral cancer detection but very few studies to date have used RNA to detect viable infections [47–54]. In one Australian study, it was demonstrated that saliva rinses could be used to detect key HPV-DNA oncogenic targets with 92.9% sensitivity. They found that 39 of 42 oral rinses from p16 positive patients had detectable HPV16-DNA [49]. Another study using oral rinse from 110 patients employed nested PCR to detect low copy numbers showed a sensitivity rate of 75% [52].

Even though p16 detection in tumour tissue is the conventional method for HPV16 testing, studies have demonstrated that the detection rates for HPV16 are comparable between tissue and oral rinses, with the prevalence of HPV DNA similar in tumour tissue (59.2%) and oral rinses (53.2%). Furthermore, antibodies specific to HPV16 E6 and E7 were present in serum at a similar rate of 51.4%. Although the sensitivity rates were low, it suggests that HPV detection in oral rinses may be comparable with the gold standard method of p16 testing in tumour tissues [51].

We note that digital PCR (ddPCR) was previously used to detect HPV16 RNA in oropharyngeal swabs. It was demonstrated that the sensitivity and specificity of ddPCR in detecting p16 were 92% and 98%, respectively. It also showed that ddPCR was able to detect E6 and E7 mRNAs with much higher sensitivity than conventional RT-qPCR. This was achieved using 20-50-fold less RNA than what is required for conventional RT-qPCR [54].

The main caveat with our approach is that the qPCR method only detects RNA, however there is the possibility of false negatives. That is, the virus is dormant and not transcriptional active. It may be possible to use this salivary qPCR as a companion diagnostic to p16 staining and a dual positive result may be more clinically informative than p16 staining alone. The future development of this assay to include other HPV16 targets may resolve these caveats. One possible approach is to identify HPV16 genes associated with viral dormancy and include these targets along with E6 and E7. The E2 gene is frequently overexpressed during viral latency and a key regulatory of both E6 and E7 [55]. The triumvirate of E2, E6, and E7 targets might be able to discern between viable and latent viral infection. Another strategy is to detect both the presence of viral DNA and RNA in the same qPCR assay. Other viral DNA targets could include the L1 and L2 genes which are highly conserved [56, 57] or non-transcribe regions of the HPV16 genome. The latter would be an ideal qPCR target as it is not transcribed and only primers designed for this region would bind and amplify the DNA sequence.

We also acknowledge that the sample size used for the salivary testing is limited and a larger cohort will be required to further assess the sensitivity and specificity of this salivary qPCR method. An additional hurdle in utilizing salivary samples is the absence of universally recognized standards for the collection and handling of such specimens. A consistent collection protocol and a reliable approach for extracting genomic material must be established to address this issue [34]. Presently, only a limited number of techniques are available that can extract both DNA and RNA from a single salivary sample [58, 59].

Overall, continued efforts towards standardisation and optimisation of saliva-based testing will be important for advancing the field of liquid biopsy and improving patient diagnosis. Despite these challenges, the use of saliva in HPV16 testing continues to show promise. Ongoing efforts to standardise salivary collection, processing, and inclusion of other RNA/DNA targets, will be critical in developing a robust qPCR liquid assay for HPV detection.

Ethical approval and consent to participate.

The research was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent for participation in the study procedures and collection of specimens. The study protocol was approved by the Sydney Local Health District Ethics Committee under the Head and Neck Tumour Bank (Protocol No X-14-0107) and the University of Technology Sydney.

Consent to publication

Not applicable

Data availability statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Conflict of interest

The authors declare no competing interests.

Funding

This project was funded by a Translational Cancer Research Network PhD Scholarship Top-Up Award, supported by the Cancer Institute NSW.

Acknowledgement

Not applicable

Author contribution

Principle investigator: NT; Conception and design: NT, FD, DS; Samples provided by: ME; Experimentation: FD, DS; Drafting of the manuscript: FD; Critical revision of the manuscript: NT, DS, MH, NHT, ME. All authors read and approved the final manuscript.

Author information (Optional)

Not applicable

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No competing interests reported.

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published 10 Apr, 2024

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You are reading this latest preprint version

A Liquid Biopsy To Detect Transcriptionally Active Human Papillomavirus 16 From Patient Saliva

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Journal Publication

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Abstract

Figures

Introduction

Materials and methods

Cell lines

Tissue specimens

Saliva specimens

RNA Isolation from Cell Lines

RNA isolation from Tissue

RNA isolation from Saliva

RNA Quantification and Quality Control

cDNA Synthesis

Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR); Multiplexing HPV16 Oncogenes E6/E7

Data Analysis

Results

HPV16 singleplex and multiplex with HPV16 positive cell lines

Minimal RNA required for detection of HPV16

Single and multiplex detection of E6 and E7 using Patient Tissue

Detection of viable HPV16 virus in patient saliva

Multiplex qPCR of E6 and E7 mRNA in Patient Saliva

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1