*Article* **Serum HPV16 E7 Oncoprotein Is a Recurrence Marker of Oropharyngeal Squamous Cell Carcinomas**

**Lucia Oton-Gonzalez <sup>1</sup> , John Charles Rotondo <sup>1</sup> , Carmen Lanzillotti <sup>1</sup> , Elisa Mazzoni <sup>1</sup> , Ilaria Bononi <sup>2</sup> , Maria Rosa Iaquinta <sup>1</sup> , Luca Cerritelli <sup>3</sup> , Nicola Malagutti <sup>3</sup> , Andrea Ciorba <sup>3</sup> , Chiara Bianchini <sup>3</sup> , Stefano Pelucchi <sup>3</sup> , Mauro Tognon 1,\* and Fernanda Martini 1,\***


**Simple Summary:** Classical markers alone, such as HPV DNA, p16 and HPV mRNA expression, are not enough to stratify HPV-positive head and neck squamous cell carcinoma (HNSCC) patients, but when combined with serological markers, the latter are strong indicators of prognosis in oropharyngeal squamous cell carcinoma (OPSCC) patients. Specifically, HPV16 E7 oncoprotein in serum at the time of diagnosis, correlates with disease recurrence and patient overall survival. To our knowledge, this is the first study to investigate HPV E7 oncoprotein in patient serum. The E7 oncoprotein detection in serum at the time of diagnosis may be useful as a non-invasive procedure for HPV-positive OPSCC patient stratification and follow-up, helping to identify patients at risk for tumor recurrence and metastasis during follow-up, and ultimately, providing a tool for clinicians to identify patients for de-escalation treatment or those to be kept under close surveillance.

**Abstract:** Despite improved prognosis for many HPV-positive head and neck squamous cell carcinomas (HNSCCs), some cases are still marked by recurrence and metastasis. Our study aimed to identify novel biomarkers for patient stratification. Classical HPV markers: HPV-DNA, p16 and HPV mRNA expression were studied in HNSCC (*n* = 67) and controls (*n* = 58) by qPCR. Subsequently, ELISA tests were used for HPV16 L1 antibody and HPV16 E7 oncoprotein detection in serum at diagnosis and follow-up. All markers were correlated to relapse-free survival (RFS) and overall survival (OS). HPV-DNA was found in HNSCCs (29.85%), HPV16-DNA in 95% of cases, HPV16 E7 mRNA was revealed in 93.75%. p16 was overexpressed in 75% of HPV-positive HNSCC compared to negative samples and controls (*p* < 0.001). Classical markers correlated with improved OS (*p* < 0.05). Serological studies showed similar proportions of HPV16 L1 antibodies in all HNSCCs (*p* > 0.05). Serum E7 oncoprotein was present in 30% HPV-positive patients at diagnosis (*p* > 0.05) and correlated to HNSCC HPV16 E7 mRNA (*p* < 0.01), whereas it was associated to worse RFS and OS, especially for oropharyngeal squamous cell carcinoma (OPSCC) (*p* < 0.01). Detection of circulating HPV16 E7 oncoprotein at diagnosis may be useful for stratifying and monitoring HPV-positive HNSCC patients for worse prognosis, providing clinicians a tool for selecting patients for treatment de-escalation.

**Keywords:** human papillomavirus; oropharyngeal squamous cell carcinoma; treatment de-escalation; patient stratification; E7 oncoprotein; HPV DNA; HPV antibodies; ELISA

**Citation:** Oton-Gonzalez, L.; Rotondo, J.C.; Lanzillotti, C.; Mazzoni, E.; Bononi, I.; Iaquinta, M.R.; Cerritelli, L.; Malagutti, N.; Ciorba, A.; Bianchini, C.; et al. Serum HPV16 E7 Oncoprotein Is a Recurrence Marker of Oropharyngeal Squamous Cell Carcinomas. *Cancers* **2021**, *13*, 3370. https://doi.org/ 10.3390/cancers13133370

Academic Editor: Fabrizio Bianchi

Received: 18 May 2021 Accepted: 2 July 2021 Published: 5 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Human papillomavirus (HPV)-related head and neck squamous cell carcinomas (HN-SCCs) are increasing worldwide [1]. Specifically, HPV-positive oropharyngeal squamous cell carcinomas (OPSCC) have increased over the past few years, with approximately 93,000 new OPSCCs diagnosed per year worldwide [2–4]. HPV-positive OPSCCs constitute a biologically distinct group of HNSCCs. Indeed, the American Joint Committee on Cancer, 8th edition, reports improved prognosis and treatment outcome for HPV-positive OPSCC patients compared to HPV-negative cases [5,6].

HPV plays an important role in OPSCC onset [7]. The main transforming activity of HPV relies on oncoproteins E6 and E7, which hamper p53 and pRb tumor suppressor protein activities, respectively. Moreover, HPV E6 and E7 oncoproteins are key players in tumor development, accounting for immune escape, angiogenesis, and the formation of a pro-proliferative microenvironment [8].

Optimizing protocols through targeted therapies and personalized treatments is paramount to increase survival rates for all patients [4]. In this context, several clinical trials, such as PATHOS (NCT02215265) or OPTIMA (NCT02258659), are now in progress [9,10] aiming to determine whether treatment de-intensification could improve quality of life for HPV-positive OPSCC patients whilst maintaining high rates of cure. Indeed, even if HPV-positive OPSCC patients usually respond to treatment de-escalation, 10–25% of HPV-positive patients present recurrences and worse prognosis [9,11–13].

Hence, correctly stratifying HPV-positive patients is necessary to select optimized treatment [14]. In an effort to improve stratification, many studies investigate HPV status, p16 overexpression which is the surrogate marker of HPV transformation [6,12], and HPV E6/E7 mRNA expression [15]. However, the current stratification system leads to several pitfalls, i.e., (i) HPV might be present as a transient infection, but not active in tumors [15]; (ii) p16 expression is not always observed in HPV-positive tumors [16]; (iii) HPV mRNA levels could be too low for detection [17,18].

Serological testing has gained interest in the past few years for HPV-positive OPSCC prognostic studies. The immune response of the host has been studied in association with both HPV-positive tumors and patient prognosis [4,19]. Serum IgG antibodies against HPV16 L1 capsid protein can be detected several years before OPSCC onset/presentation, but are also cumulative markers of viral exposure [20,21]. Antibodies to HPV16 E6 and E7 oncoproteins at the time of tumor diagnosis may be useful to predict disease-free survival in HPV-positive OPSCC patients [22,23]. However, routine testing for antibodies against HPV oncoproteins are difficult to perform due to the lack of available commercial kits.

Studies on cervical cancer have shown that detection of HPV E6 and E7 oncoproteins in cervical scrapings may constitute valuable markers for disease progression [24,25]. Moreover, the presence of HPV16 E6 and E7 oncoproteins has been demonstrated by direct ELISA in culture supernatant of HPV-positive cervical cancer cell lines SiHa and CaSki, indicating the release of viral oncoproteins from tumor cells [26].

Therefore, we have hypothesized that HPV E6 and E7 oncoproteins could be present in serum from HPV HNSCC patients, and their serum identification could be useful for prognostic purposes. The presence of HPV E6 and E7 oncoproteins in serum from HPVassociated cancer patients is yet to be investigated. Since new kits for testing serum HPV E7 protein are now commercially available new investigations can be carried out.

The aim of this study was to identify markers for HPV-positive HNSCC patient stratification. To this purpose, classical tumor markers, such as HPV DNA, p16 expression and HPV E7 mRNA were studied in different HNSCC subtypes, including OPSCC. Sera of HNSCC patients were analyzed for HPV16 L1 antibody titers and, for the first time, HPV16 E7 oncoprotein levels at the time of tumor diagnosis and during follow-up at 3, 6, 12 and 24 months. Finally, results were correlated to patient relapse-free survival (RFS) and overall survival (OS) at 24 months.

#### **2. Materials and Methods**

#### *2.1. Patient Samples*

HNSCC specimens (*n* = 67) from patients, mean age ± standard deviation [SD] 64.94 ± 10.88 years [y] old, were collected for analyses. Tumor-free tonsillar (TFT) samples (*n* = 58), from non-oncological patients who had undergone tonsillar surgery, 39 ± 15.17 y old, were used as controls. Samples were collected consecutively from 2016 to 2020 at the Ear, Nose and Throat (ENT) Clinic (University Hospital of Ferrara, Ferrara, Italy). Inclusion criteria was the histopathological detection of primary HNSCC in patients 18–95 y old, including hidden or occult SCC with lymph node cervical positive histology. Exclusion criteria were radiotherapy and/or chemotherapy treatment. The 8th edition of AJCC classification was used [5]. Tumor and TFT specimens were collected at the time of surgery. Blood specimens were also collected from HNSCC patients at the time of surgery and during patient follow-up at 3, 6, 12, and 24 months.

#### *2.2. Nucleic Acid Extraction*

DNA/RNA extractions from HNSCC (*n* = 67) and TFT tissues (*n* = 58) were carried out using the AllPrep DNA/RNA/Protein Extraction Kit (Qiagen, Milan, Italy). After quantification using the NanoDrop 2000 (Thermo Scientific, Milan, Italy), DNA and RNA samples were stored at −80 ◦C until analyses. DNA suitability for PCR analysis was assessed as before [27,28]. Total mRNA was retro-transcribed using the Improm II (Promega, Fitchburg, WI, USA) reverse transcription system [29].

#### *2.3. HPV Analysis*

HPV (GenBank: K02718.1) screening was performed by quantitative PCR (qPCR) using the GP5+/GP6+ primer pair (Table S1) [27]. DNA (50 ng) was analyzed in 10 µL reactions consisting of 2× SsoAdvanced Universal SYBR Green Supermix, Bio-Rad (Hercules, CA, USA) and 500 nM of each primer. QPCR analyses were performed in triplicate. Thermal cycling was: 95 ◦C for 5 min, 40 cycles of 95 ◦C for 15 s and 60 ◦C for 30 s. To discriminate between HPV genotypes, a final high-resolution melting (HRM) step was added from 65–95 ◦C, increasing 0.1 ◦C every 0.03 s. Recombinant plasmids containing DNA sequences from HPV types 6/11/16/18/31/33/45 were used as positive controls. An HPV-negative genomic DNA sample, and a mock sample, without DNA, were used as negative controls. HPV genotyping was done by comparing sample melting curves to the plasmid controls. Quantification of the viral DNA load was performed in comparison to the standard curve of a plasmid-HPV-type specific [29].

#### *2.4. Gene Expression Analysis*

QPCR was performed for p16 and HPV16 E7 gene expression analyses. Briefly, 50 ng of cDNA were used in 10 µL reactions using 2× SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). A final concentration of 500 nM of each primer was employed (Table S1). Samples were run in triplicate, along with mock samples used as negative controls. Thermal conditions for HPV E7 and p16 were; 95 ◦C for 5 min and 40 cycles of 95 ◦C for 15 s followed by a 60 ◦C for 30 s. Detection of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization of mRNA levels and the fold change was calculated using the 2−∆∆Ct method, as done previously [30,31]. Furthermore, data was normalized against the TFT control group.

#### *2.5. Detection of Serum HPV16 L1 Antibodies*

Upon collection, blood samples were allowed to clot for 15 min at room temperature and then centrifuged at 1300 g for 15 min. Serum HPV16 L1 IgG antibodies were evaluated in HPV-positive (*n* = 20) and HPV-negative (*n* = 8) HNSCC patients at the time of diagnosis (T0) and during follow-up at 3, 6, 12 and 24 months.

HPV16 L1 IgG antibodies were analyzed with a commercial kit (HPV16 L1, Cusabio, Houston, TX, USA). The test was performed according to the manufacturer's instructions. The signal intensity was measured as Optical Density (OD) at 450 nm (model Multiskan EX, Thermo Electron Corp., Waltham, MA, USA) [32]. The cutoff value was calculated according to the manufacturer's instructions; an OD sample/OD negative ratio, equal or greater than 2.1, was considered positive.

#### *2.6. Serum E7 Oncoprotein Level Detection*

Serum HPV16 E7 oncoprotein levels were evaluated in HPV-positive (*n* = 20) and HPV-negative (*n* = 8) HNSCC patients, using the "HPV16 E7 Oncoprotein ELISA Kit" (Cell Biolabs, San Diego, CA, USA), according to the manufacturer's instructions. The presence or absence of E7 oncoprotein was determined by considering sample absorbance above or below the cutoff value, respectively, calculated as done previously [33]. The cutoff for HPV16 E7 oncoprotein was 0.75 ng/mL. HPV16 E7 oncoprotein variation during the follow-up was assessed by the ratio between protein amount at time of relapse and at previous time point; ratios > 1 indicated increment of protein prior to relapse, while ratios < 1 indicated decrement.

#### *2.7. Statistical Analysis*

Statistical analyses were carried out using the GraphPad Prism for Windows (version 8.0, GraphPad, San Diego, CA, USA) [34]. The ANOVA test was used to compare the mean between groups for gene expression analyses. Pearson/Spearman correlation tests were used to correlate viral gene expression and HPV DNA load, and E7 oncogene and p16 expression, respectively, and to assess univariate differences of clinicopathological features according to E7 oncoprotein presence in serum. All parameters were correlated to patient's relapse-free survival (RFS) and overall survival (OS) at 24 months using the Kaplan-Meier model; statistical significance was estimated using the log-rank test. *p* values of less than 0.05 were considered statistically significant for all analyses.

#### **3. Results**

#### *3.1. HPV DNA Analysis*

HNSCCs and control samples were analyzed for HPV DNA sequences and genotype. HPV DNA was found in 20/67 (29.85%) of HNSCC samples, consisting of 2/20 (10%) oral squamous cell carcinoma (OSCC), 15/20 (75%) OPSCC, 2/20 (10%) hypopharyngeal cancer and 1/20 (5%) laryngeal cancer (Table 1). HPV-genotype was determined by high resolution melting (HRM) to be HPV16 in 19/20 (95%) of the HNSCC HPV-positive cases and HPV33 in 1/20 (5%) of the cases. Control DNAs were found to be HPV11-positive in 1/58 (1.7%) of the cases. Our further studies were hereafter focused on HPV type 16 due to high prevalence in HNSCC. Viral DNA load in cancer specimens ranged from 2.52 <sup>×</sup> <sup>10</sup>−<sup>4</sup> to 4.26 <sup>×</sup> <sup>10</sup><sup>2</sup> copies of HPV DNA per cell (Figure 1A).

