Evaluation of DNA methylation levels of SEPT9 and SHOX2 in plasma of patients with head and neck squamous cell carcinoma using droplet digital PCR
- Authors:
- Published online on: January 31, 2024 https://doi.org/10.3892/or.2024.8711
- Article Number: 52
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Copyright: © Grossi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Head and neck squamous cell carcinoma (HNSCC) is a cancer of the squamous epithelium of the oral cavity, larynx, pharynx and nasal cavity (1). HNSCC is the seventh leading cause of human malignancy, accounting for 890,000 new diagnoses and 450,000 cancer related-deaths per year worldwide. According to GLOBOCAN 2020, the global incidence of HNSCC has been increasing in recent years, and this trend is partially attributed to the growing prevalence of human papillomavirus (HPV)-related oropharyngeal carcinoma (2,3). In Italy, HNSCC accounts for ~3% of all malignancies, most observed in the male population (3). The major risk factors for HNSCC are smoking, alcohol abuse, and HPV (1). Treatments of HNSCC include surgery, radiotherapy and chemotherapy (4). However, the prognosis of HNSCC is inauspicious due to recurrent or metastatic HNSCC; in this case, curative options are very limited (1,2,4). Liquid biopsy is an important tool in molecular oncology as it is an excellent source of biomolecules, particularly circulating cell-free DNA (ccfDNA) released into the bloodstream from cell secretion or as a result of apoptosis and necrosis (5,6). In total, <1% of the ccfDNA is circulating tumor DNA (ctDNA) characterized by cancer hallmarks, such as mutations or aberrant gene methylation; their detection can serve as molecular indicators for diagnosis, prognosis, and identification of early recurrence (7,8). Alterations in the DNA methylation profile are known to occur early during cancer development, and hypermethylation of the promoter region of tumor suppressor genes is involved in cancer onset and progression (9,10). DNA methylation is a stable covalent modification that mainly occurs at the 5C position of cytosine in CpG dinucleotides to form the 5-methylcytosine and can be detected in bio-fluids by PCR-based methods (11). DNA hypermethylation of septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) has been previously described in tissues and in ccfDNA from plasma of patients with HNSCC using qPCR assay (12). SEPT9 belongs to the septin family and is involved in cytokinesis and cell cycle control (13). DNA methylation of SEPT9 has been found in different tumors and its increased methylation levels have been detected during the progression of cells to malignancy (14–17). In colon mucosa, SEPT9 methylation levels were found gradually increasing in tissues from: Controls-not-advanced adenomas-advanced adenomas-invasive adenocarcinoma (14,15). Accordingly, a significant reduction of SEPT9 protein levels was identified in adenoma and tumor tissues (15). Similarly, SEPT9 hypermethylation was observed in breast cancer (BC) tissues, but not in healthy breast tissues, and was inversely correlated with SEPT9 mRNA expression in BC cell lines and tissues (16). In BC cells, DNA hypermethylation was revealed to inhibit the expression of SEPT9, which, in turn, altered the formation of structured filaments and increased the migratory potential of tumor cells by promoting cancer progression (13,18,19). Hypermethylation of SHOX2 gene has been identified in several malignancies (20). The SHOX2 gene is a member of the SHOX gene family and encodes for a protein containing a 60-amino acid DNA-binding domain, suggesting its role as a transcriptional regulator. The exact molecular mechanism of SHOX2 or the role of SHOX2 hypermethylation during carcinogenesis has not been determined (21). However, numerous studies have clearly evidenced the strong association between SHOX2 hypermethylation and cancer progression (14,22,23). In colon mucosa, SHOX2 methylation levels gradually increased during the progression from the non-cancerous stage to the adenoma and adenocarcinoma stages (14). Similarly, SHOX2 methylation was found to be absent or low in non-malignant brain tissues and pilocytic astrocytomas, at intermediate values in lower-grade gliomas, and high in glioblastomas (23). In lung adenocarcinoma, SHOX2 methylation levels gradually increased in accordance with disease stage (from stage 0-II) and cancer invasiveness (22). Hypermethylation of circulating SHOX2 and SEPT9 has been detected in several human cancers, and they are considered promising circulating tumor liquid biopsy biomarkers (24). Methods used to detect DNA methylation are usually based on qPCR. The commonly used droplet digital PCR (ddPCR) technology provides greater sensitivity and absolute quantification of the template than conventional qPCR systems (25–28). The target templates are partitioned into 20,000 water-in-oil droplets produced by a ‘generator’, each representing a nano-sized PCR environment (29). The PCR-positive and PCR-negative droplets are automatically counted by a ‘reader’ to provide absolute quantification of the target DNA in digital form (30,31). To the best of the authors' knowledge, epigenetic studies in liquid biopsies of patients with HNSCC using ddPCR are still very limited and the combined SEPT9 and SHOX2 methylation analysis by ddPCR is lacking (32). In the present study, a ddPCR-based-assay was developed for absolute quantification of SEPT9 and SHOX2 methylation levels in ccfDNA. The ability of ddPCR-based assays to detect very low copies of methylated SEPT9 and SHOX2 was demonstrated. Finally, the feasibility of measuring SEPT9 and SHOX2 DNA methylation levels in the plasma of 20 patients with HNSCC, before curative treatment (surgery, radiotherapy, chemotherapy) and during different follow-up time points of the same patients with intervals of 3 months, was revealed. The present study is preliminary research of a larger project of liquid biopsies in HNSCC (‘Identify’ project) to assess whether DNA methylation levels of SHOX2 and SEPT9 may vary during treatment.
Materials and methods
Plasma samples from patients with HNSCC
All patients enrolled in the present study (n=20) were recruited from the Unit of Otorhinolaryngology-Head and Neck Surgery, ASST Spedali Civili, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia (Brescia, Italy). Clinical and pathological characteristics are reported in Table I for each patient. All patients with HNSCC met the following criteria: i) Histologically confirmed squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx or larynx; ii) clinical stage I–IV according to the VIII edition of the American Joint Committee on Cancer (AJCC) staging system (33) iii) aged ≥18 years and written informed consent provided. Peripheral blood samples were collected in EDTA-coated tubes. All recruited patients were screened for HPV-related disease by determining the HPV status genotyping by PANA RealTyper HPV kit CE/IVD (cat. no. PNAM-5001; HLB PANAGENE). The p16 protein expression was assessed using the CINtec p16 histological test (cat. no. 06695256001; Roche Diagnostics) with strong and widespread nuclear and cytoplasmic staining in at least 70% of cells used as a reference for positivity (Fig. S1). All these clinical characteristics were determined from the medical records of the patients; therefore they were not investigated as part of the present study. The screenings were assessed routinely in HNSCC clinic management. Peripheral blood (10 ml/patient) from patients with HNSCC was collected before the start of the first treatment (T0) and at intervals after the first treatment, including surgery, radiotherapy and chemotherapy (T1=3 months, T2=6 months, T3=12 months after treatment). Collecting liquid biopsies from patients that received the same type of treatment would have taken much longer. Plasma was obtained by centrifugation of peripheral blood at 200 × g for 10 min at 4°C in an accuSpin Micro21 centrifuge (Thermo Fisher Scientific, Inc.). The plasma was transferred to a new tube and stored at −80°C until DNA extraction. The study was approved by the Ethics Committee of Spedali Civili of Brescia (Protocol Identify, Ethics Committee approval no. NP 4551).
ccfDNA isolation from plasma and bisulfite conversion
According to the manufacturer's instructions, ccfDNA was isolated from 2 ml of plasma using MagMAX Cell-Free DNA isolation kit (cat. no. A29319; ThermoFisher Scientific, Inc.). Purified ccfDNA was eluted in a 30-µl volume, and 1 µl ccfDNA was used for ccfDNA quantification using Qubit Fluorometer and Qubit dsDNA HS (High Sensitivity) Assay kit (cat. no. Q32854; Thermo Fisher Scientific, Inc.). Following the manufacturer's instructions, the remaining ccfDNA (29 µl) was used for bisulfite conversion using EZ DNA Methylation-Lightning kit (cat. no. D5030; Zymo Research Corp). A total of 500 ng of a methylated and non-methylated human DNA standard (Human Methylated & Non-methylated DNA Set; cat. no. D5014; Zymo Research Corp.) were converted with bisulfite as positive controls. Subsequently, 13 and 10 µl of bisulfite-converted DNA were obtained from ccfDNA and methylated and non-methylated DNA, respectively, and stored at −80°C until their use.
Methylation-specific ddPCR (MS-ddPCR) assays
The MS-ddPCR assays were optimized according to the principles of MS-PCR (34,35) to detect the methylation levels of SEPT9 and SHOX2. MS-ddPCR experiments were performed using QX200™ ddPCR System (Bio-Rad Laboraties, Inc.) (36,37). The MS-ddPCR reaction mix consisted of the 2X ddPCR Supermix for Probes, and locus-specific primers and probes. For the SEPT9 assay, the primers and probe sequences were designed using Beacon Designer (Premier Biosoft International). Two sets of primers and probes were obtained and correspond to the bisulfite-modified methylated or unmethylated sequence: The set with primers and probe with the fluorescent FAM reporter for methylated SEPT9 (named SEPT9-M) and the set with primers and probe with HEX reporter for unmethylated SEPT9 (named SEPT9-U). For SHOX2, the assay was designed by Beacon Designer to detect bisulfite-modified methylated SHOX2 using a FAM-labelled probe set (named SHOX2) and to detect, after bisulfite conversion, a CpG-free region in the actin beta (ACTB) gene using a HEX-labelled probe set (named ACTB) (38). The complete list of all primer and probe sequences is provided in Table SI. The PCR mix was prepared in a 22-µl reaction volume containing 11 µl 2X ddPCR Supermix for Probes (no dUTP) (cat. no. 186-3024; Bio-Rad Laboratories, Inc.), 0.55 µl 20X PCR probe assay specific for the methylated loci (SEPT9-M or SHOX2) and 0.55 µl 20X PCR probe assay specific for the unmethylated SEPT9 (SEPT9-U) or ACTB, and bisulfite-treated DNA, as a template. Each ddPCR assay mixture (20 µl) was loaded into a disposable droplet generator cartridge (cat. no. 1864008; Bio-Rad Laboratories, Inc.). Subsequently, 70 µl of droplet generation oil for probes (cat. no. 1863005; Bio-Rad Laboratories, Inc.) was loaded into each of the eight oil wells. The cartridge was then placed inside the QX200 droplet generator (Bio-Rad Laboratories, Inc.). After droplet generation was completed, the droplets were transferred to a 96-well PCR plate (cat. no. 12001925; Bio-Rad Laboratories, Inc.) using a multichannel pipette. The plate was heat-sealed with foil and placed in a T100 Thermal Cycler (Bio-Rad Laboratories, Inc.). Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles at 94°C for 30 sec and 52°C (for the SEPT9 assay) or 57°C (for the SHOX2 assay) for 1 min (ramp rate reduced to 2%), with a final step at 98°C for 10 min and a 4°C indefinite hold. QuantaSoft software version 1.7.4 (Bio-Rad Laboratories, Inc.) was used to verify the number of total droplets and positive droplets for methylated SEPT9 or SHOX2 in the FAM channel and for the unmethylated SEPT9 or ACTB in the HEX channel. The SEPT9 methylation level was calculated as a percentage: Concentration (copies/µl) for SEPT9-M/(concentration (copies/µl) for SEPT9-M + concentration (copies/µl) for SEPT9-U). In addition, due to the lack of primers/probe set for unmethylated SHOX2, the SHOX2 methylation level was calculated as the ratio: Concentration (copies/µl) for SHOX2/concentration (copies/µl) ACTB.
Establishing the efficiency of MS-ddPCR assays
Methylated and non-methylated human DNA standards (Zymo Research Corp.) converted with bisulfite were used to verify the efficiency of MS-ddPCR assays in detecting SEPT9 and SHOX2 methylation. By following the same experimental workflow used by Yu et al (39), two-fold serial dilutions of fully methylated DNA were prepared with water. A series of samples containing 20,000, 10,000, 5,000, 2,500, 1,250, 625, 312.5, 156.25, 78.125 and 0 pg of standard bisulfite-converted DNA was assessed for SEPT9 and SHOX2 by MS-ddPCR assays, as aforementioned. The range of the standard curve comprised the expected yield of DNA isolated from 1–2 ml of plasma. To verify the ability of MS-ddPCR to discriminate methylated DNA from the DNA background, 20 ng of total DNA containing the following percentages of fully methylated DNA (99, 90, 70, 50, 30, 10 and 1%) were tested (39). A negative template control (NTC) containing all components of the reaction except for the DNA template was included in each experiment.
MS-quantitative PCR (MS-qPCR)
Commercial 100% methylated and non-methylated human DNA standards converted with bisulfite were used to verify the efficiency of MS-qPCR assays in detecting SEPT9 and SHOX2 methylation. Samples containing 20,000, 10,000, 5,000, 2,500, 1,250, 625, 312.5, 156.25, 78.125 and 0 pg of standard bisulfite-converted DNA were tested for SEPT9 and SHOX2 by MS-qPCR assays, as aforementioned. Furthermore, to verify the ability of MS-qPCR to discriminate methylated DNA from the DNA background, 20 ng of total DNA containing the following percentages of fully methylated DNA (99, 90, 70, 50, 30, 10 and 1%) were tested. The qPCR reaction (20 µl/well) contained 10 µl of Taq-Man 2X Universal PCR Master Mix (Thermo Fisher Scientific, Inc.), 0.5 µl 20X PCR probe assay specific for the methylated loci (SEPT9-M or SHOX2), and 0.5 µl 20X PCR probe assay specific for unmethylated SEPT9 (SEPT9-U) or ACTB, and bisulfite-treated DNA, as a template. The PCR reactions were incubated at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 52°C (for SEPT9 assay) or 57°C (for SHOX2 assay) for 1 min. PCRs were performed in triplicate using the QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific, Inc.).
Detection of SEPT9 and SHOX2 methylation levels in ccfDNA of patients with HNSCC by MS-ddPCR
To assess the methylation levels of SEPT9 and SHOX2 in the plasma of patients with HNSCC, 6 µl of bisulfite-converted ccfDNA were used for both MS-ddPCR assays. Multiplex ddPCR assays and relative analysis were performed as aforementioned. Each experiment included the positive control wells for the methylated and unmethylated loci containing 4 µl (20 ng) of fully methylated DNA (Zymo Research Corp.) converted with bisulfite and 4 µl (20 ng) of completely unmethylated DNA (Zymo Research Corp.) converted with bisulfite. NTC wells were also included.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 7.0 software (Dotmatics). The linear regression between the calculated percentage of the DNA methylation levels of SEPT9 and SHOX2 and the percentage of input methylated DNA was performed to establish the efficiency of MS-ddPCR assays. The experiments were performed in triplicate. One-way ANOVA or two-way ANOVA, followed by Tukey's post hoc test, was used to compare the mean values of methylation levels for SEPT9 and SHOX2 in ccfDNA among the different follow-up time points. The histograms are presented as the mean values ± standard error of the mean (SEM). The mean values of SEPT9 and SHOX2 methylation levels at each time point (T0, T1, T2, T3) were used to determine the trend, shown as the red line, for longitudinal analysis of SEPT9 and SHOX2 methylation levels during the follow-up of patients with HNSCC. P<0.05 was considered to indicate a statistically significant difference.
Results
Establishing the efficiency of MS-ddPCR assays for the detection of SEPT9 DNA methylation
In the present study, two multiplex assays were used for measuring the methylation levels of SEPT9 and SHOX2 using ddPCR technology, defined as MS-ddPCR. MS-ddPCR for SEPT9 consisted of i) a TaqMan probe-based assay designed with FAM reporter to detect the methylated bisulfite-converted DNA (SEPT9-M) and ii) a TaqMan probe-based assay with HEX reporter to detect the unmethylated bisulfite-converted DNA (SEPT9-U) (Fig. 1A). The sensitivity and specificity of the assays were assessed using commercial methylated DNA and unmethylated DNA after bisulfite conversion. The two-dimensional (2D) amplitude plot showed that the SEPT9-M set detected only the methylated template (Fig. 1B, positive droplets in blue, left) and, by contrast, the SEPT9-U set detected only the unmethylated template (Fig. 1B, positive droplets in green, right) in multiplex ddPCR experiments. Next, the performance of the MS-ddPCR assay was evaluated by considering its ability to detect the SEPT9 DNA methylation levels in samples with low amounts of DNA input, and in the presence of an unmethylated DNA background. MS-ddPCR for SEPT9 displayed a dose-dependent trend, and the methylation level was detectable using a starting input of commercial bisulfite-treated DNA as low as 78.125 pg (Fig. 1C). To assess the ability of the assay to detect methylated SEPT9 molecules in an unmethylated DNA background, the methylated DNA with unmethylated DNA was diluted at different percentages (99, 90, 70, 50, 30, 10 and 1%) and multiplex MS-ddPCR was performed on 20 ng of the bisulfite-treated DNA mixtures. The concentration of the methylated target (copies/µl, in blue) and that of the unmethylated target (copies/µl, in green) decreased and increased, respectively, according to the percentage of methylated DNA. The SEPT9-M and SEPT9-U assays detected up to 1% methylated SEPT9 and unmethylated SEPT9, with a concentration of 0.14 and 1.5 copies/µl, respectively (Fig. 1D). The level of methylated SEPT9 (expressed as percent, %) was calculated as described in the Materials and methods section. The standard curve demonstrated good linearity between the level of methylated SEPT9 (expressed as percent, %) and the percentage of commercial bisulfite-treated methylated DNA loaded in each reaction (R2=0.92; Fig. 1E).
To evaluate the efficiency of qPCR in detecting the DNA methylation levels of SEPT9, the sensitivity and specificity in the same conditions were assessed. The SEPT9-M set reached the detection limit of qPCR (cycle threshold, Ct >35) with 625 pg of input methylated DNA, and the SEPT9-U set with 1,250 pg of input unmethylated DNA (Fig. 2A). As revealed in Fig. 2B, ddPCR was able to detect positive droplets up to 78.125 pg of input DNA for both sets. Analysis of the ability of the assay to detect methylated SEPT9 molecules in an unmethylated DNA background, revealed that qPCR detected up to 30% of methylated SEPT9 but the threshold cycles for 10 and 1% of methylated SEPT9 were above the cutoff (Ct >35) (Fig. 2C). These results indicated the higher sensitivity and specificity of ddPCR-based assays than qPCR.
Establishing the efficiency of MS-ddPCR assays for the detection of SHOX2 DNA methylation
MS-ddPCR for SHOX2 consisted of i) a TaqMan probe-based assay labeled with FAM reporter for methylated SHOX2 and ii) a TaqMan probe-based assay labeled with HEX reporter for a region CpG-free in the ACTB gene (Fig. 3A). The specificity of SHOX2 assays was tested by following the procedures described for SEPT9. Only methylated DNA treated with bisulfite was amplified using the SHOX2 assay (Fig. 3B, positive droplets in blue). As expected, the ACTB assay amplified methylated and unmethylated DNA (Fig. 3B, positive droplets in green). The MS-ddPCR assay for SHOX2 displayed a dose-dependent trend and could detect methylated SHOX2 as low as 78.125 pg of commercial bisulfite-treated DNA (Fig. 3C). The concentration of ACTB (copies/µl, in green) remained stable with values between 69.1 and 78.2 copies/µl; meanwhile, the concentration of methylated SHOX2 (copies/µl, in blue) increased accordingly to the percentage of input methylated DNA with good linearity (R2=0.98; Fig. 3D and E).
To evaluate the efficiency of qPCR in detecting DNA methylation levels of SHOX2, the sensitivity and specificity in the same conditions were assessed. The SHOX2 set reached the detection limit of qPCR (Ct >35) with 156.25 pg and the ACTB set with 1,250 pg of input methylated DNA (Fig. 4A). As shown in Fig. 4B, ddPCR was able to detect positive droplets up to 78.125 pg of input DNA for both sets. Analysis of the ability of the assay to detect methylated SHOX2 molecules in unmethylated DNA background showed that qPCR detected up to 50% of methylated SHOX2 with threshold cycles below the cut-off (Ct=35) (Fig. 4C). These results indicated the higher sensitivity and specificity of ddPCR-based assays compared with qPCR.
Methylation levels of SEPT9 and SHOX2 in ccfDNA from the plasma of patients with HNSCC
Using the MS-ddPCR technology, the methylation levels of SEPT9 and SHOX2 in the plasma of 20 patients with HNSCC were assessed (Table I). The SEPT9 and SHOX2 methylation levels in the plasma of each patient before the treatment (T0) and at 3-month intervals during follow-up (T1=3 months and T2=6 months after treatment) were analyzed. Considering all the patients with 2 time points of follow-up (n=18; BS008 and BS014 developed distant metastasis or tumor persistence and thus they were excluded from the subsequent analysis), methylation of SEPT9 was detectable in 13 (72%) patients at T0 (Fig. 5A). The mean methylation level of SEPT9 (mSEPT9) decreased during follow-up, showing a reduction at T1 (mean mSEPT9 T0=1.84±2.44, mean mSEPT9 T1=0.81±1.12; fold change of 0.4) and a significant drop at T2 (P<0.05; mean mSEPT9 T2=0.377±0.519; fold change of 0.2 vs. T0). A total of 8 (44%) patients displayed SHOX2 methylation (mSHOX2) in ccfDNA at T0 (mean mSHOX2 T0=0.97±1.798), and a significant decrease in the mean methylation levels of SHOX2 at T1 and T2 follow-up time points (Fig. 5B; P<0.05; mean mSHOX2 T1=0.093±0.39, mean mSHOX2 T2=0.072±0.146; fold change of 0.09 and 0.07, respectively) was obtained. Of these 8 patients, 5 exhibited a concomitant SEPT9 methylation in ccfDNA at T0.
Longitudinal variations of methylated SEPT9 and SHOX2 in ccfDNA from the plasma of patients with HNSCC
Among the 18 patients who were followed up longitudinally, 10 reached the time point of 12 months after the treatment (T3) at the time of the writing of the present study. For the SEPT9 analysis, 2 out of 18 patients were not included due to undetectable methylation levels at all time points. By monitoring the longitudinal methylation levels of SEPT9, four different groups of patients were depicted according to SEPT9 methylation levels during follow-up. As shown in Fig. 6A, a decreasing trend was observed for the first group of patients (BS017 and BS019), with a mean of mSEPT9 in plasma from 4.89 at T0 to 0 at post-treatment time points (T1 and T2). Both of these patients presented oropharyngeal cancer, received the same type of therapy (radiotherapy and chemotherapy), and had no evidence of disease (NED) at T2/T3. A total of 4 patients exhibited a decrease in methylated SEPT9 in plasma at T1 followed by an increase in methylation levels at T2 (BS002, BS011 and BS018) or T3 (BS006) (mean mSEPT9 T0=2.4, mean mSEPT9 T1=0, mean mSEPT9 T2=0.97, mean mSEPT9 T3=1.81; Fig. 6B). A total of 3 patients had the same cancer stage (III) and received the same therapy (surgery followed by adjuvant treatment). Furthermore, 5 patients exhibited a decreasing trend of SEPT9 methylation levels at T1 and T2 (mean mSEPT9 T0=2.44, mean mSEPT9 T1=1.28, mean mSEPT9 T2=0.05, mean mSEPT9 T3=0.47; Fig. 6C). All these patients underwent surgery resection. The last group of 5 patients showed a significant increase in SEPT9 methylation levels at T1, followed by a decrease at T2 (mean mSEPT9 T0=0.31, mean mSEPT9 T1=1.66; P<0.05; mean mSEPT9 T2=0.52, mean mSEPT9 T3=0.67; Fig. 6D). All these patients had stage I or II HNSCC with NED at T2/T3. All the patients were divided according to the disease status: NED (n=13) and patients with progressive disease (PD; n=6). In the NED group, a significant decrease in the mean mSEPT9 was found at T2 vs. T0 (Fig. S2A). For SHOX2 analysis, 6 patients out of 18 were excluded because the methylation levels were undetectable at all time points. In the remaining patients, three different longitudinal trends were observed during follow-up (Fig. 7). In the first group, 5 patients displayed a high methylation level of SHOX2 at T0 (mean mSHOX2 T0=2.58), followed by a decrease at T1 (or T2 for BS013) (mean mSHOX2 T1=0.33; T0 vs. T2, P<0.05; Fig. 7A). A total of 4 out of the 5 patients shared the following clinical characteristics: Tumor site (oropharynx), cancer stage (I–II), HPV infection, therapy (radiotherapy and chemotherapy), and NED. In the second group, 3 patients exhibited different methylation levels of SHOX2 at T0 (mean mSHOX2 T0=1.52) followed by a decrease to an undetectable level at T1 and a slight increase at T2 (BS011 and BS023) or T3 (BS016) (mean mSHOX2 T2=0.22, mean mSHOX2 T3=0.19; Fig. 7B). In the third group of patients, it was revealed that the methylation levels of SHOX2 were absent at T0 and T1 in 4 patients but they increased at T2 (BS003, BS019 and BS029) and T3 (BS006) (mean mSHOX2 T2=0.16, mean mSHOX2 T3=0.03; Fig. 7C). The patients in these two groups did not share any clinical characteristics. No significant variations were found in the methylation levels of SHOX2 among the different follow-up time points in the NED and PD groups (Fig. S2B).
Discussion
The methylation levels of SEPT9 and SHOX2 in ccfDNA are considered biomarkers of diagnosis, staging, and prognosis for HNSCC and other malignancies (12,40,41). It has been demonstrated that circulating levels of methylated SEPT9 and SHOX2 are associated with some clinicopathological features of patients with HNSCC, such as tumor and nodal category, and high methylation levels were associated with an increased risk of death (12). The concentration of ccfDNA ranges from 1 to 15 ng/ml plasma in healthy individuals to 100 ng/ml plasma in patients with cancer (42,43). The total amount of ctDNA can also be <1% of total ccfDNA (44), and these low concentrations make detection challenging. Accurate and precise quantification of the genomic alterations with prognostic and predictive values can be of great importance for clinical management. Therefore, the set-up of a ddPCR-based assay was considered useful and innovative to improve the detection of the methylated SEPT9 and SHOX2 circulating levels in the plasma of patients with HNSCC. Additionally, ddPCR is a well-known end-point PCR method that allows absolute quantification of the target template without requiring standard curves. Several studies have previously reported the advantages of ddPCR, including its high sensitivity and great accuracy in assessing DNA methylation levels of low DNA input samples (27,28,39,45). However, for liquid biopsy, there is still limited data on the levels of DNA methylated molecules of cancer-associated genes using ddPCR (32,46). In the present study, the methylation-specific assay with the ddPCR technology (MS-ddPCR or MethyLight ddPCR) was combined to quantify the plasma amount of methylated SEPT9 and SHOX2 in HNSCC. Specifically, to detect the SEPT9 methylation levels in a multiplex ddPCR reaction, two TaqMan probe-based assays labeled with FAM (SEPT9-M) and HEX (SEPT9-U) were designed for the amplification of the SEPT9 sequence in bisulfite-converted methylated and unmethylated DNA, respectively. For SHOX2, an assay with a FAM-labeled probe against the bisulfite-converted methylated SHOX2 sequence was designed. Due to poor efficiency of assays amplifying the unmethylated SHOX2, primers and a HEX-labeled probe were used against a CpG-free sequence in the ACTB gene to normalize data (38). Using a set of commercial fully methylated and non-methylated DNA, the assays in the present study detected up to 78 pg of methylated DNA and quantified up to 1% of methylated DNA in a non-methylated DNA background. As aforementioned, this amount and relative percentages may reflect those detected in circulation. In the present study, the efficiency of the SEPT9 and SHOX2 methylation assays were evaluated using qPCR. The data revealed very low accuracy in detecting small amounts of methylated DNA (as low as 625–312 pg) and low percentages of methylated DNA (as low as 30–50%), making ddPCR the ideal technology for quantifying very low levels of methylated targets.
The ddPCR assay was then assessed on a discovery cohort of 18 patients with HNSCC to determine the methylation levels of SEPT9 and SHOX2 in plasma before the initiation of therapies and during monitoring of treatment response at three different follow-up time points. At the time of the writing of the present study, plasma samples up to 1 year (T3) after the end of treatment (surgical resection of the tumor, chemotherapy and radiotherapy) with 3-months intervals were collected. Most patients are still being monitored, and methylation analysis will be performed at the available follow-up time points.
A significant reduction of the mean methylation plasma levels of SEPT9 and SHOX2 in patients at T2 (SEPT9) and T1-T2 (SHOX2) monitoring times, including 3 (T1) and 6 (T2) months after the end of treatment, were found. In this context, in a previous study, the post-therapeutic plasmatic circulating SHOX2 and SEPT9 methylation ccfDNA levels were decreased in patients with colorectal cancer with localized disease, while there was no decrease in patients with distant metastases (41). Furthermore, high methylation levels of circulating SEPT9 and SHOX2 characterized patients with metastatic disease in prostate cancer (47). In HNSCC, the baseline positivity of SEPT9 and SHOX2 methylation in plasma was identified in 15 patients (15/20, 75%), and methylation levels decreased with systemic therapy (40). All the patients except one (BS002) (n=17) were disease-free at the T2 monitoring time. In the observational cohort of the present study, different trends were observed in SEPT9 methylation levels by representing the data according to the longitudinal quantification for each patient during monitoring (as detailed in Results and shown in Fig. 6). Among them, 5 patients with I–II tumor stages treated differently (Fig. 6D and Table I) did not display tumor progression, at least until T3 monitoring time, and exhibited a decrease of SEPT9 methylation at T2. Furthermore, 3 patients with the same cancer stage (III) undergoing the same treatment (surgery followed by adjuvant treatment) displayed a significant decrease of SEPT9 methylation at T1 monitoring time followed by an increase at T2 (Fig. 6B). It appears that the changes of the SEPT9 methylation level detectable at T2 may be relevant, but it is necessary to expand the cohort to attribute clinical significance to this observation. For SHOX2 (as detailed in Results and shown in Fig. 7 and Table I) 5 patients displayed a significant decrease in methylation levels at the T2 monitoring time compared with T0. A total of 4 patients out of 5 had the same clinicopathological features in terms of tumor localization (oropharynx), tumor stage (I–II), HPV p16 infection, type of treatment (chemotherapy and radiotherapy) and they were all disease-free. Therefore, this could be promising because it is known that high circulating levels of methylated SHOX2 are correlated with a worse prognosis in patients with HNSCC (48). The authors of the present study are aware that the results obtained in this study are derived from the analysis of circulating SEPT9 and SHOX2 methylation levels in small groups of patients, however promising longitudinal changes during the time points of the follow-up of the patients were revealed. In an ongoing larger study (Identify project), it will be thoroughly investigated whether these variations are potentially associated with the clinical features of the patients or response to treatments.
At present, at least to the best of the authors' knowledge, three studies have evaluated the impact of circulating SEPT9 and SHOX2 in post-therapeutic monitoring as epigenetic biomarkers of prognosis and early diagnosis of tumor recurrence in HNSCC. SEPT9 and SHOX2 methylation levels determined by qPCR were revealed to be correlated with diagnosis, prognosis, staging and monitoring of patients with HNSCC (12). The impact of SEPT9 and SHOX2 DNA methylation in the diagnosis of HNSCC and treatment response was evaluated by relative and quantitative determinations using qPCR (24,40).
The main limitation of the present study is related to the number of patients. It will be necessary to expand the cohort to confidently define the translational implication of the study. In addition, it is difficult at present to associate a clinical significance to the longitudinal DNA methylation variations detected in small groups of patients. Another limitation may be the heterogeneity of the patients recruited thus far according to major clinical features (such as tumor site, type of systemic treatment, and tumor stage). There is a lack of an association between the main risk factors for HNSCC (including smoking, alcohol abuse, and HPV infection) and methylation levels of circulating SEPT9 and SHOX2. Furthermore, the methylation levels of SEPT9 and SHOX2 in solid biopsies to compare with the corresponding circulating levels, were not analyzed.
In conclusion, a sensitive assay based on ddPCR technology was developed by the authors to detect the methylation levels of circulating SEPT9 and SHOX2 DNA. At least to the best of the authors' knowledge, the use of a performant ddPCR assay for these two epigenetic markers has not yet been developed. The use of ddPCR to detect small amounts of circulating methylated SEPT9 and SHOX2 and monitor their dynamic changes at multiple pre-established time points during clinical monitoring represents an advancement in the HNSCC field. The diagnostic accuracy of either methylated SEPT9 and SHOX2 has been previously demonstrated leading to their use as diagnostic biomarkers for lung cancer (Epi proLung) and colorectal cancer (Epi proColon) (49). For future clinical practice, the identification of the circulating methylation levels of SEPT9 and SHOX2 in patients with HNSCC in the post-treatment phase may allow for earlier diagnosis of recurrence/second primary malignancy and the definition of a personalized follow-up based on patient risk stratification. Extensive validation of SEPT9 and SHOX2 as circulating methylated biomarkers capable of stratifying groups of patients with HNSCC based on homogenous clinicopathological characteristics is necessary. For this purpose, the authors are continuing with a multicenter study, to collect liquid biopsies from eight different hospitals, to investigate the methylation levels of SEPT9 and SHOX2 in a large cohort of Italian patients.
Supplementary Material
Supporting Data
Supporting Data
Acknowledgements
We would like to thank Dr Aashni Shah (quality assurance manager and editorial consultant; Polistudium Srl, Milan, Italy) and Ms Valentina Attanasio (English specialist; Polistudium Srl, Milan, Italy) for the linguistic revision of the manuscript. We woul also like to thank the family of Ms Claudia Massoni for the support of the research.
Funding
The present study was supported by Fondazione Spedali Civili. The research was also funded by CIB (Biotechnology Interuniversity Consortium, Italy) grant no. 12/10/2020, and by the University of Brescia (local grants; nos. 60/2021 and 60/2022). The funding bodies played no role in the design of the study and in the collection, analysis, and interpretation of data, or in the writing of the manuscript.
Availability of data and materials
The data obtained and analyzed during the current study are available from the corresponding author upon reasonable request.
Authors' contributions
PB, GDP and AS conceptualized the study. AS and IG developed methodology. AS, IG and IAP conducted investigation. CA, LL, DS, CG, SG, AP, DM, CP and PB acquired and interpreted data. AS and IG wrote the original draft. AS, IG, IAP, GDP and PB wrote, reviewed and edited the manuscript. AS and PB supervised the study. PB, GDP and AS acquired funding. AS and IG confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript. All authors have agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of Spedali Civili of Brescia (Protocol Identify; Ethics Committee approval no. NP 4551). Written informed consent was obtained from each patient.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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