Clinical details

Methylation Sensitive High Resolution Melting (MS-HRM) analyses

In comparison to much of the previously published literature, no methylation or infrequent methylation was detected for the majority of loci examined. No significant methylation (0%) was detected in any of the samples for the following genes: DAPK1 (91/91), RASSF1A (49/49), MGMT (50/50), MLH1 (49/49), BRCA1 (45/45), ERCC1 (47/47) and ATM (48/48). For the following loci, the majority of samples had no methylation, but definite methylation was present in a subset of samples: RUNX3 (18/108 methylated), ABO (22/107 methylated), APC (3/105 methylated), CDH1 (13/114 methylated) and CDH13 (4/112 methylated). Both homogeneous and heterogeneous methylation, as ascertained by melting profiles, was observed for these loci.

Heterogeneous melting patterns were detected for ABO (19/107), CDH1 (13/114), CDH13 (3/112) and RUNX3 (18/108). Homogeneous melting patterns were observed for APC in three samples (3/105), all of which had a methylation level of greater than 25%. Homogeneous methylation of ABO was also observed for three samples, which all demonstrated methylation levels of 10 to 25%. Including the samples with heterogeneous methylation, the frequency of methylation events for ABO and RUNX3 were similar to the literature although numerically less [21, 26].

Sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP)

On the basis of reports suggesting frequencies of methylation greater than 20% for both DAPK1 and RASSF1A[8, 9, 68], sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP) analyses were used to confirm a subset of the MS-HRM findings [97]. Utilizing this quantitative modification of MSP permitted the concurrent investigation of possible reasons for discordance between MS-HRM results and the MSP-based literature. SMART-MSP incorporates the use of a DNA intercalating dye, which permits both real-time quantification and melting curve analysis (MCA), which can minimize the number of false positives [97].

The promoter status of DAPK1 for 50 samples was investigated using SMART-MSP. On quantification, the median level of methylation present was 0.0015% (range 0 to 1.3%), with only one sample demonstrating a level of methylation above 1%. However, despite the low levels of methylation quantified, on MCA analyses 41/50 samples demonstrated a pattern consistent with the presence of methylation, emphasizing the sensitivity of MSP. Gel electrophoresis of SMART-MSP products was also performed (Figure 1), and positive bands were detected for samples with calculated quantification of methylation below 0.2%. False positive, similar sized bands were also observed for some samples that failed to amplify during PCR and also for samples with a MCA pattern consistent with unmethylated DNA. SMART-MSP analysis for RASSF1A was performed for ten samples, and no (0%) methylation was detected in these.

Thus, we confirmed with a second quantitative methodology that no significant level of DNA promoter methylation for DAPK1 and RASSF1A was found in the cohort examined. Gel electrophoresis revealed similar sized bands for samples with extremely low levels of methylation or no methylation, illustrating a likely cause in the literature for overestimation of significant methylation events.

Clinical correlation between methylation sensitive high resolution melting (MS-HRM) analyses and patient outcome

Analyses correlating the presence of promoter methylation with patient outcome was possible only for the following genes for which methylation was detected; RUNX3, CDH13, APC, ABO and CDH1. Patients with promoter methylation detected for RUNX3 (18/108, all heterogeneously methylated) had a significantly worse overall survival (OS) compared with patients without methylation, with a five-year OS rate of 32% versus 57% (HR = 2.22, 95% CI: 1.15-4.29, P value = 0.01). This effect was maintained for progression-free survival (PFS) (33% versus 51% five-year PFS; HR = 2.07, 95% CI: 1.03-4.18, P value = 0.04).

While a much smaller number of events was observed for samples demonstrating ABO promoter hypermethylation, for the 3/87 patient samples that demonstrated homogeneous methylation, this significantly correlated with a worse five-year OS rate of 0% versus 57% (HR = 6.51, 95% CI: 1.94-21.8, P value = 0.014). Similar results were found for PFS. The effect was maintained when samples with heterogeneous methylation were included in the analysis (3/107, 10 to 25% homogeneously methylated and 19/107 heterogeneously methylated), with a worse five-year OS rate of 32% versus 57% for methylated samples (HR = 1.98, 95% CI: 1.06-3.70, P value = 0.03). Significant correlation with PFS was not observed.

Bisulfite pyrosequencing analyses and correlation with patient outcome

Given the statistically significant correlation with outcome for patient samples with methylation of RUNX3 and ABO, which included unquantified heterogeneously methylated samples, we sought to investigate this further with bisulfite pyrosequencing.

For the RUNX3 pyrosequencing analysis, 83 samples had sufficient bisulfite modified DNA available for assessment. These samples included the 16/18 samples with heterogeneous methylation identified by MS-HRM. Five samples that failed to produce an adequate signal on pyrosequencing were not included in the analysis. On pyrosequencing, only four samples (4/78) demonstrated mean methylation quantities greater than 15%. The rest of the cohort (74/78), including samples that previously had heterogeneous methylation identified on MS-HRM, had mean quantities of methylation below 12% and thus were scored as negative according to our predefined criteria (Figure 2). A significantly worse outcome was found for the four patients with RUNX3 promoter hypermethylation greater than 15%, with a five-year OS rate of 0% versus 56% (HR = 10.2, 95% CI: 3.23-32.4, P value = 0.001). Given the small number of observed events, firm conclusions could not be made despite the significance of the P value, and further analyses were not pursued.

For the ABO pyrosequencing analysis, 85 samples had sufficient bisulfite modified DNA available. Of these, 20/85 samples demonstrated levels of mean methylation greater than 15%. For the statistical analysis, two samples that had demonstrated quantities of methylation between 10 to 25% on MS-HRM but had insufficient DNA for pyrosequencing were also included. For the 22 patients with significant methylation of ABO identified, no statistically significant clinical correlation with survival was found (HR = 1.43, 95% CI: 0.73-2.80, P value = 0.29).

Thus, we demonstrate the impact of quantification of methylation levels. For the investigated loci, though heterogeneous methylation was identified on MS-HRM, the vast majority of these indicated low level methylation events. Of particular note, the initial MS-HRM analysis indicated a frequency of (heterogeneous) RUNX3 methylation of 17% of samples consistent with the literature. However, when quantified by pyrosequencing, the actual frequency of significant methylation events for RUNX3 was considerably less (5%). The presence of RUNX3 promoter methylation was found to correlate with a worse patient outcome, although due to the small numbers robust conclusions cannot be made. In contrast, when ABO promoter methylation was quantified with pyrosequencing, correlation with patient outcome was lost with a minor alteration in the frequency of observed events (22/107, 21% for MS-HRM, and 22/85, 26% for pyrosequencing). These observations highlight the impact of methodology and sample size on correlative analyses.

External validation of results through interrogation of the Cancer Genome Atlas (TCGA) database

To externally validate our findings, which suggested that the frequency of significant methylation events of selected loci was less common than the literature suggested, the Cancer Genome Atlas (TCGA) head and neck cohort were examined. At the time of download (24 June 2013; clinical data download date 22 July 2013), there were 373 TCGA head and neck tumor samples with publically available Infinium HumanMethylation450 beadchip array (HM450K) data available. A total of 26 patients were excluded due to the presence of metastatic disease or an unknown “M-category” (metastasis-category), a history of previous malignancy, or primary site documented as the ‘lip’. Of the remaining 346 patients, 305 patients had both clinical details and methylation data available. Of these 305 patients, 86 patients had OTSCC.

The summary of significant methylation events (β value >0.2) for the examined loci within the TCGA HNSCC cohort is presented in Table 3. For the HM450K probes that overlapped the MS-HRM amplicons, a similar frequency of methylation was observed (see Additional file 1: Figure S1). Methylation levels from probes that flanked the MS-HRM amplicons also indicated a similar frequency of methylation events (see Additional file 1: Figure S2), although comparison across different regions is difficult.

Interestingly, DAPK1 methylation for the probes flanking the MS-HRM amplicon demonstrated a frequency of methylation events of 15% in the heterogeneous HNSCC TCGA cohort and 9% in the TCGA OTSCC subset. Samples with DAPK1 methylation in the whole HNSCC cohort were distributed between multiple head and neck subsites. Methylation of RASSF1A was identified in 4% of samples in the HNSCC TCGA cohort, but none of these were OTSCC samples.

To assess whether RUNX3 promoter methylation had prognostic value in the TCGA cohort, methylation reported from the probes flanking the MS-HRM amplicon was correlated with patient outcome for the whole TCGA HNSCC cohort, and for the subset of patients with OTSCC. There were no statistically significant relationships between methylation and survival identified, when methylation from individual probes or methylation averaged across the two probes were used (HR = 1.08, 95% CI: 0.65-1.82, P = 0.76 for averaged methylation for the whole TCGA cohort, and HR = 1.27, 95% CI: 0.56-2.88, P = 0.57 for averaged methylation for only the OTSCC TCGA subset).

Thus, in an independent, external heterogeneous HNSCC cohort assessed by an orthogonal quantitative methodology, we confirmed the infrequent methylation events for the loci examined. RUNX3 methylation was not a prognostic marker in the TCGA cohort.

Patient information

The patient cohort of 131 OTSCC patients has been described previously [96]. Briefly, patients included consisted of those who we could confirm had OTSCC, had comprehensive clinicopathological and outcome data available, and had pre-treatment FFPE blocks containing invasive squamous cell carcinoma. Of these, 115/131 patient samples had sufficient DNA for bisulfite modification and methylation analyses.

DNA extraction, bisulfite modification and preparation of control DNA

A hematoxylin and eosin stained slide representative of each pretreatment tumor specimen was labeled by an expert oral pathologist for areas of invasive tumor. Two millimeter representative cores were punched to extract DNA. After deparaffinization with xylene and daily proteinase K digestion over three days at 56°C, DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). A pre-analytical quality-assurance polymerase chain reaction (QA-PCR) [107] was performed that demonstrated the presence of 300 to 600-bp fragments, confirming the quality of DNA was adequate for methylation assessment. For the generation of a dilution series of control DNA standards, fully methylated human genomic DNA (FMD, Merck Millipore, Billerica, US) was purchased, and secondary whole genome amplification (WGA) product was used as unmethylated DNA. The primary WGA product was generated using the Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Little Chalfont, UK) and then purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), according to the manufacturers’ instructions. Tumor DNA and control DNA were quantified using the Qubit 2.0 Fluorometer (Life Technologies, Invitrogen, Oregon, US). Bisulfite modification of one microgram of input DNA was performed according to the manufacturer’s instructions with the Methyl Easy Xceed kit (Human Genetic Signatures, Sydney, Australia) and eluted into two aliquots of 50 μL, to make a theoretical concentration of 10 ng/μL.

Methylation sensitive high resolution melting (MS-HRM)

Methylation was assessed in samples initially with MS-HRM [108]. Amplifiable bisulfite modified FMD was normalized against bisulfite modified unmethylated DNA (WGA) using a real-time quantitative PCR assay that amplified a CpG dinucleotide free region of the COL2A1 gene. Standard dilutions of FMD and WGA were made to 50%, 25%, 10% and 5%. Primers, reaction mixtures and conditions are listed for each promoter region interrogated in Additional file 2: Table S1. MS-HRM analyses were performed on each tumor sample at least in duplicate, with duplicates of the DNA methylation standards, FMD, WGA and non-template control (NTC). Initially, 50 patient samples were assessed for each gene, and then the analysis was extended to the rest of the cohort if any methylation was identified. Heterogeneous methylation was determined as previously described [14, 15, 100]. Briefly, heterogeneous methylation refers to the overall different patterns of methylation that can be observed on any given allele [14], similar to that of genetic mosaicism [109]. When homogeneous methylation is assessed by MS-HRM, melting curves replicate that observed for the FMD or the dilution series of standards. In contrast, when heterogeneous methylation is present, melting curve analysis reveals complex melting patterns as a consequence of heteroduplex formation [14]. If methodology is not specifically chosen to assess the presence of heterogeneous methylation, it will remain undetected. Thus in these instances, the quantity of methylation reported actually represents the mean level of methylation averaged across the entire number of CpG dinucleotides assessed within the amplicon. Importantly, MS-HRM can identify but not quantify heterogeneous methylation.

Sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP)

We sought to confirm a subset of our findings with a second methodology, and concurrently investigate potential reasons for the discordance between our MS-HRM findings and reported literature. SMART-MSP is a quantitative adaption of MSP, which is readily performed with the added benefit of a melting curve analysis step for quality control [97]. The primers for DAPK1 were located within the same region interrogated by the MS-HRM primers and produced a 61-bp amplicon. Due to the requirements of primer design, the SMART-MSP primers were located downstream of the MS-HRM primers. PCR cycling and HRM analysis were performed on a Rotor-Gene Q (Qiagen). Each assay was performed with duplicates of the bisulfite modified standard dilution series of FMD and WGA (50%, 25%, 10%, 5%, 1% and 0.1%), tumor samples, NTC and unmodified genomic DNA. The primer sequences, reaction mixtures and conditions are listed in Additional file 2: Table S2.

Quantification of methylation using Sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP)

The quantity of input DNA of the FMD, WGA and each tumor sample was calibrated against a CpG dinucleotide free region of the COL2A1 gene. The raw run data for COL2A1, DAPK1 and RASSF1A were generated by the Rotor-Gene Q (Qiagen) and exported into LinRegPCR (version 2012.3.0.0) for the estimation of amplification efficiency, after accounting for bias introduced by variable baseline fluorescence [110]. The proportion of methylated templates was then quantified according to methodology previously described [111].

Gel electrophoresis analysis

Four microliters of the SMART-MSP product were resolved on a 3.5% agarose gel stained with SYBR Safe (Invitrogen), at 80 mV over 1.5 hours.

Bisulfite pyrosequencing

The preliminary statistical analysis of the MS-HRM results indicated a significant correlation with worse outcome for samples demonstrating mostly heterogeneous methylation of RUNX3 and ABO. Thus, as MS-HRM cannot quantify heterogeneous methylation, bisulfite pyrosequencing analyses were also performed for these genes. Bisulfite pyrosequencing can quantify the averaged methylation level for each individual CpG dinucleotide within a region of interest [14]. MS-HRM was repeated with the reverse MS-HRM primer containing a 5′-biotin label, and products of duplicates were pooled for bisulfite pyrosequencing [100]. Forward pyrosequencing primers, the interrogated sequence and dispensation order are listed in Additional file 2: Table S3. Analyses were performed on a PyroMark Q96 instrument (Qiagen, Hilden, Germany) with PyroMark Gold Q96 SQA Reagents (Qiagen) according to manufacturer’s instructions, and data was analyzed with the Pyro Q-CpG software (Qiagen, version 1.0.9).

TCGA data and pre-processing

The TCGA database of head and neck cancers was accessed for publically available methylation data assessed by the Infinium HumanMethylation450 beadchip array (HM450K, Illumina inc., San Diego, US).

Methylation data from Level 1 raw IDAT files were obtained from the heterogeneous TCGA HNSCC samples (http://cancergenome.nih.gov/; HM450K download date 24 June 2013; clinical data download date 22 July 2013). Utilizing R software (version 3.0.1; http://cran.r-project.org/), background correction and probe specific correction with SWAN normalization was performed utilizing Methylumi and Minfi packages found in Bioconductor [112–114]. Probes mapping to common variant SNPs or non-unique mappings were removed from the analysis, leaving 330,557 probes for the downstream analysis [115]. Our MS-HRM amplicons interrogating the APC, BRCA1, CDH1, CDH13 and MLH1 genes, overlapped with CpG probes assessed by the HM450K. For the seven genes (ABO, ATM, DAPK1, RASSF1A, MGMT, ERCC1 and RUNX3) that did not have overlapping CpGs on the HM450K, the two probes immediately flanking upstream and downstream of our MS-HRM amplicon were assessed. A β-value greater than 0.2 was used to define the presence of significant methylation [116].

Statistical analysis

In order to account for the sensitivity of assays utilized and background methylation from normal stroma, significant methylation was defined a priori as a quantity 10% and greater for MS-HRM, and 15% and greater for pyrosequencing. A higher cut-off was chosen for pyrosequencing to ensure that only significant quantities of methylation were included on the basis that unmethylated DNA controls (WGA) demonstrated quantities of methylation up to 8% at individual CpG sites (see Additional file 2: Table S4).

As heterogeneous methylation can be identified but not quantified on MS-HRM analysis [100], two analyses were performed: one for samples with an estimated level of methylation 10% and greater while excluding samples with heterogeneous methylation, and the second analysis included all heterogeneously methylated samples in the same category.

Following bisulfite pyrosequencing which can quantify the average methylation at individual CpG dinucleotides for both homogenously and heterogeneously methylated samples, correlation with patient outcome was investigated. The analysis included only samples that had 15% and greater mean methylation on pyrosequencing, or for samples with insufficient DNA for pyrosequencing, those with 10% and greater methylation on MS-HRM were also included.

The Kaplan-Meier product-limit method was used to estimate OS and PFS. OS was defined as the time (in years) from the date of diagnosis to the date of death from any cause. PFS was considered only for patients treated with curative intent and was defined as the time (in years) from the date of diagnosis to the date of progression (loco-regional or distant) or death from any cause. A close-out date of 31 January, 2011, was set for the purpose of analysis. Associations between the presence of methylation and survival outcome were assessed using the exact Log-rank test and Cox proportional hazards regression, to estimate hazard ratios (HR) and associated 95% confidence intervals (95% CIs). P values <0.05 were regarded as statistically significant.