Molecular staging of surgical margins in head and neck ...
Molecular staging of surgical margins in oral squamous cell carcinoma using promoter methylation of p16INK4A, cytoglobin, E-cadherin and TMEFF2Richard J Shawa,b, Andrew J Hobkirka,b, George Nikolaidisa, Julia A Woolgarc,d, Asterios Triantafyllouc,d, James S. Brownb, Triantafillos Lilogloua,d, Janet M Riska,d* Department of Molecular & Clinical Cancer Medicine, University of Liverpool. UK. L69 3BX. Regional Maxillofacial Unit, Aintree University Hospitals NHS Trust, Liverpool. UK. L9 7ALCellular Pathology, Aintree University Hospitals NHS Foundation Trust, Liverpool. UK. L9 7ALSchool of Dentistry, University of Liverpool, UK. L69 3GN*: corresponding author:Department of Molecular & Clinical Cancer MedicineSchool of Dentistry Research Wing,Daulby Street,LiverpoolL69 3GNE: j.m.risk@liverpool.ac.ukT: 0151 706 5265F: 0151 706 5809Running Head:Methylation biomarker staging of OSCC margins Synopsis:Prognostic molecular staging of surgical margins in a series of prospectively-collected, fresh frozen tissue from oral squamous cell carcinoma using DNA methylation biomarkers, determined by quantitative methylation-specific PCR. Comparison with more usual, pathologically derived, indicators of recurrence.Word count: 2631 excluding abstract/figuresAbstractBackground: Local recurrence in oral squamous cell carcinoma (OSCC) despite clear surgical margins may indicate the presence of residual, sub-microscopic disease. Molecular assessment of surgical margins may provide a greater prognostic sensitivity compared to histopathology. We aim to determine whether promoter methylation in deep and mucosal resection margins can predict recurrence in OSCC.Methods: 48 consecutive OSCC cases were recruited and a 5mm3 tumour sample plus 5 deep and 5 mucosal margin samples snap frozen. Clinical, pathological, adjuvant therapy and outcome data were recorded. Tumours were informative if >5% promoter methylation was found for ≥1 of 4 genes using qMSP. Margins were declared molecularly positive if >1% promoter methylation was found in any margin.Results: 30/48 (63%) cases were methylation-informative. Mucosal margin samples were largely positive for methylation (26/30; 87%) indicating the presence of field cancerisation. Methylation at ≥1 gene promoters in ≥1 deep margin correlated with the presence of close/involved mucosal margins (P=0.027) and increased pT status (P=0.027) but not the status of deep margins, recurrence or survival.Conclusions: The current gene panel did not add prognostic information to histopathological reporting of resection margins. Future efforts should concentrate on improving gene selection, informativity and assay performance in the patient group with intermediate indications for adjuvant therapy.IntroductionThe principal aim of surgical ablation for malignancy is to achieve clear resection margins. These are routinely defined by histopathology , where an additional 5mm of ‘normal’ tissue beyond the tumour should be identified in three dimensions ADDIN EN.CITE <EndNote><Cite><Author>Heliwell T R</Author><Year>2011</Year><RecNum>53</RecNum><DisplayText>[1]</DisplayText><record><rec-number>53</rec-number><foreign-keys><key app="EN" db-id="axztatz0nw0pzueprv7pxsaexzdvr5xw22xx">53</key></foreign-keys><ref-type name="Electronic Article">43</ref-type><contributors><authors><author>Heliwell T R, Woolgar J A</author></authors></contributors><titles><title>Royal College of Pathologists: Dataset for histopathology reporting of mucosal malignancies
of the oral cavity</title><tertiary-title> </tertiary-title></titles><dates><year>2011</year></dates><urls></urls></record></Cite></EndNote>[1]. This requirement is based on the assumption that histopathologically invisible cancer cells exist within this margin and might explain the common finding of local recurrence despite histopathologically defined clear margins PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5XZWlqZXJzPC9BdXRob3I+PFllYXI+MjAwNDwvWWVhcj48
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ADDIN EN.CITE.DATA [2]. In head and neck cancer (HNSCC), the temptation to increase excision margins to minimise local recurrence must be tempered against the concern of unjustified and irreversible loss of function. For this reason, the novel staging methodologies, such as molecular staging, have been explored to a greater extent in head and neck surgery than other surgical disciplines ADDIN EN.CITE <EndNote><Cite><Author>Braakhuis</Author><Year>2010</Year><RecNum>2</RecNum><DisplayText>[3]</DisplayText><record><rec-number>2</rec-number><foreign-keys><key app="EN" db-id="avzrr5pw1d00v2ea5xexepr8ffsz0pxvd5rd">2</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Braakhuis, B. J.</author><author>Bloemena, E.</author><author>Leemans, C. R.</author><author>Brakenhoff, R. H.</author></authors></contributors><auth-address>Department of Otolaryngology/Head-Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands. bjm.braakhuis@vumc.nl <bjm.braakhuis@vumc.nl></auth-address><titles><title>Molecular analysis of surgical margins in head and neck cancer: more than a marginal issue</title><secondary-title>Oral Oncology</secondary-title><alt-title>Oral Oncol.</alt-title></titles><periodical><full-title>Oral Oncology</full-title></periodical><pages>485-91</pages><volume>46</volume><number>7</number><edition>2010/03/02</edition><keywords><keyword>*Carcinoma, Squamous Cell/genetics/pathology/surgery</keyword><keyword>*Head and Neck Neoplasms/genetics/pathology/surgery</keyword><keyword>Humans</keyword><keyword>Immunohistochemistry</keyword><keyword>*Neoplasm Recurrence, Local</keyword><keyword>Neoplasm, Residual</keyword><keyword>Tumor Markers, Biological/genetics</keyword></keywords><dates><year>2010</year><pub-dates><date>Jul</date></pub-dates></dates><isbn>1368-8375 (Print)
1368-8375 (Linking)</isbn><accession-num>20189442</accession-num><urls><related-urls><url>(10)00032-1 [pii]
10.1016/j.oraloncology.2010.01.019</electronic-resource-num><language>eng</language></record></Cite></EndNote>[3] with a view to providing a greater sensitivity for the detection of local recurrence. The technical and theoretical hurdles to be overcome are, however, substantial. Firstly, the assay should be robust and clinically applicable. Ideally, the molecular aberration should be observed with high specificity, i.e. in the tumour but not in normal tissues, and informativity, i.e. in all or a high percentage of cases. Additionally, an assay needs to be developed that identifies this aberration with high sensitivity, and the technical platform should be reliable, reproducible, inexpensive and applicable in routine clinical practice. Secondly, it must be clear that an appropriate and effective intervention is available for cases with molecularly involved but histologically clear margins. It is known that involved and close margins are associated with other markers of biological aggression PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TdXR0b248L0F1dGhvcj48WWVhcj4yMDAzPC9ZZWFyPjxS
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ADDIN EN.CITE.DATA [7, 8] but there are many sites where mutation has been shown to occur in this large gene with multiple exons. As such, the potentially attractive, sensitive PCR assay for any individual mutation offers very poor informativity, and the complex plaque phage functional assays for p53 are expensive and difficult to implement clinically. The common sites for chromosomal allelic loss in HNSCC have been explored as alternative molecular biomarkers of margin involvement PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5HcmF2ZWxhbmQ8L0F1dGhvcj48WWVhcj4yMDExPC9ZZWFy
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ADDIN EN.CITE.DATA [9, 10], but here the potential for highly sensitive assays is limiting. In contrast promoter hypermethylation is common ADDIN EN.CITE <EndNote><Cite><Author>Shaw</Author><Year>2006</Year><RecNum>11</RecNum><DisplayText>[11]</DisplayText><record><rec-number>11</rec-number><foreign-keys><key app="EN" db-id="avzrr5pw1d00v2ea5xexepr8ffsz0pxvd5rd">11</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Shaw, R.</author></authors></contributors><auth-address>Regional Maxillofacial Unit, University Hospital Aintree, Liverpool, UK. freedna@</auth-address><titles><title>The epigenetics of oral cancer</title><secondary-title>International journal of oral and maxillofacial surgery</secondary-title><alt-title>Int J Oral Maxillofac Surg</alt-title></titles><periodical><full-title>International journal of oral and maxillofacial surgery</full-title><abbr-1>Int J Oral Maxillofac Surg</abbr-1></periodical><alt-periodical><full-title>International journal of oral and maxillofacial surgery</full-title><abbr-1>Int J Oral Maxillofac Surg</abbr-1></alt-periodical><pages>101-8</pages><volume>35</volume><number>2</number><edition>2005/09/13</edition><keywords><keyword>Acetylation</keyword><keyword>CpG Islands/genetics</keyword><keyword>DNA/blood</keyword><keyword>*DNA Methylation</keyword><keyword>Epigenesis, Genetic/*genetics</keyword><keyword>Gene Silencing/physiology</keyword><keyword>Genes, Tumor Suppressor/physiology</keyword><keyword>Genomic Imprinting/genetics/physiology</keyword><keyword>Histones/metabolism</keyword><keyword>Humans</keyword><keyword>Mouth Neoplasms/*genetics</keyword></keywords><dates><year>2006</year><pub-dates><date>Feb</date></pub-dates></dates><isbn>0901-5027 (Print)
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ADDIN EN.CITE.DATA [13, 14]. Previous analyses of promoter hypermethylation in HSNCC PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Hb2xkZW5iZXJnPC9BdXRob3I+PFllYXI+MjAwNDwvWWVh
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ADDIN EN.CITE.DATA [15-19], as well as resection of liver ADDIN EN.CITE <EndNote><Cite><Author>Yang</Author><Year>2005</Year><RecNum>20</RecNum><DisplayText>[20]</DisplayText><record><rec-number>20</rec-number><foreign-keys><key app="EN" db-id="avzrr5pw1d00v2ea5xexepr8ffsz0pxvd5rd">20</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Yang, B.</author><author>Gao, Y. T.</author><author>Du, Z.</author><author>Zhao, L.</author><author>Song, W. Q.</author></authors></contributors><auth-address>Department of Genetics, College of Life Science, Nankai University, Tianjin 300071, PR China.</auth-address><titles><title>Methylation-based molecular margin analysis in hepatocellular carcinoma</title><secondary-title>Biochem Biophys Res Commun</secondary-title></titles><periodical><full-title>Biochem Biophys Res Commun</full-title></periodical><pages>1353-8</pages><volume>338</volume><number>3</number><edition>2005/11/05</edition><keywords><keyword>Adult</keyword><keyword>Aged</keyword><keyword>Base Sequence</keyword><keyword>Carcinoma, Hepatocellular/genetics/*metabolism/*pathology/surgery</keyword><keyword>Cyclin-Dependent Kinase Inhibitor p16/genetics/*metabolism</keyword><keyword>Female</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Methylation</keyword><keyword>Middle Aged</keyword></keywords><dates><year>2005</year><pub-dates><date>Dec 23</date></pub-dates></dates><isbn>0006-291X (Print)
0006-291X (Linking)</isbn><accession-num>16269133</accession-num><urls><related-urls><url>(05)02351-X [pii]
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ADDIN EN.CITE.DATA [21] malignancies, are characterised by small cohort size, analysis of a limited number of margin samples, and a lack of distinction between mucosal and deep margins. Importantly there have been a variety of methylation detection assays used with little consensus or justification for the cut-off at which a margin might be declared reliably positive. The aims of this study are to evaluate DNA hypermethylation in the analysis of surgical margins using a consecutive cohort of oral squamous cell carcinoma (OSCC) patients treated by primary surgery. We will focus on quantitative methylation analysis using real time quantitative methylation-specific PCR (qMSP) assays in DNA derived from multiple fresh-frozen margin samples from each case. The overall informativity of a panel of gene promoter methylation markers, as well as their individual contribution will be defined. The ability of methylation status of deep margins, and separately, mucosal margins, in predicting recurrence will be defined and compared with the histopathologically reported margin status and other clinicopathological features. MethodsClinical53 consecutive OSCC patients were selected for inclusion in this study over the period 1st April 2007 – 30th April 2008, and all gave informed consent under a specific institutional ethical approval (REC 07/Q1505/15). No power calculation was undertaken as this was designed as a pilot study. Inclusion criteria were histologically confirmed stage T2-T4 OSCC with a treatment decision for primary surgery. FiveT1 tumours presenting over this time period were excluded from the study in order to enrich for cases likely to show either involved margins or local recurrence, leaving 48 tumours for analysis. Following surgical resection, thorough irrigation of the tumour bed was carried out with 1000ml of 0.9% NaCl applied though a pressurised giving set. Subsequently, five mucosal (peripheral) and five deep margin samples, 5mm3, were randomly selected and surgically excised prior to reconstruction. Intra-operative frozen sections were not taken, as is the norm for UK practice. These samples were placed in 10 separately pre-labeled containers and immediately frozen and stored at -80C until DNA preparation. An additional frozen sample was taken from the primary tumour and stored similarly. Detailed histopathological analysis was recorded for each surgical resection according to standardized protocols, together with details of adjuvant therapy, and the clinical outcome for each patient was recorded for a minimum of 24 months.LaboratoryDNA was extracted from 2mm3 of each tissue sample using a DNeasy tissue kit (Qiagen Ltd, UK). DNA concentration was measured by spectrophotometry and subsequently adjusted to 40ng/ml. Bisulphite treatment of 1?g of each sample was undertaken using the EZ DNA Methylation Kit (Zymo Research Corporation, Orange, CA, USA) and the converted DNA eluted in 30?l of 0.1 TE buffer. Human genomic DNA (4?g) was artificially methylated as a positive control using SssI (CpG) Methylase (New England Biolabs, UK).qMSP assays were used to determine DNA methylation in the promoters of p16INK4A (CDKN2A), cytoglobin (CYGB), E-cadherin (CDH1) and TMEFF2. The incidence of promoter methylation at these genes in a similar OSCC cohort had been previously shown to exceed 25% [12, 22, Risk et al, unpublished data]. qMSP assays were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA) (primer and probe sequences and PCR conditions used available on request). A total reaction volume of 25?l contained Taqman Universal Master Mix II (Applied Biosystems), 500nM of each primer, 250nM of probe and 100ng of bisulphite-treated DNA. A separate assay utilising a methylation-independent primer/probe set specific for the ?-actin gene (ACTB) was used to normalise for the DNA input in each sample. Real-time PCR reactions were performed on an Applied Biosystems 7500 FAST system. Dilutions (5%, 1%, 0.5%, 0.1% and 0%) of in vitro methylated (SssI) human lymphocyte DNA made in untreated lymphocyte DNA were used as a reference. ΔΔCT values were generated for each target after normalisation by ACTB values. The RQ values were subsequently calculated (2-ΔΔCT) referenced to the artificially methylated samples for statistical analysis. All analysed data were the mean of duplicate reactions.For tumour specimens, a threshold of 5% methylation was used to define a sample that was methylated at that particular gene promoter, and hence the case was deemed to be informative for that marker. This threshold was based on our previous methylation data using a variety of techniques and HNSCC tumour types [12, 23] and has been used in other similar studies [17]. Both 1% and 0.1% methylation were considered as possible thresholds for a positive margin, irrespective of whether deep or mucosal in origin. A threshold of 1% was chosen for the analyses presented in this paper as this was the lowest value for which reproducible assignment of methylation positivity could be obtained in the present series of samples containing variable numbers of tumour cells diluted with ‘normal’ cells.Statistical analysisThe Statistical Package for the Social Sciences (SPSS, v 18, Chicago) was used to undertake statistical analysis, including Chi-square test for categorical data and Kaplan-Meier survival analysis.ResultsClinical characteristics of the cohortOf the 48 OSCC patients used in this study, 5 were lost to follow up and 6 had incomplete pathological information. 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ADDIN EN.CITE.DATA [5]. The only significant differences between the two populations were an increased incidence of higher pathological stage (p=0.022), the use of post-operative, adjuvant treatment (p=0.008) and presence of neck dissection (p=0015) in the cohort for the current study. These are a direct consequence of excluding T1 tumors in this cohort. Promoter methylation Thirty of 48 tumours (63%) demonstrated ≥5% promoter methylation at ≥1 gene. Thirteen tumours (26%) were methylated at TMEFF2, 11 (22%) at p16, 9 (18%) at CDH1 and 8 (16%) at CYGB. The ‘promoter positive’ cohort had a younger profile than the ‘promoter negative’ cohort (p=0.048), while the ‘promoter negative’ cohort contained more large (p=0.018), well differentiated (p=0.043) tumours than the ‘promoter positive’ cohort (Table 1). No other statistically significant differences in clinical characteristics or demographic data were observed between these two groups, although the ‘promoter positive’ cohort showed a trend towards improved 2 year survival (3/26 [12%] vs 5/17 [29%]DOD; Table 1).At least one mucosal margin from 26 of the 30 informative tumours (87%) showed promoter methylation at ≥1 gene, while in 19 /30 (63%) at least one deep margin showed methylation at ≥1 gene (Supplementary table). As gene promoter methylation at mucosal margins did not appear to be discriminatory, this data was not included for further analysis. CDH1 promoter methylation was observed in ≥1 mucosal margin in all the positive tumours (9/9) and in ≥1deep margin from 8/9 positive tumours and was thus deemed to be not discriminatory and removed from further analyses. Thus, 26 tumours remained for correlation of promoter methylation at 3 genes in deep margin samples with clinicopathological data.Correlation of margin methylation with clinicopathological features Methylation at ≥1 out of 3 gene promoters in ≥1 deep margin correlated with the presence of close/involved mucosal margins (P=0.027), an absence of dysplasia at the surgical margin (P=0.024) and with tumour stage (P=0.027), most notably an increase in pT4 tumours (Table 2). There was no correlation with the histopathologically documented presence of deep margin involvement, pattern of invasion, nodal involvement, ECS, recurrence, pattern of recurrence or survival. Indeed, close or involved pathological margins were superior to methylation of ≥1 gene promoters at predicting recurrence in methylation positive tumours (5/7 recurrences were in patients with close/involved surgical margins vs 3/7 recurrences in patients with methylation positive deep surgical margins). Interestingly, histopathological assessment of margins was not such a good prognostic indicator for methylation negative tumours, where only 3/9 recurrences were in patients with close/involved margins (not significant: Supplementary table).Of the 11 tumours with p16 promoter methylation, 7 showed concordant methylation in ≥1 deep margin tissues. This correlated with the presence of a non-cohesive invasive tumour front (P=0.015) and showed some association with the presence of histopathologically close or involved deep margins (Table 2). Although the numbers are small, there was some indication that patients with p16 positive margins presented with recurrence earlier than those with p16 negative margins and had a shorter survival period after recurrence (not significant: patients 3329,3338, 3371 & 3363, Supplementary table).DiscussionIn this study, we investigated the surgical margins in OSCC for promoter methylation as a predictor of clinical outcome. 30/48 tumours showed promoter methylation at ≥1 of 4 genes, with the incidence of individual gene promoter methylation comparable to those previously described using similar, quantitative assays PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UYW48L0F1dGhvcj48WWVhcj4yMDA4PC9ZZWFyPjxSZWNO
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ADDIN EN.CITE.DATA [19, 22].Analysis of data for individual genes provided some insight as to why observations using data from all three genes was not a good prognostic indicator. p16 promoter methylation at deep margins was observed to be associated with pattern of invasion and possibly with close deep pathological margins and early recurrence. Three of the four recurrences from tumours demonstrating p16 methylation also showed p16 promoter methylation in the deep margins. However, four additional patients with methylation of this gene promoter in deep margin tissue did not recur. Conversely, TMEFF2 promoter methylation in deep margins showed an association with smaller tumour size. These data suggest a possible role of p16 downregulation in tumour recurrence, while TMEFF2 may be a bystander event.The advantages of the present study over many previous reports [15-19] are that we have obtained snap frozen tissue with detailed pathology and at least 2 year follow-up. Furthermore, we have used a quantitative, real time MSP methodology with a pre-determined cutoff. We have also investigated mucosal and deep margins separately, finding mucosal margins show lack of specificity with extensive methylation in presumed field change. Limitations of the present study include lack of informativity (30/48 tumours positive for promoter methylation at ≥1 genes) and the choice of biomarkers. This was unexpected given our previously determined incidence of informativity for these genes, but may reflect the different methodologies employed [12, 22]. However, previous published reports reflect even fewer informative cases - a cumulative total of only 70 informative tumours have been previously published amongst five previous series using methylation assays in surgical margins PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Hb2xkZW5iZXJnPC9BdXRob3I+PFllYXI+MjAwNDwvWWVh
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ADDIN EN.CITE.DATA [15-19]Given the intensive nature of sample collection and analysis of 11 samples per tumour involved in this study, some conclusions regarding sample selection/pooling should be drawn from our data before embarking on a larger series. We have found it difficult to use DNA methylation biomarkers to distinguish field change or premalignant lesions [24] at the mucosal margin from residual tumour. Further, it may be more appropriate to sample the deep margins at several sites, but to then pool the DNA to create a single sample for prognostic purposes. Similarly, the selection of the panel of biomarkers used in the present study was unfortunate in that one marker (CDH1) was largely uninformative, while the sensitivity of one other marker (CYGB) appeared to be lower than the other two genes, as shown by the low number of positive margins associated with CYGB positive tumours. The identification of further markers with suitable sensitivity for inclusion in a methylation biomarker panel is required and candidates may yet emerge with improving genome-wide array techniques. Lastly, it is worthwhile to reflect on the clinical context for which molecular margin analysis may be of greatest therapeutic value. In those cases defined as the intermediate risk group [25] where the role for adjuvant therapy remains unproven, the value of molecular margin analysis might be best highlighted. This may be particularly the case for those resections with close margins as a sole adverse prognostic feature. Concentrating future efforts on improving informativity and the utility of assay performance in this group seems logical. This would clarify if these, as yet unproven, techniques can be translated into clinical practice.AcknowledgementsThis work was funded by a CRUK Pilot Project Award [C23033/A8332] and by the British Association of Oral and Maxillofacial SurgeonsReferences ADDIN EN.REFLIST 1.Heliwell T R WJA. Royal College of Pathologists: Dataset for histopathology reporting of mucosal malignancies of the oral cavity. M, Snow GB, Bezemer DP, van dr Wal JE, van der Waal I. The status of the deep surgical margins in tongue and floor of mouth squamous cell carcinoma and risk of local recurrence; an analysis of 68 patients. Int J Oral Maxillofac Surg 2004; 33(2):146-9.3.Braakhuis BJ, Bloemena E, Leemans CR, Brakenhoff RH. Molecular analysis of surgical margins in head and neck cancer: more than a marginal issue. Oral Oncology 2010; 46(7):485-91.4.Sutton DN, Brown JS, Rogers SN, Vaughan ED, Woolgar JA. The prognostic implications of the surgical margin in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2003; 32(1):30-4.5.Rogers SN, Brown JS, Woolgar JA, et al. Survival following primary surgery for oral cancer. Oral Oncol 2009; 45(3):201-11.6.Shaw RJ, Lowe D, Woolgar JA, et al. Extracapsular spread in oral squamous cell carcinoma. Head Neck 2010; 32(6):714-22.7.Brennan JA, Mao L, Hruban RH, et al. Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck. New England Journal of Medicine 1995; 332(7):429-35.8.Huang X, Pateromichelakis S, Hills A, et al. p53 mutations in deep tissues are more strongly associated with recurrence than mutation-positive mucosal margins. Clin Cancer Res 2007; 13(20):6099-106.9.Graveland AP, Golusinski PJ, Buijze M, et al. Loss of heterozygosity at 9p and p53 immunopositivity in surgical margins predict local relapse in head and neck squamous cell carcinoma. Int. J. Cancer 2011; 128(8):1852-9.10.Temam S, Casiraghi O, Lahaye JB, et al. Tetranucleotide microsatellite instability in surgical margins for prediction of local recurrence of head and neck squamous cell carcinoma. Clin Cancer Res 2004; 10(12 Pt 1):4022-8.11.Shaw R. The epigenetics of oral cancer. Int J Oral Maxillofac Surg 2006; 35(2):101-8.12.Shaw RJ, Liloglou T, Rogers SN, et al. Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer 2006; 94(4):561-8.13.Eads CA, Danenberg KD, Kawakami K, et al. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 2000; 28(8):E32.14.Shaw RJ, Akufo-Tetteh EK, Risk JM, Field JK, Liloglou T. Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing. Nucleic Acids Res 2006; 34(11):e78.15.Goldenberg D, Harden S, Masayesva BG, et al. Intraoperative molecular margin analysis in head and neck cancer. Arch. Otolaryngol.Head Neck Surg. 2004; 130(1):39-44.16.Martone T, Gillio-Tos A, De Marco L, et al. Association between hypermethylated tumor and paired surgical margins in head and neck squamous cell carcinomas. Clin Cancer Res 2007; 13(17):5089-94.17.Shaw RJ, Hall GL, Woolgar JA, et al. Quantitative methylation analysis of resection margins and lymph nodes in oral squamous cell carcinoma. Br J Oral Maxillofac Surg 2007; 45(8):617-22.18.Sinha P, Bahadur S, Thakar A, et al. Significance of promoter hypermethylation of p16 gene for margin assessment in carcinoma tongue. Head Neck 2009; 31(11):1423-30.19.Tan HK, Saulnier P, Auperin A, et al. Quantitative methylation analyses of resection margins predict local recurrences and disease-specific deaths in patients with head and neck squamous cell carcinomas. Br J Cancer 2008; 99(2):357-63.20.Yang B, Gao YT, Du Z, Zhao L, Song WQ. Methylation-based molecular margin analysis in hepatocellular carcinoma. Biochem Biophys Res Commun 2005; 338(3):1353-8.21.Guo M, House MG, Hooker C, et al. Promoter hypermethylation of resected bronchial margins: a field defect of changes? Clin. Cancer Res. 2004; 10(15):5131-6.22.Shaw RJ, Hall GL, Lowe D, et al. CpG island methylation phenotype (CIMP) in oral cancer: associated with a marked inflammatory response and less aggressive tumour biology. Oral oncology 2007; 43(9):878-86.23.Schache AG, Hall G, Woolgar JA, et al. Quantitative promoter methylation differentiates carcinoma ex pleomorphic adenoma from pleomorphic salivary adenoma. Br J Cancer 2010; 103(12):1846-51.24.Hall GL, Shaw RJ, Field EA, et al. p16 Promoter methylation is a potential predictor of malignant transformation in oral epithelial dysplasia. Cancer Epidemiol Biomarkers Prev 2008; 17(8):2174-9.25.Brown JS, Blackburn TK, Woolgar JA, et al. A comparison of outcomes for patients with oral squamous cell carcinoma at intermediate risk of recurrence treated by surgery alone or with post-operative radiotherapy. Oral Oncology 2007; 43(8):764-73.Table 1 Clinical and demographic characteristics of the patient cohort used in this studyAll tumours(n=48)me+ tumoura (n=30: 63%)me– tumoursb (n=18: 38%)Gender: Male Female33 (72%)13 (28%)20 (71%) 8 (29%)13 (72%) 5 (28%)Age: < 55 55-64 65-74 75+13 (28%)21 (45%)8 (17%)5 (11%)12 (41%)10 (34%) 5 (17%) 2 (7%) 1 (6%)11 (61%) 3 (17%) 3 (17%)*Tumour site: Buccal Lower gum Tongue (ant 2/3) Floor of mouth Other5 (11%)7 (15%)16 (34%)11 (23%)8 (17%)2 (7%)5 (17%)9 (31%)6 (21%)7 (24%)3 (17%)2 (11%)7 (39%)5 (28%)1 (6%)Tumour differentiation: Poor Moderate Well 2 (5%)28 (67%)12 (29%) 1 (4%)21 (81% 4 (15%) 1 (6%) 7 (44%) 8 (50%)*Invasive frontc: Cohesive Non-cohesive11 (24%)35 (76%) 9 (31%)20 (69%) 2 (12%)15 (88%)Mucosal margins: Clear ≥ 5mm Close < 5mm Involved24 (51%)16 (34%) 7 (15%)14 (48%) 9 (31%) 6 (21%)10 (56%) 7 (39%) 1 (6%)Deep margins: Clear ≥ 5mm Close < 5mm Involved22 (51%)17 (40%) 4 (9%)11 (42%)12 (46%) 3 (12%)11 (65%) 5 (29%) 1 (6%)pT: T1/T2 T3/T424 (51%)23 (49%)15 (52%)14 (48%)9 (50%)9 (50%)pN: 0 1 2-324 (52%) 6 (13%)16 (35%)18 (62%) 3 (10%) 8 (28%) 6 (35%) 3 (18%) 8 (47%)p stage: 2 3 4 11 (24%) 7 (15%)28 (61%) 8 (28%) 4 (14%)17 (59%) 3 (18%) 3 (18%) 11 (65%)Nodal status: N0 N+ ECS – N+ ECS +24 (52%)10 (22%)12 (26%)18 (62%) 6 (21%) 5 (17%) 6 (35%) 4 (24%) 7 (41%)Adjuvant radiotherapy Yes No26 (60%)17 (40%)17 (65%) 9 (35%)9 (53%)8 (47%)Recurrence: Yes No16 (37%)27 (63%)7 (27%)19 (73%)9 (53%)8 (47%)2 yr Survival: Disease free DOD Died (other)32 (74%)8 (19%)3 (7%)21 (81%) 3 (12%)2 (8%)11 (65%)5 (29%)1 (6%)a me+ tumours: tumours showing ≥5% methylation at ≥ 1 gene promoterb me- tumours: tumours showing <5% methylation at all gene promotersc invasive front classified into cohesive and non-cohesive patternsECS: Extracapsular spread; DOD: Died of disease* p=0.05 Table 2 Correlation of methylation at deep margins with clinicopathological parametersGene Promoter Methylation at:p16 and/or TMEFF2 and/or CYGBP16 aloneTMEFF aloneCYGB aloneClose/involved deep marginsns0.071nsnsClose/involved mucosal margins0.027nsnsnsInvasive frontans0.015nsnsDepthnsnsnsnsNodes/ECSbnsnsnsnsECSnsnsnsnsNodesnsnsnsnsStage0.074nsnsnspT (pT2 v pT3 v pT4) (pT2/3 v pT4)0.0270.035nsns0.043nsnsnsAnneroth scorensns0.032nsRecurrencensnsnsnsDSScnsnsnsnsOSdnsnsnsns a invasive front classified into cohesive and non-cohesiveb ECS: extracapsular spread c DSS: Disease-specific survivald OS: Overall survival ................
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