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Quantitative methylation analysis of resection margins and lymph nodes in oral squamous cell carcinoma
Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery 45 (2007) 617–622
Quantitative methylation analysis of resection margins and lymph nodes in oral squamous cell carcinoma Richard J. Shaw a,b,∗ , Gillian L. Hall a,c , Julia A. Woolgar c , Derek Lowe b , Simon N. Rogers b , John K. Field a , Triantafillos Liloglou d , Janet M. Risk a,1 a
Molecular Genetics & Oncology Group, School of Dental Sciences, University of Liverpool, Liverpool L69 3GN, UK Regional Maxillofacial Unit, University Hospital Aintree, Longmoor Lane, Liverpool L9 7AL, UK c Department of Oral Pathology, University of Liverpool, UK d University of Liverpool Cancer Research Centre, Roy Castle Lung Cancer Research Programme, 200 London Rd, Liverpool L3 9TA, UK b
Accepted 15 April 2007 Available online 7 June 2007
Abstract Resection is the preferred treatment for oral squamous cell carcinoma, and pathological staging of the resected specimen is crucial. The role of molecular biology in the diagnosis of minimal residual disease has not been fully investigated and may improve staging. Multiple adjacent specimens were taken from the tumour, the invasive front, the surgical margin, and the lymph nodes of 20 specimens from patients with oral cancer. Bisulphite-treated DNA from these specimens was assayed quantitatively with pyrosequencing methylation assays (PMA) of CpG islands within the gene promoters of the p16 and CYGB genes. Results were recorded with histopathological results, and compared with clinical outcome. Biological and technical replicates confirmed the reliability of the techniques. PMA upgraded 13 of the 20 surgical margins, 6 of which subsequently had a recurrent tumour. Not all of these recurrences were predicted and the effects of adjuvant treatment make firm conclusions difficult. © 2007 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Methylation; Oral squamous cell carcinoma; Surgical margin; Pathology; Pyrosequencing
Introduction Oral squamous cell carcinoma is the commonest form of head and neck cancer in the UK1 and is also common in other parts of the world.2 Primary resection is the commonest treatment of oral squamous cell carcinoma (SCC). Postoperative radiotherapy (PORT) is indicated in about half of such resections, decided by factors such as cervical lymph node status,3 pattern of tumour invasion4,5 and the status of the
∗ Corresponding author at: University Hospital Aintree, Department of Oral and Maxillofacial Surgery, Lower Lane, Liverpool L9 7AL, UK. Tel.: +44 151 529 5290. E-mail addresses: [email protected] (R.J. Shaw), [email protected] (J.M. Risk). 1 Tel.: +44 151 706 5265; fax: +44 151 706 5809.
surgical margin. However, the exact histopathological criteria for prescription of PORT remain controversial,6 and so it is logical to investigate the potential role of molecular biomarkers. The issues about surgical margins are particularly relevant to oral resections. The decision to increase the “safety margin” around a tumour might result in a more favourable histological report and a reduction in the need for adjuvant treatment, but could damage oral functions such as speech and swallowing. The role that epigenetic aberrations take in cancer is being increasingly realised with promoter hypermethylation being the best understood.7–9 Molecular resection margin analysis using methylation assays have been described in single reports in hepatocellular carcinoma,10 non-small cell lung cancer11 as well as head and neck cancer.12 Quantitative pyrosequencing methylation assays (PMAs) have been
0266-4356/$ – see front matter © 2007 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2007.04.015
618
R.J. Shaw et al. / British Journal of Oral and Maxillofacial Surgery 45 (2007) 617–622
described13,14 including those for the p16 and Cytoglobin (CYGB) gene promoters. Pyrosequencing methylation analysis has several crucial advantages in methylation analysis, but does not offer particularly high sensitivity (1:20 to 1:50) and may be open to question in the identification of the small proportion of methylated alleles expected in samples from surgical margins. Whether a quantitative assay such as PMA is sensitive enough to be of clinical value in this context has not been previously investigated. In this study, we apply quantitative pyrosequencing assays to the diagnosis of minimal residual disease using two gene promoters. This focuses on surgical margins as well as evaluation of lymph nodes in a cohort of 20 patients whose pTNM, full treatment details, and two-year outcome data were known.
Methods Patients and tissue Twenty patients newly diagnosed with biopsy-confirmed oral squamous cell carcinoma that had not been treated previously were selected because promoter methylation of either the p16 or CYGB promoter was present, and they were part of a larger series, the methylation status of which has been reported previously. Clinical staging and operative details were recorded. Fresh tumour samples of 5 mm3 were excised at operation from resected specimens within the tumour mass that did not involve the margin. These were coded, snap-frozen in liquid nitrogen and stored at −85 ◦ C. Sections with haematoxylin and eosin stained from each resected tumour were reviewed. One block from each tumour was selected that had histological features that represented the tumour as a whole, and also contained a strip of tissue from surface to deep margin. The strip to be removed was marked out on the slide and used as a template to guide macroscopic dissection of a strip of tissue from the block 5 mm wide. This was then divided into 4–6 equally sized cubes (Fig. 1). Care was taken to avoid cross-contamination within and between tissue blocks by using fresh scalpel blades on each occasion. The boundaries of the blocks were measured and marked on to the original slide. The slide was re-examined to allow esti-
mation of the proportion (to the nearest 10%) of tumour cells in relation to stroma or infiltrate of inflammatory host cells in the more superficially dissected blocks containing tumour, and identification of those that were histologically free of carcinoma. Areas of lymph nodes that were free of tumour and those containing solid deposits of metastatic tumour were obtained in a similar way. Preparation of samples DNA was extracted from the 20 fresh, snap frozen tissue samples using a DNeasyTM tissue kit (Qiagen Ltd.) and treated with bisulphite using the EZ DNA Methylation KitTM (Zymo Research) as previously described.14 The preparation of fixed tissues differed in that the DNA extraction was preceded by two xylene paraffin extractions and two 100% ethanol washes before lysis. In contrast to preparation of fresh tissue, double the recommended concentration of proteinase K, and 24 h of lysis were required for the fixed tissues. Fixed tissue samples were obtained from the 20 paraffin blocks: 60 from tumour, 52 from (histologically) tumour-free surgical margins, and 16 from lymph nodes. For fixed tissue, DNA was prepared using 2 mm3 samples from within the 5 mm3 specimens. Additional 2 mm3 samples were taken from 12 of these specimens to act as representative biological replicates. In total, 140 fixed specimens were prepared for molecular assay. Quantitative pyrosequencing methylation analysis (PMA) Hot Start (inhibition of polymerase activity during preparation of reaction) polymerase chain reaction (PCR) was done on all 20 fresh and 140 fixed tumour samples (including the 12 biological replicates) as described previously.12 Confirmation of the quality of the PCR and freedom from contamination was established on 2% agarose gels stained with ethidium bromide. Pyrosequencing was done using the PSQ96MA System (Biotage), and a mean methylation index was calculated as described previously.14 Statistical analysis We used the Statistical Package for the Social Sciences (SPSS, v 11, Chicago) to help with statistical analysis, including the intraclass correlation coefficient (ICC), which has a range of 0–1, 1 being complete agreement.
Results Clinical data
Fig. 1. Indication of where samples were removed from resection. T: specimen for snap freezing in liquid nitrogen, T1 → T4: 4× tumour, and M1 → M2: 2× margin specimens removed from formalin fixed tissue block.
Of the 20 tumours resected, 15 were staged pT2, 1 pT3, and 4 pT4. Eight contained metastases to the cervical lymph nodes; 6 staged pN2b and 2 pN2c. Seven of the patients were given PORT and eight had developed either local or regional recur-
R.J. Shaw et al. / British Journal of Oral and Maxillofacial Surgery 45 (2007) 617–622
rence at the time of their latest review (minimum 26 months, range 26–37). When the deep margins were re-examined, all the resections had at least 5 mm (range 5–20 mm) of resection margins free of tumour on histopathological examination, and none of them had frank cut-through of tumour at the mucosal or other deep margin. Pyrosequencing methylation analysis of fixed compared with snap-frozen tissue Methylation of the p16 promoter in fixed tumour tissue correlated with that of the fresh frozen samples of tumour in 18 of the 20 cases. The remaining two cases had different results. However, it is clear from Fig. 1 that the tumour tissue samples taken for this comparison were geographically separate so it remains possible that molecular heterogeneity within the tumour might explain the discrepancy. The level of agreement between fresh and fixed specimens was assessed, and using the ICC reliability module (two way mixed effects), the ICC for p16 was 0.75 (95% confidence interval (CI) 0.48 to 0.89). Methylation of the CYGB promoter was similar in 18 of the 20 cases using the two tissue sources and the ICC was 0.81 (95% CI 0.58 to 0.92). The two discordant cases in this instance differed from the p16 discordant cases. Biological and technical replicates Additional DNA samples were prepared from 12 of the fixed tissue blocks for use as biological replicates. Samples were chosen for this analysis to give the mean methylation index from 0 to >25 for each gene. These were subject to p16 and CYGB PMA, which was compared with the original data. The ICC for p16 was 0.97 (95% CI 0.91 to 0.99) and for CYGB was 0.71 (95% CI 0.27 to 0.91). Some of this variation may, again, potentially be the result of heterogeneity of the tumours. Technical replicates were also done on these 12 samples by repeating the PCR and pyrosequencing steps on existing DNA templates treated with bisulphite. These data gave an ICC for p16 of 0.87 (95% CI 0.60 to 0.96) and for CYGB of 0.89 (95% CI 0.68 to 0.97). These results reassured us about the reliability and repeatability of the assays. We did a kappa analysis with the 12 additional samples mentioned to test the ability of the assay to discriminate between those with greater than 5% methylation and those with less than 5%. The kappa values of agreement for technical replicates were 1.00 for p16, 0.83 for CYGB and the biological replicates were 1.00 for p16 and −0.13 for CYGB. Kappa values of 0.60 are good, above 0.80 is very good, whereas 0 or less than 0 is poor. The confidence intervals were wide, which reflects the small sample size. Methylation at surgical margins p16 promoter methylation Eleven of the 20 snap-frozen specimens of tumour showed p16 promoter methylation by PMA (range 1–45%), while
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11 of the 20 fixed tumour tissue specimens that contained ≥10% tumour cells showed p16 promoter methylation ≥5% in at least one of the serial tumour samples (range 5–53%). A mean methylation index of 1% is considered to be a valid result for snap-frozen tissue based on our previous analysis of variance,15 while 5% is used as the cut-off for fixed tissue. Evidence of p16 promoter methylation within specimens of surgical margins (all 0 tumour on histopathological examination) was found in 11/20 cases using PMA (range 2–11%), however, only 4 of these give methylation indexes of ≥5%, two of which showed local recurrence despite having less aggressive tumours with no pathological lymph nodes involved. Three of the remaining seven with methylation indexes ≥5% were present in cases in which no p16 methylation had been seen in the tumour on analysis of either fixed or fresh specimens, and may indicate false positive results, or perhaps some heterogeneity within the tumour. However, one of these subsequently developed a local recurrence. Of the eight cases with paired positive and negative lymph nodes available for analysis, three had p16 promoter methylation in their primary tumour. All three cases had PMA results ≥5% in invaded lymph nodes but no significant methylation in the clear lymph node. Two of the three patients had locoregional recurrence. None of the remaining five showed p16 promoter methylation in either the invaded or clear lymph nodes. Two of the five with recurrent neck disease had borderline evidence of p16 promoter methylation in a histologically clear lymph node (3% and 2%, respectively), while none of the three disease-free nodes gave similar results. CYGB promoter methylation Eighteen of the 20 snap-frozen tumour tissues showed CYGB methylation using PMA (range 4–54%), while all 20 fixed specimens of tumour tissue containing ≥10% tumour cells showed CYGB promoter methylation of ≥5% in at least one of the serial tumour samples (range 5–79%). Evidence of tumour within surgical margins was found in 17/20 cases using PMA (range 2–32%), 14 of which gave methylation indexes of ≥5%, and five of whom developed local recurrences. In two of the three apparently less aggressive tumours (T2N0) that recurred, there was consistent evidence of CYGB promoter methylation of ≥5% by PMA in all of the specimens of margins tested however, this was also seen in three other ‘less aggressive’ tumours which did not recur, although one of these patients was given postoperative radiotherapy. Seven of eight cases with invaded lymph nodes on histopathological examination had evidence of CYGB promoter methylation in nodes shown to be invaded by PMA. Two of eight cases had evidence of methylation in nodes histologically by PMA. Four of five patients with nodal involvement and neck recurrence were in this group.
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Figs. 2–4. Co-registration of pathology with methylation data. The upper row shows photomicrographs spanning the tumour, invasive front and surgical margin. The second row indicates the sample codes as in Fig. 1. The third row shows the percentage of tumour cells as estimated by pathologist. The fourth and fifth rows indicate corresponding methylation indices for each sample for genes p16 and cygb. (2) A favourable tumour with neither pathological nor molecular evidence of disease beyond the invasive front. (3 and 4) Molecular evidence beyond the tumour margin not apparent on histopathology and both suffered local or regional recurrence.
R.J. Shaw et al. / British Journal of Oral and Maxillofacial Surgery 45 (2007) 617–622
Summary Using the 5% methylation index as a threshold, in 13 of the 20 patients the surgical margins were upgraded; subsequently 6 of these 13 had either a local or regional recurrence. A further two patients had recurrences that did not show positive molecular margins within our analysis of the tissue studied. Illustrations of results for three of these patients are shown in Fig. 2–4.
Discussion This exploratory study brings together clinical, histopathological and molecular data in resection of oral squamous carcinoma to clarify their role. The conclusions are limited by the number of patients and also by the use of fixed tissue, so we are cautious about making any recommendations of the clinical value of these assays. However, the data show that the assays can identify molecular evidence in “clear” tissues using routine histopathological techniques, and in some patients who ultimately develop recurrences. This was apparent in the assays used for histologically clear lymph nodes and also for tissues at the edge of resection margins. We feel these assays offer sufficient potential to merit further investigation, optimisation and clinical validation in larger trials. It may add greatly to the sensitivity in any particular patient to process several samples from each resection margin. Our study has presented data from 2 mm2 , only a small fraction of the surface area of any margin. Radiological or histopathological assessments in clinical practice would not routinely take such a limited view in the staging of a tumour. It would be feasible to take 6–10 small samples beyond the resection margin for molecular analysis, both in the deep and mucosal margins. Some epigenetic changes may be present in premalignant oral epithelium and these might confound the findings at mucosal margins, but our previous data with p16 and CYGB are reassuring about this.14 The selection of lymph nodes for molecular analysis might be improved by adopting techniques used for identification of sentinel nodes.16 Contamination of samples, either in the operating theatre or in the laboratory, may be important if more sensitive assays are used (such as methylation-specific PCR). The principles behind the selection of methylation as a biomarker have been well-defined. Promoter methylation is common, tumour-specific, and there are a number of methods for detection, some of which are highly sensitive. Which gene or combination of genes, might be best is less clear. We have selected cases that had known promoter methylation at either p16 or CYGB, a finding true in 80 of 92 (87%) specimens of oral malignant tumours that we examined. The addition of other genes to a panel might bring the percentage of “informative” cases closer to 100%. It remains possible that low level CYGB methylation exists even in normal oral tissues, which would explain some of the apparent false positives. Extensive investigations of any likely biomarker gene in var-
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ious normal tissues are necessary. The search for other genes that are commonly silenced by promoter methylation continues, using techniques such as pharmacological unmasking and promoter tiling arrays. These assays may require further optimisation, although fresh clinical samples rather than fixed tissue are preferable. Assays that perform well in laboratory dilutions of high quality DNA may not be as reliable in clinical samples in which various contaminants, necrosis of the tumour, and degradation of DNA, can have an impact. Any test used clinically may need to be done in duplicate or even triplicate to eliminate mispriming artefacts or other causes of false positive results. Other methylation assays such as nested MSP and real-time MSP may also be considered for comparison. We have recently described methylation enrichment pyrosequencing15 a new assay that combines the potentially high sensitivity of methylation-specific PCR (MSP) with a second, confirmatory pyrosequencing step. This second step is rapid, inexpensive, and offers advantages over previously described sequencing or restriction enzyme methods. We have reported the potential benefits of methylation enrichment pyrosequencing in clinical samples from the head and neck,15 and speculate this might be a promising technique for future studies of surgical margins.
Acknowledgements RJS is in receipt of a Royal College of Surgeons of England Research Fellowship; research was funded by the British Association of Oral and Maxillofacial Surgeons; TL is funded by the Roy Castle Foundation.
References 1. Office of National Statistics, Welsh Cancer Intelligence Surveillance Unit, 2000; 2004. 2. Shah JP BJJJ. Oral Cancer. Martin Dunitz, Taylor & Francis; 1999. 3. Woolgar JA, Rogers SN, Lowe D, Brown JS, Vaughan ED. Cervical lymph node metastasis in oral cancer: the importance of even microscopic extracapsular spread. Oral Oncol 2003;39(2):130–7. 4. Shaw RJ, Brown JS, Woolgar JA, Lowe D, Rogers SN, Vaughan ED. The influence of the pattern of mandibular invasion on recurrence and survival in oral squamous cell carcinoma. Head Neck 2004 Oct;26(10):861. 5. Soo KC, Carter RL, O’Brien CJ, Barr L, Bliss JM, Shaw HJ. Prognostic implications of perineural spread in squamous carcinomas of the head and neck. Laryngoscope 1986;96(10):1145–8. 6. Blackburn TK, Bakhtawar S, Brown JS, Lowe D, Vaughan ED, Rogers SN. A questionnaire survey of current UK practice for adjuvant radiotherapy following surgery for oral and oropharyngeal squamous cell carcinoma. Oral Oncol 2007;43(2):143–9. 7. Esteller M, Fraga MF, Paz MF, Campo E, Colomer D, Novo FJ, et al. Cancer epigenetics and methylation. Science 2002;297(5588):1807–8. 8. Momparler RL. Cancer epigenetics. Oncogene 2003;22(42):6479–83. 9. Shaw R. The epigenetics of oral cancer. Int J Oral Maxillofac Surg 2006;35(2):101–8. 10. 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.
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11. Guo M, House MG, Hooker C, Han Y, Heath E, Gabrielson E, et al. Promoter hypermethylation of resected bronchial margins: a field defect of changes? Clin Cancer Res 2004;10(15):5131–6. 12. Goldenberg D, Harden S, Masayesva BG, Ha P, Benoit N, Westra WH, et al. Intraoperative molecular margin analysis in head and neck cancer. Arch Otolaryngol Head Neck Surg 2004;130(1):39–44. 13. Colella S, Shen L, Baggerly KA, Issa JP, Krahe R. Sensitive and quantitative universal pyrosequencing methylation analysis of CpG sites. Biotechniques 2003;35(1):146–50.
INTERESTING CASE: Walter Whitehead: A brief history of the man and his varnish Whitehead’s varnish is popular in oral and maxillofacial surgical practice, where it has a long history of use as an intraoral or buried dressing pack. Despite its longstanding and common use few surgeons will know of its inventor and how it was originally applied. Whitehead’s first recorded description of his varnish is in a paper in the British Medical Journal in 1891 entitled “A hundred cases of entire excision of the tongue”.1 In this paper, Walter Whitehead states that he first started using the dressing 10 years previously. A varnish is a transparent, hard, protective finish or film primarily used in finishing wood but also other materials. Varnishes are traditionally a combination of a drying oil, a resin, and a thinner or solvent. The varnish that Whitehead made was composed of iodoform, (a compound tincture of benzoin Friar’s balsam which is commonly used as an antiseptic, or for inhalation with steam as an expectorant) and turpentine. He wrote that the preparation had both antiseptic and anaesthetic properties, the turpentine component having a “very marked influence in promptly checking capillary oozing”. In addition to these properties, he went on to say that “it lessened the discomfort which follows surgery and also enables the patient to take food in the ordinary manner”. Today Whitehead’s varnish (pigmentum iodoform compound: BPC) consists of: iodoform10 g, benzoin10 g, prepared storax (a resinous exudate of the Sweetgum tree) 7.5 g, Balsam of Tolu 5 g (a resin obtained from South American balsam trees) and solvent ether mixed to 100 ml. Although turpentine is no longer used as the volatile liquid, Whitehead’s varnish is still advocated for many uses including dressing of skin graft donor sites,2 packing after exposure of unerupted canine teeth,3 jaw cavities,4 and nasal packing.5 Whitehead’s varnish has a benefit over a number of other dressings in that it remains ‘clean’ and uninfected in potentially contaminated environments. It contains a number of aromatic resins, which are slowly broken down to produce benzoic acid. It is this slow release of a potent antiseptic, together with the waterproofing property of the compound, which many surgeons suggest makes it superior to other media.6 Walter Whitehead was born in 1840 at Halsam Hey, Bury, where his family had lived for over 200 years. At the age of 16, he entered his father’s business and began to train as a bleacher, dyer, and finisher. This is probably when he learned about chemicals and is perhaps what led him to develop his varnish. One day, aged 19, and still an ordinary workman, he was invited by a medical student to observe some operations at the local infirmary. It was while watching these operations that Walter Whitehead decided that he wanted to practice medicine. He had already provided emergency dental extractions for his brothers and sisters in the past, and was eager to operate from an early age. On the day he decided to enrol in medical school, he did not even inform his parents. He borrowed some money from his cousin instead to pay his way. After qualifying from medicine and finishing his residence in 1864, he bought a general practice in Nottinghamshire, but surgery was his real passion and he became a fellow of the Royal College of Surgeons of Edinburgh in 1866. He was honorary surgeon at St Mary’s Hospital, Manchester from 1867 to 1873 when he joined the Manchester Royal
14. Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D, 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. 15. 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. 16. Ross GL, Shoaib T. Role of sentinel node biopsy in the management and staging of the N0 neck. Odontology 2005;93(1):1–6.
Infirmary, first as assistant honorary surgeon, and he became consulting surgeon in 1879. In 1894, he became professor of clinical surgery at the Victoria University, Manchester. Walter Whitehead was acknowledged as one of the most distinguished surgeons of his day. His operating style was certainly bold, and he was known to have said that “the greatest drawback under which a surgeon can suffer is knowledge of anatomy – it makes him timid”. Like many surgeons of his time, Walter Whitehead did not practice antiseptic surgery. He did however practice clean surgical techniques with use of soap and water. Once when the apron of his assistant touched the leg he was operating on, he “damned and blasted him to hell”!7 He is most remembered for two operations that he devised, which for many years were associated with his name. The first, “The surgical treatment of haemorrhoids”, he described in the British Medical Journal in 1882. The second was the remarkable series mentioned above, “A hundred cases of entire excision of the tongue”, published in the British Medical Journal in 1891.1 This latter operation was said to have been invented one morning when he arrived at the hospital with a hangover. He looked with despair and disfavour at the selection of instruments that the nurse had put in front of him and said “for God’s sake give me a pair of scissors” The operation he described used scissors only to excise carcinoma from the mobile tongue and of the 100 cases he presented in the paper, 20 patients died postoperatively. Walter Whitehead retired from practice in 1903 and went to live at Colwyn Bay, where he spent the rest of his days yachting, gardening, and entertaining friends. He died in 1913 aged 73. He was survived by his second wife and daughter. Although his surgical techniques are now largely obsolete, his varnish is still going (and smelling) strong.
References 1. Whitehead W. A hundred cases of entire excision of the tongue. BMJ 1891:961–4. 2. Stanley D, Emerson DJ, Daley JC. Whitehead’s varnish and Jelonet – a better dressing for skin graft donor sites than Jelonet alone. Ann R Coll Surg Engl 1988;70:369–71. 3. Iramaneerat S, Cunningham SJ, Horrocks EN. The effect of two alternative methods of canine exposure upon subsequent duration of orthodontic treatment. Int J Paediatr Dent 1998;8:123–9. 4. Lee SMG, Cooper JC. Noonan syndrome with giant cell lesions. Int J Paediatr Dent 2005;15:140–5. 5. Lim M, Lew-Gor S, Sandhu G, Howard D, Lund VJ. Whitehead’s varnish nasal pack. J Laryng Otol 2007;121:592–4. 6. Banks P, Brown A. Fractures of the facial skeleton. Edinburgh: Wright; 2001. p. 134. 7. Brockbank W. Honorary staff of the Manchester Royal Infirmary 1830–1948. Manchester: Manchester University Press; 1965. Roshi Frafjord Luke Cascarini Andrew E. Brown
British Journal of Oral and Maxillofacial Surgery 45 (2007) 617–622
Quantitative methylation analysis of resection margins and lymph nodes in oral squamous cell carcinoma Richard J. Shaw a,b,∗ , Gillian L. Hall a,c , Julia A. Woolgar c , Derek Lowe b , Simon N. Rogers b , John K. Field a , Triantafillos Liloglou d , Janet M. Risk a,1 a
Molecular Genetics & Oncology Group, School of Dental Sciences, University of Liverpool, Liverpool L69 3GN, UK Regional Maxillofacial Unit, University Hospital Aintree, Longmoor Lane, Liverpool L9 7AL, UK c Department of Oral Pathology, University of Liverpool, UK d University of Liverpool Cancer Research Centre, Roy Castle Lung Cancer Research Programme, 200 London Rd, Liverpool L3 9TA, UK b
Accepted 15 April 2007 Available online 7 June 2007
Abstract Resection is the preferred treatment for oral squamous cell carcinoma, and pathological staging of the resected specimen is crucial. The role of molecular biology in the diagnosis of minimal residual disease has not been fully investigated and may improve staging. Multiple adjacent specimens were taken from the tumour, the invasive front, the surgical margin, and the lymph nodes of 20 specimens from patients with oral cancer. Bisulphite-treated DNA from these specimens was assayed quantitatively with pyrosequencing methylation assays (PMA) of CpG islands within the gene promoters of the p16 and CYGB genes. Results were recorded with histopathological results, and compared with clinical outcome. Biological and technical replicates confirmed the reliability of the techniques. PMA upgraded 13 of the 20 surgical margins, 6 of which subsequently had a recurrent tumour. Not all of these recurrences were predicted and the effects of adjuvant treatment make firm conclusions difficult. © 2007 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Methylation; Oral squamous cell carcinoma; Surgical margin; Pathology; Pyrosequencing
Introduction Oral squamous cell carcinoma is the commonest form of head and neck cancer in the UK1 and is also common in other parts of the world.2 Primary resection is the commonest treatment of oral squamous cell carcinoma (SCC). Postoperative radiotherapy (PORT) is indicated in about half of such resections, decided by factors such as cervical lymph node status,3 pattern of tumour invasion4,5 and the status of the
∗ Corresponding author at: University Hospital Aintree, Department of Oral and Maxillofacial Surgery, Lower Lane, Liverpool L9 7AL, UK. Tel.: +44 151 529 5290. E-mail addresses: [email protected] (R.J. Shaw), [email protected] (J.M. Risk). 1 Tel.: +44 151 706 5265; fax: +44 151 706 5809.
surgical margin. However, the exact histopathological criteria for prescription of PORT remain controversial,6 and so it is logical to investigate the potential role of molecular biomarkers. The issues about surgical margins are particularly relevant to oral resections. The decision to increase the “safety margin” around a tumour might result in a more favourable histological report and a reduction in the need for adjuvant treatment, but could damage oral functions such as speech and swallowing. The role that epigenetic aberrations take in cancer is being increasingly realised with promoter hypermethylation being the best understood.7–9 Molecular resection margin analysis using methylation assays have been described in single reports in hepatocellular carcinoma,10 non-small cell lung cancer11 as well as head and neck cancer.12 Quantitative pyrosequencing methylation assays (PMAs) have been
0266-4356/$ – see front matter © 2007 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2007.04.015
618
R.J. Shaw et al. / British Journal of Oral and Maxillofacial Surgery 45 (2007) 617–622
described13,14 including those for the p16 and Cytoglobin (CYGB) gene promoters. Pyrosequencing methylation analysis has several crucial advantages in methylation analysis, but does not offer particularly high sensitivity (1:20 to 1:50) and may be open to question in the identification of the small proportion of methylated alleles expected in samples from surgical margins. Whether a quantitative assay such as PMA is sensitive enough to be of clinical value in this context has not been previously investigated. In this study, we apply quantitative pyrosequencing assays to the diagnosis of minimal residual disease using two gene promoters. This focuses on surgical margins as well as evaluation of lymph nodes in a cohort of 20 patients whose pTNM, full treatment details, and two-year outcome data were known.
Methods Patients and tissue Twenty patients newly diagnosed with biopsy-confirmed oral squamous cell carcinoma that had not been treated previously were selected because promoter methylation of either the p16 or CYGB promoter was present, and they were part of a larger series, the methylation status of which has been reported previously. Clinical staging and operative details were recorded. Fresh tumour samples of 5 mm3 were excised at operation from resected specimens within the tumour mass that did not involve the margin. These were coded, snap-frozen in liquid nitrogen and stored at −85 ◦ C. Sections with haematoxylin and eosin stained from each resected tumour were reviewed. One block from each tumour was selected that had histological features that represented the tumour as a whole, and also contained a strip of tissue from surface to deep margin. The strip to be removed was marked out on the slide and used as a template to guide macroscopic dissection of a strip of tissue from the block 5 mm wide. This was then divided into 4–6 equally sized cubes (Fig. 1). Care was taken to avoid cross-contamination within and between tissue blocks by using fresh scalpel blades on each occasion. The boundaries of the blocks were measured and marked on to the original slide. The slide was re-examined to allow esti-
mation of the proportion (to the nearest 10%) of tumour cells in relation to stroma or infiltrate of inflammatory host cells in the more superficially dissected blocks containing tumour, and identification of those that were histologically free of carcinoma. Areas of lymph nodes that were free of tumour and those containing solid deposits of metastatic tumour were obtained in a similar way. Preparation of samples DNA was extracted from the 20 fresh, snap frozen tissue samples using a DNeasyTM tissue kit (Qiagen Ltd.) and treated with bisulphite using the EZ DNA Methylation KitTM (Zymo Research) as previously described.14 The preparation of fixed tissues differed in that the DNA extraction was preceded by two xylene paraffin extractions and two 100% ethanol washes before lysis. In contrast to preparation of fresh tissue, double the recommended concentration of proteinase K, and 24 h of lysis were required for the fixed tissues. Fixed tissue samples were obtained from the 20 paraffin blocks: 60 from tumour, 52 from (histologically) tumour-free surgical margins, and 16 from lymph nodes. For fixed tissue, DNA was prepared using 2 mm3 samples from within the 5 mm3 specimens. Additional 2 mm3 samples were taken from 12 of these specimens to act as representative biological replicates. In total, 140 fixed specimens were prepared for molecular assay. Quantitative pyrosequencing methylation analysis (PMA) Hot Start (inhibition of polymerase activity during preparation of reaction) polymerase chain reaction (PCR) was done on all 20 fresh and 140 fixed tumour samples (including the 12 biological replicates) as described previously.12 Confirmation of the quality of the PCR and freedom from contamination was established on 2% agarose gels stained with ethidium bromide. Pyrosequencing was done using the PSQ96MA System (Biotage), and a mean methylation index was calculated as described previously.14 Statistical analysis We used the Statistical Package for the Social Sciences (SPSS, v 11, Chicago) to help with statistical analysis, including the intraclass correlation coefficient (ICC), which has a range of 0–1, 1 being complete agreement.
Results Clinical data
Fig. 1. Indication of where samples were removed from resection. T: specimen for snap freezing in liquid nitrogen, T1 → T4: 4× tumour, and M1 → M2: 2× margin specimens removed from formalin fixed tissue block.
Of the 20 tumours resected, 15 were staged pT2, 1 pT3, and 4 pT4. Eight contained metastases to the cervical lymph nodes; 6 staged pN2b and 2 pN2c. Seven of the patients were given PORT and eight had developed either local or regional recur-
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rence at the time of their latest review (minimum 26 months, range 26–37). When the deep margins were re-examined, all the resections had at least 5 mm (range 5–20 mm) of resection margins free of tumour on histopathological examination, and none of them had frank cut-through of tumour at the mucosal or other deep margin. Pyrosequencing methylation analysis of fixed compared with snap-frozen tissue Methylation of the p16 promoter in fixed tumour tissue correlated with that of the fresh frozen samples of tumour in 18 of the 20 cases. The remaining two cases had different results. However, it is clear from Fig. 1 that the tumour tissue samples taken for this comparison were geographically separate so it remains possible that molecular heterogeneity within the tumour might explain the discrepancy. The level of agreement between fresh and fixed specimens was assessed, and using the ICC reliability module (two way mixed effects), the ICC for p16 was 0.75 (95% confidence interval (CI) 0.48 to 0.89). Methylation of the CYGB promoter was similar in 18 of the 20 cases using the two tissue sources and the ICC was 0.81 (95% CI 0.58 to 0.92). The two discordant cases in this instance differed from the p16 discordant cases. Biological and technical replicates Additional DNA samples were prepared from 12 of the fixed tissue blocks for use as biological replicates. Samples were chosen for this analysis to give the mean methylation index from 0 to >25 for each gene. These were subject to p16 and CYGB PMA, which was compared with the original data. The ICC for p16 was 0.97 (95% CI 0.91 to 0.99) and for CYGB was 0.71 (95% CI 0.27 to 0.91). Some of this variation may, again, potentially be the result of heterogeneity of the tumours. Technical replicates were also done on these 12 samples by repeating the PCR and pyrosequencing steps on existing DNA templates treated with bisulphite. These data gave an ICC for p16 of 0.87 (95% CI 0.60 to 0.96) and for CYGB of 0.89 (95% CI 0.68 to 0.97). These results reassured us about the reliability and repeatability of the assays. We did a kappa analysis with the 12 additional samples mentioned to test the ability of the assay to discriminate between those with greater than 5% methylation and those with less than 5%. The kappa values of agreement for technical replicates were 1.00 for p16, 0.83 for CYGB and the biological replicates were 1.00 for p16 and −0.13 for CYGB. Kappa values of 0.60 are good, above 0.80 is very good, whereas 0 or less than 0 is poor. The confidence intervals were wide, which reflects the small sample size. Methylation at surgical margins p16 promoter methylation Eleven of the 20 snap-frozen specimens of tumour showed p16 promoter methylation by PMA (range 1–45%), while
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11 of the 20 fixed tumour tissue specimens that contained ≥10% tumour cells showed p16 promoter methylation ≥5% in at least one of the serial tumour samples (range 5–53%). A mean methylation index of 1% is considered to be a valid result for snap-frozen tissue based on our previous analysis of variance,15 while 5% is used as the cut-off for fixed tissue. Evidence of p16 promoter methylation within specimens of surgical margins (all 0 tumour on histopathological examination) was found in 11/20 cases using PMA (range 2–11%), however, only 4 of these give methylation indexes of ≥5%, two of which showed local recurrence despite having less aggressive tumours with no pathological lymph nodes involved. Three of the remaining seven with methylation indexes ≥5% were present in cases in which no p16 methylation had been seen in the tumour on analysis of either fixed or fresh specimens, and may indicate false positive results, or perhaps some heterogeneity within the tumour. However, one of these subsequently developed a local recurrence. Of the eight cases with paired positive and negative lymph nodes available for analysis, three had p16 promoter methylation in their primary tumour. All three cases had PMA results ≥5% in invaded lymph nodes but no significant methylation in the clear lymph node. Two of the three patients had locoregional recurrence. None of the remaining five showed p16 promoter methylation in either the invaded or clear lymph nodes. Two of the five with recurrent neck disease had borderline evidence of p16 promoter methylation in a histologically clear lymph node (3% and 2%, respectively), while none of the three disease-free nodes gave similar results. CYGB promoter methylation Eighteen of the 20 snap-frozen tumour tissues showed CYGB methylation using PMA (range 4–54%), while all 20 fixed specimens of tumour tissue containing ≥10% tumour cells showed CYGB promoter methylation of ≥5% in at least one of the serial tumour samples (range 5–79%). Evidence of tumour within surgical margins was found in 17/20 cases using PMA (range 2–32%), 14 of which gave methylation indexes of ≥5%, and five of whom developed local recurrences. In two of the three apparently less aggressive tumours (T2N0) that recurred, there was consistent evidence of CYGB promoter methylation of ≥5% by PMA in all of the specimens of margins tested however, this was also seen in three other ‘less aggressive’ tumours which did not recur, although one of these patients was given postoperative radiotherapy. Seven of eight cases with invaded lymph nodes on histopathological examination had evidence of CYGB promoter methylation in nodes shown to be invaded by PMA. Two of eight cases had evidence of methylation in nodes histologically by PMA. Four of five patients with nodal involvement and neck recurrence were in this group.
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Figs. 2–4. Co-registration of pathology with methylation data. The upper row shows photomicrographs spanning the tumour, invasive front and surgical margin. The second row indicates the sample codes as in Fig. 1. The third row shows the percentage of tumour cells as estimated by pathologist. The fourth and fifth rows indicate corresponding methylation indices for each sample for genes p16 and cygb. (2) A favourable tumour with neither pathological nor molecular evidence of disease beyond the invasive front. (3 and 4) Molecular evidence beyond the tumour margin not apparent on histopathology and both suffered local or regional recurrence.
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Summary Using the 5% methylation index as a threshold, in 13 of the 20 patients the surgical margins were upgraded; subsequently 6 of these 13 had either a local or regional recurrence. A further two patients had recurrences that did not show positive molecular margins within our analysis of the tissue studied. Illustrations of results for three of these patients are shown in Fig. 2–4.
Discussion This exploratory study brings together clinical, histopathological and molecular data in resection of oral squamous carcinoma to clarify their role. The conclusions are limited by the number of patients and also by the use of fixed tissue, so we are cautious about making any recommendations of the clinical value of these assays. However, the data show that the assays can identify molecular evidence in “clear” tissues using routine histopathological techniques, and in some patients who ultimately develop recurrences. This was apparent in the assays used for histologically clear lymph nodes and also for tissues at the edge of resection margins. We feel these assays offer sufficient potential to merit further investigation, optimisation and clinical validation in larger trials. It may add greatly to the sensitivity in any particular patient to process several samples from each resection margin. Our study has presented data from 2 mm2 , only a small fraction of the surface area of any margin. Radiological or histopathological assessments in clinical practice would not routinely take such a limited view in the staging of a tumour. It would be feasible to take 6–10 small samples beyond the resection margin for molecular analysis, both in the deep and mucosal margins. Some epigenetic changes may be present in premalignant oral epithelium and these might confound the findings at mucosal margins, but our previous data with p16 and CYGB are reassuring about this.14 The selection of lymph nodes for molecular analysis might be improved by adopting techniques used for identification of sentinel nodes.16 Contamination of samples, either in the operating theatre or in the laboratory, may be important if more sensitive assays are used (such as methylation-specific PCR). The principles behind the selection of methylation as a biomarker have been well-defined. Promoter methylation is common, tumour-specific, and there are a number of methods for detection, some of which are highly sensitive. Which gene or combination of genes, might be best is less clear. We have selected cases that had known promoter methylation at either p16 or CYGB, a finding true in 80 of 92 (87%) specimens of oral malignant tumours that we examined. The addition of other genes to a panel might bring the percentage of “informative” cases closer to 100%. It remains possible that low level CYGB methylation exists even in normal oral tissues, which would explain some of the apparent false positives. Extensive investigations of any likely biomarker gene in var-
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ious normal tissues are necessary. The search for other genes that are commonly silenced by promoter methylation continues, using techniques such as pharmacological unmasking and promoter tiling arrays. These assays may require further optimisation, although fresh clinical samples rather than fixed tissue are preferable. Assays that perform well in laboratory dilutions of high quality DNA may not be as reliable in clinical samples in which various contaminants, necrosis of the tumour, and degradation of DNA, can have an impact. Any test used clinically may need to be done in duplicate or even triplicate to eliminate mispriming artefacts or other causes of false positive results. Other methylation assays such as nested MSP and real-time MSP may also be considered for comparison. We have recently described methylation enrichment pyrosequencing15 a new assay that combines the potentially high sensitivity of methylation-specific PCR (MSP) with a second, confirmatory pyrosequencing step. This second step is rapid, inexpensive, and offers advantages over previously described sequencing or restriction enzyme methods. We have reported the potential benefits of methylation enrichment pyrosequencing in clinical samples from the head and neck,15 and speculate this might be a promising technique for future studies of surgical margins.
Acknowledgements RJS is in receipt of a Royal College of Surgeons of England Research Fellowship; research was funded by the British Association of Oral and Maxillofacial Surgeons; TL is funded by the Roy Castle Foundation.
References 1. Office of National Statistics, Welsh Cancer Intelligence Surveillance Unit, 2000; 2004. 2. Shah JP BJJJ. Oral Cancer. Martin Dunitz, Taylor & Francis; 1999. 3. Woolgar JA, Rogers SN, Lowe D, Brown JS, Vaughan ED. Cervical lymph node metastasis in oral cancer: the importance of even microscopic extracapsular spread. Oral Oncol 2003;39(2):130–7. 4. Shaw RJ, Brown JS, Woolgar JA, Lowe D, Rogers SN, Vaughan ED. The influence of the pattern of mandibular invasion on recurrence and survival in oral squamous cell carcinoma. Head Neck 2004 Oct;26(10):861. 5. Soo KC, Carter RL, O’Brien CJ, Barr L, Bliss JM, Shaw HJ. Prognostic implications of perineural spread in squamous carcinomas of the head and neck. Laryngoscope 1986;96(10):1145–8. 6. Blackburn TK, Bakhtawar S, Brown JS, Lowe D, Vaughan ED, Rogers SN. A questionnaire survey of current UK practice for adjuvant radiotherapy following surgery for oral and oropharyngeal squamous cell carcinoma. Oral Oncol 2007;43(2):143–9. 7. Esteller M, Fraga MF, Paz MF, Campo E, Colomer D, Novo FJ, et al. Cancer epigenetics and methylation. Science 2002;297(5588):1807–8. 8. Momparler RL. Cancer epigenetics. Oncogene 2003;22(42):6479–83. 9. Shaw R. The epigenetics of oral cancer. Int J Oral Maxillofac Surg 2006;35(2):101–8. 10. 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.
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11. Guo M, House MG, Hooker C, Han Y, Heath E, Gabrielson E, et al. Promoter hypermethylation of resected bronchial margins: a field defect of changes? Clin Cancer Res 2004;10(15):5131–6. 12. Goldenberg D, Harden S, Masayesva BG, Ha P, Benoit N, Westra WH, et al. Intraoperative molecular margin analysis in head and neck cancer. Arch Otolaryngol Head Neck Surg 2004;130(1):39–44. 13. Colella S, Shen L, Baggerly KA, Issa JP, Krahe R. Sensitive and quantitative universal pyrosequencing methylation analysis of CpG sites. Biotechniques 2003;35(1):146–50.
INTERESTING CASE: Walter Whitehead: A brief history of the man and his varnish Whitehead’s varnish is popular in oral and maxillofacial surgical practice, where it has a long history of use as an intraoral or buried dressing pack. Despite its longstanding and common use few surgeons will know of its inventor and how it was originally applied. Whitehead’s first recorded description of his varnish is in a paper in the British Medical Journal in 1891 entitled “A hundred cases of entire excision of the tongue”.1 In this paper, Walter Whitehead states that he first started using the dressing 10 years previously. A varnish is a transparent, hard, protective finish or film primarily used in finishing wood but also other materials. Varnishes are traditionally a combination of a drying oil, a resin, and a thinner or solvent. The varnish that Whitehead made was composed of iodoform, (a compound tincture of benzoin Friar’s balsam which is commonly used as an antiseptic, or for inhalation with steam as an expectorant) and turpentine. He wrote that the preparation had both antiseptic and anaesthetic properties, the turpentine component having a “very marked influence in promptly checking capillary oozing”. In addition to these properties, he went on to say that “it lessened the discomfort which follows surgery and also enables the patient to take food in the ordinary manner”. Today Whitehead’s varnish (pigmentum iodoform compound: BPC) consists of: iodoform10 g, benzoin10 g, prepared storax (a resinous exudate of the Sweetgum tree) 7.5 g, Balsam of Tolu 5 g (a resin obtained from South American balsam trees) and solvent ether mixed to 100 ml. Although turpentine is no longer used as the volatile liquid, Whitehead’s varnish is still advocated for many uses including dressing of skin graft donor sites,2 packing after exposure of unerupted canine teeth,3 jaw cavities,4 and nasal packing.5 Whitehead’s varnish has a benefit over a number of other dressings in that it remains ‘clean’ and uninfected in potentially contaminated environments. It contains a number of aromatic resins, which are slowly broken down to produce benzoic acid. It is this slow release of a potent antiseptic, together with the waterproofing property of the compound, which many surgeons suggest makes it superior to other media.6 Walter Whitehead was born in 1840 at Halsam Hey, Bury, where his family had lived for over 200 years. At the age of 16, he entered his father’s business and began to train as a bleacher, dyer, and finisher. This is probably when he learned about chemicals and is perhaps what led him to develop his varnish. One day, aged 19, and still an ordinary workman, he was invited by a medical student to observe some operations at the local infirmary. It was while watching these operations that Walter Whitehead decided that he wanted to practice medicine. He had already provided emergency dental extractions for his brothers and sisters in the past, and was eager to operate from an early age. On the day he decided to enrol in medical school, he did not even inform his parents. He borrowed some money from his cousin instead to pay his way. After qualifying from medicine and finishing his residence in 1864, he bought a general practice in Nottinghamshire, but surgery was his real passion and he became a fellow of the Royal College of Surgeons of Edinburgh in 1866. He was honorary surgeon at St Mary’s Hospital, Manchester from 1867 to 1873 when he joined the Manchester Royal
14. Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D, 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. 15. 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. 16. Ross GL, Shoaib T. Role of sentinel node biopsy in the management and staging of the N0 neck. Odontology 2005;93(1):1–6.
Infirmary, first as assistant honorary surgeon, and he became consulting surgeon in 1879. In 1894, he became professor of clinical surgery at the Victoria University, Manchester. Walter Whitehead was acknowledged as one of the most distinguished surgeons of his day. His operating style was certainly bold, and he was known to have said that “the greatest drawback under which a surgeon can suffer is knowledge of anatomy – it makes him timid”. Like many surgeons of his time, Walter Whitehead did not practice antiseptic surgery. He did however practice clean surgical techniques with use of soap and water. Once when the apron of his assistant touched the leg he was operating on, he “damned and blasted him to hell”!7 He is most remembered for two operations that he devised, which for many years were associated with his name. The first, “The surgical treatment of haemorrhoids”, he described in the British Medical Journal in 1882. The second was the remarkable series mentioned above, “A hundred cases of entire excision of the tongue”, published in the British Medical Journal in 1891.1 This latter operation was said to have been invented one morning when he arrived at the hospital with a hangover. He looked with despair and disfavour at the selection of instruments that the nurse had put in front of him and said “for God’s sake give me a pair of scissors” The operation he described used scissors only to excise carcinoma from the mobile tongue and of the 100 cases he presented in the paper, 20 patients died postoperatively. Walter Whitehead retired from practice in 1903 and went to live at Colwyn Bay, where he spent the rest of his days yachting, gardening, and entertaining friends. He died in 1913 aged 73. He was survived by his second wife and daughter. Although his surgical techniques are now largely obsolete, his varnish is still going (and smelling) strong.
References 1. Whitehead W. A hundred cases of entire excision of the tongue. BMJ 1891:961–4. 2. Stanley D, Emerson DJ, Daley JC. Whitehead’s varnish and Jelonet – a better dressing for skin graft donor sites than Jelonet alone. Ann R Coll Surg Engl 1988;70:369–71. 3. Iramaneerat S, Cunningham SJ, Horrocks EN. The effect of two alternative methods of canine exposure upon subsequent duration of orthodontic treatment. Int J Paediatr Dent 1998;8:123–9. 4. Lee SMG, Cooper JC. Noonan syndrome with giant cell lesions. Int J Paediatr Dent 2005;15:140–5. 5. Lim M, Lew-Gor S, Sandhu G, Howard D, Lund VJ. Whitehead’s varnish nasal pack. J Laryng Otol 2007;121:592–4. 6. Banks P, Brown A. Fractures of the facial skeleton. Edinburgh: Wright; 2001. p. 134. 7. Brockbank W. Honorary staff of the Manchester Royal Infirmary 1830–1948. Manchester: Manchester University Press; 1965. Roshi Frafjord Luke Cascarini Andrew E. Brown