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Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours

Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a... Background: Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Results: Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Conclusions: Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard. Keywords: MGMT, Methylation, Glioblastoma, Glioma, Immunohistochemistry, Double-labelling, Cocktail, MLPA, Biomarker * Correspondence: leslie.bridges@stgeorges.nhs.uk Cellular Pathology, St George’s Hospital, Blackshaw Road, London SW17 0QT, UK Full list of author information is available at the end of the article © 2013 Burke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 2 of 10 http://www.actaneurocomms.org/content/1/1/22 Background same turnaround time as standard immunohistochemis- Clinical trials have shown that determination of the try and at little extra cost. methylation status of glioblastomas is an important pre- We have used such a platform to develop a novel dictor of outcome and response to treatment [1–4]. A technique, namely double-labelling immunostaining of range of molecular genetic techniques are available for de- MGMT and a "cocktail" of non-tumour antigens (CD34, termining methylation status in tumours [5]. However, CD45 and CD68). Our aim was to produce a visual separ- these are generally expensive and somewhat slow in ation of tumour cells and non-tumour cell elements on application. It would certainly be of great help if immuno- the same histological slide and thereby greatly simplify the staining were available to infer the methylation status of assessment of MGMT immunostaining in tumour cell the tumour. In fact, immunostaining for MGMT, on the nuclei. face of it, should provide an answer. Antibodies against MGMT are readily available which stain tumour cell nu- Results clei positive in unmethylated tumours and negative in A comparison of the results for double-labelling versus methylated tumours (this opposite relationship between single-labelling and MLPA (the gold standard) is shown MGMT staining and methylation status is due to the fact in Table 2. Some representative examples of single- that methylation of the MGMT gene switches off produc- labelling and double-labelling are shown in Figure 1. tion of the MGMT protein) [6]. However, there is a major problem in interpretation of Agreement in assessment of the double-labelling MGMT immunostaining in brain tumours due to the fact There was good inter-observer and intra-observer agree- that normal non-tumourous elements within the tumour ment in the assessment of the double-labelling. For the (including endothelial cells, lymphocytes and macro- three independent "blind" observers (EB, LB and KE) all phages) normally express MGMT [6–8]. This makes it appraisers’ assessments agreed with each other for 31/40 difficult to determine whether any MGMT positivity (77.50%) of the cases. The Fleiss’ kappa statistic was 0.76 within the tumour is due to tumour cells or non-tumour and Kendall’s coefficient of concordance for ordinal cells. Not surprisingly, the literature therefore contains scores was 0.93 indicating a good level of agreement. much controversy about the value or otherwise of MGMT The intra-observer agreement was 36/40 (90%) of cases immunostaining as a clinical biomarker in brain tumours matched with a kappa of 0.84 and a Kendall of 0.97. (please see Table 1). For example, Preusser et al. [6] state that observer Sensitivity, specificity, positive predictive value and variability and lack of association with patient survival negative predictive value of double-labelling impedes the use of MGMT immunostaining in glioblast- The results of the neuropathologist (LB) were compared omas. On the other hand, Watanabe et al. [9] (on the with the gold standard, MLPA. For this, cases assessed basis of eight years clinical experience of MGMT immu- as largely MGMT-negative were considered methylated nostaining, using serial sections stained for non-tumour (i.e. test result positive) and cases assessed as largely markers such as CD45 and CD68 and a careful algorith- MGMT-positive were considered unmethylated (i.e. test mic approach) consider that MGMT immunohisto- result negative). For MLPA we use the cut-off used by chemistry correlates with outcomes in patients with Jeuken et al. [10]. According to this, all cases with an glioblastomas. MLPA ratio greater than 0.25 are at least mildly methyl- Although the use of serial sections stained with non- ated (i.e. true positives) and cases with an MLPA ratio of tumour markers for comparison with sections stained for between 0 and 0.25 are unmethylated (i.e. true negatives). MGMT is undoubtedly helpful, it is not always easy to Of the 40 cases, there were 11 negative matches, 20 compare cellular identities between serial sections. To positive matches, 4 false-positives and 5 false-negatives. some extent it is possible to identify tumour cells in the The test results showed significant association (chi-square, MGMT-stained section directly (for example, when p = 0.001) between the double-labelling and MLPA tumour cell nuclei are very large and bizarre). However, results. Sensitivity was 80%, specificity 73.33%, positive reactive elements within tumours may themselves develop predictive value (PPV) 83.33% and negative predictive a degree of nuclear enlargement and irregularity which value (NPV) 68.75%. For the 30 glioblastomas apart, sensi- makes distinction from tumour cells impossible at times. tivity was 66.7%, specificity 73.33%, PPV 71.43% and NPV Double-labelling immunohistochemistry is an estab- 68.75%. All 10 of the oligodendroglial tumours were cor- lished technique which is now available on some auto- rectly called. These values indicate a good all-round per- mated immunohistochemistry platforms such as the formance by the double-labelling. Leica platform in use in our laboratory. This means that The other assessors’ results were also compared with double-labelling protocols can readily be incorporated MLPA. The results for specificity, sensitivity, PPV and into the routine work of the laboratory, with much the NPV were 88%, 46.67%, 73.33% and 70% for KE and 88%, Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 3 of 10 http://www.actaneurocomms.org/content/1/1/22 Table 1 Previous studies Author and year Tumour type Cut off value Low MGMT protein MGMT IHC compared Molecular test Correlation between for IHC expression predictive of: to molecular gold IHC and molecular standard test Progression free Overall survival (PFS) survival (OS) Nakasu et al. 2004 [7] 69 high-grade gliomas 10% - Yes No - - (grades III and IV) Brell et al. 2005 [11] 93 anaplastic gliomas: 75 AAs and 18 5% No Yes Yes MS-PCR No with oligodendroglial component Chinot et al. 2007 [21] 28 GBMs 35% Yes Yes No - - Capper et al. 2008 [22] 75 primary GBMs 15% Yes – median survival No - - Preusser et al. 2008 [6] 164 GBMs 10% - No Yes MS-PCR Poor agreement Rodriguez et al. 2008 [23] 50 GBMs 10% No No Yes MS-PCR No Karayan-Tapon et al. 2010 [24] 81 GBMs 15% No No Yes MS-PCR - SQ-PCR No Pyrosequencing Yes Q-RT-PCR Yes Quillien et al.2012[5] 100 GBMs 23% Yes Yes No - - The table shows previous studies where MGMT IHC has been performed. The cut-off value was the percentage of MGMT-positive tumour cell nuclei (assessed by eye). The correlation of IHC with progression-free survival (PFS) and overall survival (OS) and comparison with a molecular gold standard (where applied) are also shown. Abbreviations: AA anaplastic astrocytoma, GBM glioblastoma multiforme, MS-PCR methylation-specific polymerase chain reaction, Q-RT-PCR quantitative real-time polymerase chain reaction, SQ-PCR semi-quantitative polymerase chain reaction. Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 4 of 10 http://www.actaneurocomms.org/content/1/1/22 Table 2 Cases case ID type LI SL MLPA MLPA 0-3 MLPA >0.25 DL 1 oligo 7.567 1 0.6275 2 1 1 2 oligo 13.700 1 0.675 2 1 1 3 oligo 12.600 1 0.8075 3 1 1 4 oligo 24.667 1 0.4725 1 1 1 5 oligo 17.100 1 0.8525 3 1 1 6 oligo 13.600 1 0.8025 3 1 1 7 oligo 19.567 1 0.6425 2 1 1 8 oligo 14.267 1 0.6925 2 1 1 9 oligo 8.800 1 0.6525 2 1 1 10 oligo 26.133 1 0.5925 2 1 *1 11 gbm 56.367 0 0.1125 0 0 0 12 gbm 19.167 1 0.8475 3 1 1 13 gbm 84.400 0 0.09 0 0 0 14 gbm 17.967 1 0.2625 1 1 *1 15 gbm 57.700 0 0.255 1 1 0 16 gbm 89.900 0 0.4075 1 1 0 17 gbm 72.267 0 0.2075 0 0 0 18 gbm 19.533 1 0.3525 1 1 *1 19 gbm 54.133 0 0.42 1 1 *1 20 gbm 53.900 0 0.1375 0 0 0 21 gbm 21.967 1 0.485 1 1 1 22 gbm 54.167 0 0.5475 2 1 1 23 gbm 33.000 1 0.085 0 0 0 24 gbm 76.300 0 0.2725 1 1 0 25 gbm 96.733 0 0.24 0 0 0 26 gbm 49.567 1 0.4275 1 1 1 27 gbm 43.967 1 0.1375 0 0 *1 28 gbm 40.533 1 0.1025 0 0 0 29 gbm 7.233 1 0.1275 0 0 1 30 gbm 39.067 1 0.5125 2 1 1 31 gbm 25.833 1 0.2075 0 0 *1 32 gbm 33.900 1 0.13 0 0 *1 33 gbm 28.000 1 0.365 1 1 0 34 gbm 45.000 1 0.275 1 1 1 35 gbm 84.100 0 0.205 0 0 0 36 gbm 85.100 0 0.2975 1 1 0 37 gbm 64.933 0 0.11 0 0 0 38 gbm 53.400 0 0.17 0 0 0 39 gbm 6.800 1 0.825 3 1 1 40 gbm 22.500 1 0.2425 0 0 0 Case ID (identification) is the case number we assigned in this study. Tumour types were oligodendroglial tumours (oligo) and glioblastomas (gbm). The oligodendroglial tumours were oligodendroglioma WHO grade II (cases 4, 5 and 6), anaplastic oligodendroglioma WHO grade III (cases 1, 2, 3, 7 and 9), oligoastrocytoma WHO grade II (case 10) and anaplastic oligoastrocytoma WHO grade III (case 8). The glioblastomas were all glioblastoma WHO grade IV except cases 13 and 18 which were glioblastoma with oligodendroglioma component WHO grade IV. LI is the nuclear labelling index for single-labelled MGMT as determined by computerised image analysis. SL is the binarised score for the single-labelling (0 = LI > 50%, 1 = LI from 0 to 50%). MLPA is the MLPA ratio (please see text). MLPA 0–3 is the score for MLPA according to cut-offs for the MLPA ratio provided by Jeuken et al. 2007 [10] (0 = unmethylated, 1 = mildly methylated, 2 = moderately methylated and 3 = extensively methylated). MLPA > 0.25 is the binarised methylation score for MLPA (0 = unmethylated, 1 =methylated). DLis the binarised score for double labelling (0 = mainly MGMT-positive in tumour cells, 1 = mainly MGMT-negative in tumour cells). The asterisked cases were initially assigned a score of 1 in the tripartite system (please see text for explanation). Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 5 of 10 http://www.actaneurocomms.org/content/1/1/22 40%, 70.97% and 66.67% for EB. The results are similar to Figure 1 Photomicrographs comparing single-labelling and LB’s apart from sensitivity which scored less well for these double-labelling for MGMT. This figure shows the advantage of assessors. This may reflect the particular approach of the double-immunolabelling over single-immunolabelling in the interpretation of MGMT immunostaining. Four glioblastomas are assessors; the neuropathologist appears to have been more shown, one per row, with the single-labelling for MGMT on the left willing to ignore low levels of MGMT staining than the and double-labelling for MGMT and "cocktail" (combined CD34, two laboratory scientists. In this paper we have carried CD45 and CD68) on the right. All images were photographed at an forward the neuropathologist’s scores on the basis that the objective lens magnification of 80x (scale bar = 50 microns). For all pathologist carries out the everyday reporting in practice. images, nuclear immunostaining for MGMT is seen as brown due to the DAB-peroxidase product. For images on the right, cytoplasmic staining for CD34, CD45 and CD68 is seen as red due to the Fast False-negatives and false-positives Red-Alkaline Phosphatase product used in the double-labelling Of the 5 false-negatives it is interesting to note that all system. The counterstain is haematoxylin (nuclei unstained for were only mildly methylated on MLPA. The slides were MGMT appear blue). The case in row 1 (case 25 in Table 2) was reviewed and all 5 cases were confirmed as showing un- unmethylated by MLPA. Double-labelling (B) is more informative than single-labelling (A) because it highlights the cytoplasm of the doubted MGMT-positivity suggestive of an unmethylated non-tumourous elements (endothelial cells, lymphocytes and status. It is therefore of interest that although these cases macrophages) as red. The remaining cells can therefore be positively were false negatives, the result of MLPA was only mild identified as tumour cells and the presence of undoubted nuclear methylation – the closest category to unmethylated. Fur- immunostaining for MGMT in these tumour cells correctly indicates ther clinicopathological correlation may help develop a an unmethylated status. The case in row 2 (C and D; case 39 in Table 2) was extensively methylated by MLPA. The case was easily better understanding of the significance of the "mildly" assessed as MGMT-negative (methylated) on double-labelling (D). methylated category on MLPA. The case in row 3 (E and F; case 18 in Table 2) illustrates the issue of The slides of the 4 false-positives were also reviewed. "equivocal" staining as seen a number of cases (asterisked in One case was undoubtedly MGMT-negative suggestive of Table 2). Please see Results for further commentary on this phenomenon. The case in row 4 (case 22 in Table 2) illustrates a situation where single-labelling (G) shows a high labelling index for MGMT due to a high content of non-tumourous cells (endothelial cells, lymphocytes and macrophages). Double-labelling (H) provides an easy "one-look" diagnosis of MGMT-negative (methylated). a methylated status but MLPA was unmethylated. The other 3 cases showed equivocal MGMT-immunostaining and we had chosen by convention to call these MGMT- negative (please see below). Equivocal MGMT-immunostaining Table 2 shows 7/40 cases (asterisked) with equivocal (focal or faint) MGMT-immunostaining of tumour cell nuclei on double-labelling. For illustrations of equivocal staining please see Figure 1 (E and F). These cases were difficult to classify as MGMT-negative (methylated) or MGMT-positive (unmethylated). We therefore decided on a convention by which such cases were interpreted as MGMT-negative (suggestive of a methylated status). Of the 7 such cases, 4 cases (3 glioblastomas and 1 oligo- dendroglial tumour) turned out to be correctly thus- called (i.e. they were methylated by MLPA). Three cases (all glioblastomas) were incorrectly called (i.e. they were unmethylated). Since there is no clear indication at present from these figures as to how the equivocal cases should be assessed (roughly equal numbers were methylated and unme- thylated) we suggest - until more data is available - treating these cases as MGMT-negative (suggestive of a methylated status) as we have here. It may be worth not- ing in the pathology report that such cases - in particular - Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 6 of 10 http://www.actaneurocomms.org/content/1/1/22 are liable to a change in status when molecular testing is through. This approach would seem most consistent with the default clinical position whereby patients with glio- blastomas in which the methylation status is unknown are managed as if the tumour were methylated. We are gath- ering further data on the issue of equivocal staining which, as indicated, accounts for about a fifth of our assessments. Comparison with single-labelling As mentioned, Figure 1 shows how quantitative assess- ment of double-labelling is quick and easy compared to single-labelling. We wondered how the automated as- sessment of the labelling index for MGMT on single- labelling performed against the qualitative assessment on double-labelling. For this we set a convenient cut-off of 50% for the labelling index (close to the actual median of 47.28 for the 40 cases in our series). Cases with a la- Figure 2 Chart of MGMT single-labelling, MGMT double- labelling and MLPA status. The chart shows the labelling index for belling index of up to 50% were considered methylated MGMT from the single-labelled slides on the Y-axis for the 40 cases (i.e. a positive test result) and cases with a labelling plotted against the results of double-labelling and MLPA status. The index greater than 50% were considered unmethylated 4 cases that were false-positive by double-labelling are seen in the (i.e. a negative test result) and this was compared with second column from the left. All of these cases have a labelling MLPA using the 0.25 cut-off of Jeuken et al. [10] as pre- index of less than 50% by single-labelling suggestive of a methylated status. The results of double-labelling and single- viously detailed. Please note that our choice of a median labelling are therefore concordant and combined analysis would not cut-off for the single-labelled MGMT labelling index have avoided calling these cases (falsely) positive. The 5 cases that may at first sight appear to be a departure from previous were false-negative by double-labelling are seen in the third column studies (e.g. Preusser et al. [6]). Generally these other from the left. Of these, 4 have a labelling index of greater than 50% studies have used assessment "by eye” and mentally by single-labelling suggestive of an unmethylated status. The results of double-labelling and single-labelling of these 4 cases are subtracted tumour-only elements whereas our data is therefore concordant and combined analysis would not have truly quantitative and applied to both tumourous and avoided calling these (falsely) negative. Only one of the 5 false- non-tumourous elements since the machine cannot sep- negative cases has a labelling index of less than 50% by single- arate the two. We feel that the median cut-off is appro- labelling suggestive of a methylated status. In this case the labelling priate in our context. index might have been helpful in avoiding calling this (falsely) negative on double-labelling. Overall however the extra effort For all cases (n = 40) for the single-labelling MGMT la- involved in measuring the labelling index by automated quantitative belling index versus MLPA sensitivity was 76%, specificity image analysis on the single-labelled slides may not be worthwhile, 55.33%, PPV 73.08% and NPV 57.14%. For the glioblast- particularly in labs not set up for image analysis. omas (n = 30) sensitivity was 60%, specificity 53.33%, PPV 56.25% and NPV 57.14%. Compared to double-labelling these results for quantitative single-labelling are not as (for endothelium), CD45 (for lymphocytes) and CD68 (for good and there are also other disadvantages compared to macrophages). The end result was a histology slide in double-labelling (please see Discussion). which we could easily distinguish between tumourous and We then wondered whether the single-labelling and non-tumourous components and thereby quickly come to double-labelling could be combined in some way to give a conclusion as to whether the tumour cell nuclei were a better result and the analysis is shown in Figure 2. MGMT-positive or MGMT-negative (and thence to a conclusion as to their likely methylation status). Discussion We found good agreement between observers (kappa We have developed a novel protocol for the analysis of 0.76) and within observer (kappa 0.84). Furthermore, MGMT-immunostaining using double-labelling. The aim double-labelling showed good specificity (80%), sensitiv- of our approach is to simplify the assessment of MGMT- ity (73.33%), positive predictive value (83.33%) and immunostaining by creating a visual separation of the negative predictive value (68.75%) compared to MLPA tumourous and non-tumourous elements. We did this by (the gold-standard). using the usual brown chromogen (DAB-peroxidase) for Compared to qualitative assessment of single-labelling nuclear MGMT staining and a red chromogen (Fast Red- MGMT-immunostaining, we found double-labelling to Alkaline Phosphatase) for a “cocktail" of non-tumourous be much quicker and easier to assess (Figure 1). Further- cytoplasmic elements. The cocktail is composed of CD34 more, double-labelling outperformed even quantitative Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 7 of 10 http://www.actaneurocomms.org/content/1/1/22 computerised image analysis of MGMT single-labelling raised the question of whether or not a strict correlation in terms of sensitivity, specificity, PPV and NPV. Further- should really be expected between MGMT staining and more, we did not find that much benefit would accrue methylation status. For example, Kreth and colleagues from combining the results of double-labelling with [12] found that patients with discordant results for quantitative single-labelling. Since quantitation is some- MGMT expression (determined by mRNA expression) what time-consuming we would therefore not necessarily and methylation status did worse than concordant coun- recommend using both double-labelling with quantita- terparts. A molecular basis for MGMT expression inde- tive single-labelling on a routine basis: we consider pendent of methylation may apply in some situations [13]. double-labelling alone to be sufficient for everyday Our double-labelling technique offers a possible new ap- purposes. proach to investigate such questions. We are aware that the double-labelling as with any test is not perfect and that certain cautions are required in Conclusions interpretation. The cocktail of three reagents (CD34, We have developed a novel method for double-labelling CD45 and CD68) should stain a good proportion of for MGMT, which is quick and easy to apply and might non-tumourous elements but not necessarily all. For ex- readily be subsumed into routine reporting for glioblast- ample, there may also be non-neoplastic astrocytes and omas (and other tumours). There is good inter-observer oligodendrocytes which are not identified by the cock- and intra-observer agreement and, compared to a mo- tail. For this reason we would recommend that interpret- lecular genetic gold-standard, the method yields good ation of the double-labelling be focussed on areas of sensitivity, specificity, positive predictive value and nega- tumour proper and caution exercised in areas where tive predictive value as a predictor of methylation status. tumour is infiltrating largely normal tissues. We are also aware that CD34 occasionally stains glioblastoma Methods tumour cells and in such situations tumour cells will be The study was carried out in compliance with local re- stained red by the red chromagen and will thus be ex- search ethics committee (LREC) guidelines as a service cluded from analysis. However, we feel the CD34 stain- development. ing by glioblastomas cells is usually just focal so that it This was a retrospective study. Forty formalin-fixed should be possible in most cases to find interpretable paraffin-embedded (FFPE) samples were obtained from areas in the slide. patients diagnosed with gliomas (10 oligodendroglial tu- In our own current practice, we carry out double- mours and 30 glioblastomas) from the archives of the labelling for MGMT and "cocktail" (CD34, CD45 and Cellular Pathology department at St George’s Hospital, CD68) to come to an initial conclusion as to the likely Tooting. The oligodendroglial tumours were from 2005 methylation status of the tumour. Our report contains a to 2010 and the glioblastomas were all from 2011. A caveat that this result may be overturned by MLPA. haematoxylin and eosin (H&E) stained section from each Nevertheless, because of the quicker turnaround time of sample was reviewed to ensure the specimen comprised immunohistochemistry compared to molecular genetic sufficient tumour for diagnosis and further testing. testing, the clinicians find this helpful with their initial management pending the definitive result from MLPA. Immunohistochemistry At present, all cases are followed up with MLPA for de- Immunohistochemistry (IHC) staining was carried out finitive diagnosis of methylation status. It may be pos- on 4 μm sections heated for 30 min at 60°C using Bond sible in time, when more information has been gathered III fully automated staining system with their Bond Poly- about double-labelling, to phase out some of the MLPA mer Refine detection system and associated reagents testing. supplied by Leica Microsystems, Newcastle-Upon-Tyne, In future studies we would like to compare the results UK. of double-labelling for MGMT and a “cocktail” of non- Antigen retrieval and dilution was carried out according tumourous elements to clinical outcomes. Quantitation of to antibody: CD34 clone QBEND10 (Novocastra, Leica MGMT expression in tumour cells in double-labelled im- Microsystems, UK) 1:100, CD45 clone 2B11&PD7/26 ages will require special thresholding protocols but should (DakoCytomation, Ely, UK) 1:500, MGMT clone 3.1 provide useful comparison with the methylation ratios (Millipore, Thermo Fisher, UK) 1:100 with Epitope obtained from MLPA. Although MLPA is a relatively new Retrieval Solution 1 (pH6) at 100°C for 30 min, CD68 technique for assessment of methylation status, in this re- clone 514H12 (Novocastra, Leica Microsystems, UK) gard it has the advantage of providing a quantitative result. 1:100 with Epitope Retrieval Solution 2 (pH9) at 100°C for The double-labelling-cocktail method may also be useful 20 min. All primary antibodies were applied to the section in the study of heterogeneity of MGMT expression in for 15 min except MGMT that was applied for 30 min. brain tumours. A number of recent studies [11–18] have The MGMT antibody was optimized using tonsil as a Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 8 of 10 http://www.actaneurocomms.org/content/1/1/22 positive control. Endothelial cell staining also acted as an Validation and scoring internal positive control. Quantitation of the labelling index for single-labelled Double-labelling utilized a sequential IHC staining MGMT (number of stained nuclei divided by total number method, incorporating a “cocktail” of CD34, CD45 and of nuclei expressed as percentage) was carried out in the CD68 as the first antibody visualized with the Bond software program imageJ version 1.46e from the National Polymer Red Refine detection system (supplied by Leica Institute of Health (Rasband, W.S., http://rsbweb.nih. Microsystems, Newcastle-upon-Tyne, UK) and MGMT gov/ij/). Three photomicrographs were taken by the clone 3.1 as the second antibody. neuropathologist (LB) as representative of the tumour, avoiding non-informative areas such as cortex and necrosis Methylation-specific multiplex ligand-dependent probe and without knowledge of the methylation status. We use amplification (MS-MLPA) the ImmunoRatio plug-in, which has been validated for the Ten x 10 μm sections were used for DNA extraction assessment of the labelling index of nuclear markers in and subsequent MS-MLPA testing. The area of tumour breast cancer [19]. In our material we found good correl- was manually micro-dissected to enrich the sample. ation of the labelling index for MGMT single-labelling DNA extraction was performed using an in-house obtained using ImageJ and ImmunoRatio compared to a method using heat for de-waxing and Chelex-100 direct cell count (Pearson correlation coefficient r = 0 .90) and sodium acetate for DNA purification. The probe and a semiquantitative manual assessment by group of mix was prepared by MRC Holland (Amsterdam, observers (r = 0.80) in 11 oligodendroglial tumours [20]. The Netherlands) and the procedure was carried out Please note that one of these cases was omitted from our according to the guidelines supplied. MS-MLPA was final study due to insufficient tissue. performed on the Beckman Coulter CEQ 8000. DNA Scoring of the double-labelling was carried out from yields were quantified using a NanoDrop (Thermo-Sci- photomicrographs. Slides were photographed “blind” entific, USA). (i.e. without knowledge of the methylation status) by A methylation ratio was produced by using an average the neuropathologist (LB) and the resulting 120 images methylation from 4 analysed probes. In fact 6 probes are (three representative images from each of 40 cases) provided in the kit but the manufacturers have recently were evaluated by LB and by two laboratory scientists recommended that only 4 are used because 2 of the EB and KE. The scheme followed was that images with probes are not particularly informative. A result of either mainly negative staining in tumour cell nuclei scored 0, methylated or unmethylated was obtained for the MS- images with equivocal staining scored 1 and images MLPA test. A ratio of less than or equal to 0.25 was used with mainly positive staining scored 2. The final score as a cut-off for unmethylated cases in accordance with for each observer was the modal average of their scores Jeuken et al. [10]. It was also possible, according to for the three images. One observer (LB) carried out a Jeuken et al. [10] to subcategorise the results as follows: second set of scores after 72 hours. Inter-observer and 0 to 0.25 unmethylated, 0.26 to 0.50 mildly methylated, intra-observer kappa statistics were calculated using the 0.51 to 0.75 moderately methylated and 0.76 to 1.00 ex- attribute agreement analysis functioninthe statistical tensively methylated. The analysis was performed using program Minitab 16. Photomicrographs were used to the software program Coffalyser v7 (MRC Holland, standardise the comparison bearing in mind the differ- Netherlands). ent skill-sets of the assessors (please note however that LB works directly from the double-labelled glass slides Photography in everyday diagnosis). The slides were viewed on a Nikon Eclipse 80i microscope Specificity, sensitivity, positive predictive value and with a Nikon DS Ri1 12 megapixel camera attached. Im- negative predictive value were calculated according to ages were captured with NIS-Elements BR 3.2 software on standard formulae in comparison with the gold-standard a Dell precision T7500 PC. All of the images were cap- MLPA after cross-tabulating the results in Minitab 16. tured under standard conditions of illumination. Tiff For this, the tripartite double-labelling scores were images (1280 ×1024 pixels) were taken at an objective lens converted to a binary scoring system (0 and 1). Original magnification of 40×. At the outset of the session, part of scores of 0 and 1 were mapped to 1 in the new system and the slide with no tissue (blankfield) was viewed in the original scores of 2 were mapped to 0. This "reverse" scor- microscope and a white balance carried out. The level of ing was adopted in order to correspond to the scoring of illumination was monitored as being between 204 and 206 the MLPA (i.e. unmethylated = 0, methylated = 1). After for the blankfield (between photographs the illumination due consideration, we adopted a convention by which MGMT-equivocal immunostaining was interpreted as level was checked as being within the same range). Photo- graphs were taken with a fixed exposure time and after MGMT-negative (i.e. presumptive of a methylated status; using the inbuilt focusing device. please see Results). Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 9 of 10 http://www.actaneurocomms.org/content/1/1/22 We wondered whether the "cocktail" (CD34, CD45 and (MGMT) immunohistochemistry in glioblastoma multiforme: observer variability and lack of association with patient survival impede its use as CD68) used in the double-labelling gave a comparable re- clinical biomarker. Brain Pathol 2008, 18:520–532. sult to the component antigens used singly. Using a 7. Nakasu S, Fukami T, Baba K, Matsuda M: Immunohistochemical study thresholding method in image J we measured the area for O6-methylguanine-DNA methyltransferase in the non-neoplastic and neoplastic components of gliomas. JNeurooncol 2004, fraction of staining in 10 oligodendroglial tumours for the 70:333–340. cocktail and for CD34, CD45 and CD68 stained singly. 8. Stupp R, Hegi ME: Methylguanine methyltransferase testing in There was good correlation between the area fraction for glioblastoma: when and how? J Clin Oncol 2007, 25:1459–1460. 9. Watanabe R, Nakasu Y, Tashiro H, Mitsuya K, Ito I, Nakasu S, Nakajima T: the cocktail (range 3 to 8%) and the sum of the area frac- O6-methylguanine DNA methyltransferase expression in tumor cells tions for CD34, CD45 and CD68 stained singly (range 3 to predicts outcome of radiotherapy plus concomitant and adjuvant 9%; Spearman's rho = 0 .76). The area fraction of the cock- temozolomide therapy in patients with primary glioblastoma. Brain Tumor Pathol 2011, 28:127–135. tail was somewhat lower than the combined area fractions 10. Jeuken JW, Cornelissen SJ, Vriezen M, Dekkers MM, Errami A, Sijben A, of the single labels consistent with a degree of overlap Boots-Sprenger SH, Wesseling P: MS-MLPA: an attractive alternative in antigenicity of target cells (e.g. CD68-positive macro- laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas. Lab Invest 2007, phages may also stain with CD45). 87:1055–1065. 11. Brell M, Tortosa A, Verger E, Gil JM, Vinolas N, Villa S, Acebes JJ, Caral L, Pujol Competing interests T, Ferrer I, et al: Prognostic significance of O6-methylguanine-DNA The authors declare that they have no competing interests. methyltransferase determined by promoter hypermethylation and immunohistochemical expression in anaplastic gliomas. Clin Cancer Res Authors’ contributions 2005, 11:5167–5174. EB to study design and immunohistochemistry. MG to study design and 12. Kreth S, Thon N, Eigenbrod S, Lutz J, Ledderose C, Egensperger R, Tonn JC, molecular genetics. KE to study design and immunohistochemistry. FB to Kretzschmar HA, Hinske LC, Kreth FW: O-methylguanine-DNA molecular genetics. RT to study design and molecular genetics. MC to methyltransferase (MGMT) mRNA expression predicts outcome in clinical aspects. LRB to study design, image analysis, statistics and drafting of malignant glioma independent of MGMT promoter methylation. manuscript. All authors read and approved the final manuscript. PLoS One 2011, 6:e17156. 13. Kreth S, Limbeck E, Hinske LC, Schutz SV, Thon N, Hoefig K, Egensperger R, Acknowledgments Kreth FW: In human glioblastomas transcript elongation by alternative Thanks are due to Prof. Anthony Bell and Mr. Henry Marsh for funding polyadenylation and miRNA targeting is a potent mechanism of MGMT and Dr. Frank Saran for helpful discussions. silencing. Acta Neuropathol 2013, 125:671–681. 14. Uno M, Oba-Shinjo SM, Camargo AA, Moura RP, Aguiar PH, Cabrera HN, Author details Begnami M, Rosemberg S, Teixeira MJ, Marie SK: Correlation of MGMT Cellular Pathology, St George’s Hospital, Blackshaw Road, London SW17 promoter methylation status with gene and protein expression levels in 0QT, UK. Molecular Genetics, St George’s Hospital, Blackshaw Road, London glioblastoma. Clinics (Sao Paulo) 2011, 66:1747–1755. SW17 0QT, UK. Neurosurgery, St George’s Hospital, Blackshaw Road, London 15. Pollack IF, Hamilton RL, Sobol RW, Burnham J, Yates AJ, Holmes EJ, Zhou T, SW17 0QT, UK. Finlay JL: O6-methylguanine-DNA methyltransferase expression strongly correlates with outcome in childhood malignant gliomas: results from Received: 10 April 2013 Accepted: 28 May 2013 the CCG-945 Cohort. J Clin Oncol 2006, 24:3431–3437. Published: 10 June 2013 16. Lalezari S, Chou AP, Tran A, Solis OE, Khanlou N, Chen W, Li S, Carrillo JA, Chowdhury R, Selfridge J, et al: Combined analysis of O6-methylguanine References -DNA methyltransferase protein expression and promoter methylation 1. Gorlia T, van den Bent MJ, Hegi ME, Mirimanoff RO, Weller M, Cairncross JG, provides optimized prognostication of glioblastoma outcome. Eisenhauer E, Belanger K, Brandes AA, Allgeier A, et al: Nomograms for Neuro Oncol 2013, 15:370–381. predicting survival of patients with newly diagnosed glioblastoma: 17. Cao VT, Jung TY, Jung S, Jin SG, Moon KS, Kim IY, Kang SS, Park CS, Lee KH, prognostic factor analysis of EORTC and NCIC trial 26981-22981/CE.3. Chae HJ: The correlation and prognostic significance of MGMT promoter Lancet Oncol 2008, 9:29–38. methylation and MGMT protein in glioblastomas. Neurosurgery 2009, 2. Hegi ME, Diserens AC, Godard S, Dietrich PY, Regli L, Ostermann S, Otten P, 65:866–875. Van MG, De TN, Stupp R: Clinical trial substantiates the predictive value of 18. Ingold B, Schraml P, Heppner FL, Moch H: Homogeneous MGMT O-6-methylguanine-DNA methyltransferase promoter methylation in immunoreactivity correlates with an unmethylated MGMT promoter glioblastoma patients treated with temozolomide. Clin Cancer Res 2004, status in brain metastases of various solid tumors. PLoS One 2009, 10:1871–1874. 4:e4775. 3. Herrlinger U, Rieger J, Koch D, Loeser S, Blaschke B, Kortmann RD, Steinbach 19. Tuominen VJ, Ruotoistenmaki S, Viitanen A, Jumppanen M, Isola J: JP, Hundsberger T, Wick W, Meyermann R, et al: Phase II trial of lomustine ImmunoRatio: a publicly available web application for quantitative plus temozolomide chemotherapy in addition to radiotherapy in newly image analysis of estrogen receptor (ER), progesterone receptor (PR), diagnosed glioblastoma: UKT-03. J Clin Oncol 2006, 24:4412–4417. and Ki-67. Breast Cancer Res 2010, 12:R56. 4. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, 20. Burke E, Grobler M, Elderfield K, Butler S, Taylor R, Crocker M, Bridges LR: Ludwin SK, Allgeier A, Fisher B, Belanger K, et al: Effects of radiotherapy Computerized image analysis in the assessment of MGMT with concomitant and adjuvant temozolomide versus radiotherapy immunostaining. Neuropathol Appl Neurobiol 2012, alone on survival in glioblastoma in a randomised phase III study: 5-year 38(Suppl. 1):30–31. analysis of the EORTC-NCIC trial. Lancet Oncol 2009, 10:459–466. 21. Chinot OL, Barrie M, Fuentes S, Eudes N, Lancelot S, Metellus P, Muracciole 5. Quillien V, Lavenu A, Karayan-Tapon L, Carpentier C, Labussiere M, Lesimple X, Braguer D, Ouafik L, Martin PM, et al: Correlation between O6- T, Chinot O, Wager M, Honnorat J, Saikali S, et al: Comparative assessment methylguanine-DNA methyltransferase and survival in inoperable newly of 5 methods (methylation-specific polymerase chain reaction, diagnosed glioblastoma patients treated with neoadjuvant MethyLight, pyrosequencing, methylation-sensitive high-resolution temozolomide. J Clin Oncol 2007, 25:1470–1475. melting, and immunohistochemistry) to analyze O6-methylguanine -DNA-methyltranferase in a series of 100 glioblastoma patients. Cancer 22. Capper D, Mittelbronn M, Meyermann R, Schittenhelm J: Pitfalls in the 2012, 118:4201–4211. assessment of MGMT expression and in its correlation with survival in 6. Preusser M, Charles JR, Felsberg J, Reifenberger G, Hamou MF, Diserens AC, Stupp diffuse astrocytomas: proposal of a feasible immunohistochemical R, Gorlia T, Marosi C, Heinzl H, et al: Anti-O6-methylguanine-methyltransferase approach. Acta Neuropathol 2008, 115:249–259. Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 10 of 10 http://www.actaneurocomms.org/content/1/1/22 23. Rodriguez FJ, Thibodeau SN, Jenkins RB, Schowalter KV, Caron BL, O'Neill BP, James CD, Passe S, Slezak J, Giannini C: MGMT immunohistochemical expression and promoter methylation in human glioblastoma. Appl Immunohistochem Mol Morphol 2008, 16:59–65. 24. Karayan-Tapon L, Quillien V, Guilhot J, Wager M, Fromont G, Saikali S, Etcheverry A, Hamlat A, Loussouarn D, Campion L, et al: Prognostic value of O6-methylguanine-DNA methyltransferase status in glioblastoma patients, assessed by five different methods. J Neurooncol 2010, 97:311–322. doi:10.1186/2051-5960-1-22 Cite this article as: Burke et al.: Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours. Acta Neuropathologica Communications 2013 1:22. 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Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours

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Copyright © 2013 by Burke et al.; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Pathology; Neurology
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Abstract

Background: Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Results: Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Conclusions: Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard. Keywords: MGMT, Methylation, Glioblastoma, Glioma, Immunohistochemistry, Double-labelling, Cocktail, MLPA, Biomarker * Correspondence: leslie.bridges@stgeorges.nhs.uk Cellular Pathology, St George’s Hospital, Blackshaw Road, London SW17 0QT, UK Full list of author information is available at the end of the article © 2013 Burke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 2 of 10 http://www.actaneurocomms.org/content/1/1/22 Background same turnaround time as standard immunohistochemis- Clinical trials have shown that determination of the try and at little extra cost. methylation status of glioblastomas is an important pre- We have used such a platform to develop a novel dictor of outcome and response to treatment [1–4]. A technique, namely double-labelling immunostaining of range of molecular genetic techniques are available for de- MGMT and a "cocktail" of non-tumour antigens (CD34, termining methylation status in tumours [5]. However, CD45 and CD68). Our aim was to produce a visual separ- these are generally expensive and somewhat slow in ation of tumour cells and non-tumour cell elements on application. It would certainly be of great help if immuno- the same histological slide and thereby greatly simplify the staining were available to infer the methylation status of assessment of MGMT immunostaining in tumour cell the tumour. In fact, immunostaining for MGMT, on the nuclei. face of it, should provide an answer. Antibodies against MGMT are readily available which stain tumour cell nu- Results clei positive in unmethylated tumours and negative in A comparison of the results for double-labelling versus methylated tumours (this opposite relationship between single-labelling and MLPA (the gold standard) is shown MGMT staining and methylation status is due to the fact in Table 2. Some representative examples of single- that methylation of the MGMT gene switches off produc- labelling and double-labelling are shown in Figure 1. tion of the MGMT protein) [6]. However, there is a major problem in interpretation of Agreement in assessment of the double-labelling MGMT immunostaining in brain tumours due to the fact There was good inter-observer and intra-observer agree- that normal non-tumourous elements within the tumour ment in the assessment of the double-labelling. For the (including endothelial cells, lymphocytes and macro- three independent "blind" observers (EB, LB and KE) all phages) normally express MGMT [6–8]. This makes it appraisers’ assessments agreed with each other for 31/40 difficult to determine whether any MGMT positivity (77.50%) of the cases. The Fleiss’ kappa statistic was 0.76 within the tumour is due to tumour cells or non-tumour and Kendall’s coefficient of concordance for ordinal cells. Not surprisingly, the literature therefore contains scores was 0.93 indicating a good level of agreement. much controversy about the value or otherwise of MGMT The intra-observer agreement was 36/40 (90%) of cases immunostaining as a clinical biomarker in brain tumours matched with a kappa of 0.84 and a Kendall of 0.97. (please see Table 1). For example, Preusser et al. [6] state that observer Sensitivity, specificity, positive predictive value and variability and lack of association with patient survival negative predictive value of double-labelling impedes the use of MGMT immunostaining in glioblast- The results of the neuropathologist (LB) were compared omas. On the other hand, Watanabe et al. [9] (on the with the gold standard, MLPA. For this, cases assessed basis of eight years clinical experience of MGMT immu- as largely MGMT-negative were considered methylated nostaining, using serial sections stained for non-tumour (i.e. test result positive) and cases assessed as largely markers such as CD45 and CD68 and a careful algorith- MGMT-positive were considered unmethylated (i.e. test mic approach) consider that MGMT immunohisto- result negative). For MLPA we use the cut-off used by chemistry correlates with outcomes in patients with Jeuken et al. [10]. According to this, all cases with an glioblastomas. MLPA ratio greater than 0.25 are at least mildly methyl- Although the use of serial sections stained with non- ated (i.e. true positives) and cases with an MLPA ratio of tumour markers for comparison with sections stained for between 0 and 0.25 are unmethylated (i.e. true negatives). MGMT is undoubtedly helpful, it is not always easy to Of the 40 cases, there were 11 negative matches, 20 compare cellular identities between serial sections. To positive matches, 4 false-positives and 5 false-negatives. some extent it is possible to identify tumour cells in the The test results showed significant association (chi-square, MGMT-stained section directly (for example, when p = 0.001) between the double-labelling and MLPA tumour cell nuclei are very large and bizarre). However, results. Sensitivity was 80%, specificity 73.33%, positive reactive elements within tumours may themselves develop predictive value (PPV) 83.33% and negative predictive a degree of nuclear enlargement and irregularity which value (NPV) 68.75%. For the 30 glioblastomas apart, sensi- makes distinction from tumour cells impossible at times. tivity was 66.7%, specificity 73.33%, PPV 71.43% and NPV Double-labelling immunohistochemistry is an estab- 68.75%. All 10 of the oligodendroglial tumours were cor- lished technique which is now available on some auto- rectly called. These values indicate a good all-round per- mated immunohistochemistry platforms such as the formance by the double-labelling. Leica platform in use in our laboratory. This means that The other assessors’ results were also compared with double-labelling protocols can readily be incorporated MLPA. The results for specificity, sensitivity, PPV and into the routine work of the laboratory, with much the NPV were 88%, 46.67%, 73.33% and 70% for KE and 88%, Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 3 of 10 http://www.actaneurocomms.org/content/1/1/22 Table 1 Previous studies Author and year Tumour type Cut off value Low MGMT protein MGMT IHC compared Molecular test Correlation between for IHC expression predictive of: to molecular gold IHC and molecular standard test Progression free Overall survival (PFS) survival (OS) Nakasu et al. 2004 [7] 69 high-grade gliomas 10% - Yes No - - (grades III and IV) Brell et al. 2005 [11] 93 anaplastic gliomas: 75 AAs and 18 5% No Yes Yes MS-PCR No with oligodendroglial component Chinot et al. 2007 [21] 28 GBMs 35% Yes Yes No - - Capper et al. 2008 [22] 75 primary GBMs 15% Yes – median survival No - - Preusser et al. 2008 [6] 164 GBMs 10% - No Yes MS-PCR Poor agreement Rodriguez et al. 2008 [23] 50 GBMs 10% No No Yes MS-PCR No Karayan-Tapon et al. 2010 [24] 81 GBMs 15% No No Yes MS-PCR - SQ-PCR No Pyrosequencing Yes Q-RT-PCR Yes Quillien et al.2012[5] 100 GBMs 23% Yes Yes No - - The table shows previous studies where MGMT IHC has been performed. The cut-off value was the percentage of MGMT-positive tumour cell nuclei (assessed by eye). The correlation of IHC with progression-free survival (PFS) and overall survival (OS) and comparison with a molecular gold standard (where applied) are also shown. Abbreviations: AA anaplastic astrocytoma, GBM glioblastoma multiforme, MS-PCR methylation-specific polymerase chain reaction, Q-RT-PCR quantitative real-time polymerase chain reaction, SQ-PCR semi-quantitative polymerase chain reaction. Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 4 of 10 http://www.actaneurocomms.org/content/1/1/22 Table 2 Cases case ID type LI SL MLPA MLPA 0-3 MLPA >0.25 DL 1 oligo 7.567 1 0.6275 2 1 1 2 oligo 13.700 1 0.675 2 1 1 3 oligo 12.600 1 0.8075 3 1 1 4 oligo 24.667 1 0.4725 1 1 1 5 oligo 17.100 1 0.8525 3 1 1 6 oligo 13.600 1 0.8025 3 1 1 7 oligo 19.567 1 0.6425 2 1 1 8 oligo 14.267 1 0.6925 2 1 1 9 oligo 8.800 1 0.6525 2 1 1 10 oligo 26.133 1 0.5925 2 1 *1 11 gbm 56.367 0 0.1125 0 0 0 12 gbm 19.167 1 0.8475 3 1 1 13 gbm 84.400 0 0.09 0 0 0 14 gbm 17.967 1 0.2625 1 1 *1 15 gbm 57.700 0 0.255 1 1 0 16 gbm 89.900 0 0.4075 1 1 0 17 gbm 72.267 0 0.2075 0 0 0 18 gbm 19.533 1 0.3525 1 1 *1 19 gbm 54.133 0 0.42 1 1 *1 20 gbm 53.900 0 0.1375 0 0 0 21 gbm 21.967 1 0.485 1 1 1 22 gbm 54.167 0 0.5475 2 1 1 23 gbm 33.000 1 0.085 0 0 0 24 gbm 76.300 0 0.2725 1 1 0 25 gbm 96.733 0 0.24 0 0 0 26 gbm 49.567 1 0.4275 1 1 1 27 gbm 43.967 1 0.1375 0 0 *1 28 gbm 40.533 1 0.1025 0 0 0 29 gbm 7.233 1 0.1275 0 0 1 30 gbm 39.067 1 0.5125 2 1 1 31 gbm 25.833 1 0.2075 0 0 *1 32 gbm 33.900 1 0.13 0 0 *1 33 gbm 28.000 1 0.365 1 1 0 34 gbm 45.000 1 0.275 1 1 1 35 gbm 84.100 0 0.205 0 0 0 36 gbm 85.100 0 0.2975 1 1 0 37 gbm 64.933 0 0.11 0 0 0 38 gbm 53.400 0 0.17 0 0 0 39 gbm 6.800 1 0.825 3 1 1 40 gbm 22.500 1 0.2425 0 0 0 Case ID (identification) is the case number we assigned in this study. Tumour types were oligodendroglial tumours (oligo) and glioblastomas (gbm). The oligodendroglial tumours were oligodendroglioma WHO grade II (cases 4, 5 and 6), anaplastic oligodendroglioma WHO grade III (cases 1, 2, 3, 7 and 9), oligoastrocytoma WHO grade II (case 10) and anaplastic oligoastrocytoma WHO grade III (case 8). The glioblastomas were all glioblastoma WHO grade IV except cases 13 and 18 which were glioblastoma with oligodendroglioma component WHO grade IV. LI is the nuclear labelling index for single-labelled MGMT as determined by computerised image analysis. SL is the binarised score for the single-labelling (0 = LI > 50%, 1 = LI from 0 to 50%). MLPA is the MLPA ratio (please see text). MLPA 0–3 is the score for MLPA according to cut-offs for the MLPA ratio provided by Jeuken et al. 2007 [10] (0 = unmethylated, 1 = mildly methylated, 2 = moderately methylated and 3 = extensively methylated). MLPA > 0.25 is the binarised methylation score for MLPA (0 = unmethylated, 1 =methylated). DLis the binarised score for double labelling (0 = mainly MGMT-positive in tumour cells, 1 = mainly MGMT-negative in tumour cells). The asterisked cases were initially assigned a score of 1 in the tripartite system (please see text for explanation). Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 5 of 10 http://www.actaneurocomms.org/content/1/1/22 40%, 70.97% and 66.67% for EB. The results are similar to Figure 1 Photomicrographs comparing single-labelling and LB’s apart from sensitivity which scored less well for these double-labelling for MGMT. This figure shows the advantage of assessors. This may reflect the particular approach of the double-immunolabelling over single-immunolabelling in the interpretation of MGMT immunostaining. Four glioblastomas are assessors; the neuropathologist appears to have been more shown, one per row, with the single-labelling for MGMT on the left willing to ignore low levels of MGMT staining than the and double-labelling for MGMT and "cocktail" (combined CD34, two laboratory scientists. In this paper we have carried CD45 and CD68) on the right. All images were photographed at an forward the neuropathologist’s scores on the basis that the objective lens magnification of 80x (scale bar = 50 microns). For all pathologist carries out the everyday reporting in practice. images, nuclear immunostaining for MGMT is seen as brown due to the DAB-peroxidase product. For images on the right, cytoplasmic staining for CD34, CD45 and CD68 is seen as red due to the Fast False-negatives and false-positives Red-Alkaline Phosphatase product used in the double-labelling Of the 5 false-negatives it is interesting to note that all system. The counterstain is haematoxylin (nuclei unstained for were only mildly methylated on MLPA. The slides were MGMT appear blue). The case in row 1 (case 25 in Table 2) was reviewed and all 5 cases were confirmed as showing un- unmethylated by MLPA. Double-labelling (B) is more informative than single-labelling (A) because it highlights the cytoplasm of the doubted MGMT-positivity suggestive of an unmethylated non-tumourous elements (endothelial cells, lymphocytes and status. It is therefore of interest that although these cases macrophages) as red. The remaining cells can therefore be positively were false negatives, the result of MLPA was only mild identified as tumour cells and the presence of undoubted nuclear methylation – the closest category to unmethylated. Fur- immunostaining for MGMT in these tumour cells correctly indicates ther clinicopathological correlation may help develop a an unmethylated status. The case in row 2 (C and D; case 39 in Table 2) was extensively methylated by MLPA. The case was easily better understanding of the significance of the "mildly" assessed as MGMT-negative (methylated) on double-labelling (D). methylated category on MLPA. The case in row 3 (E and F; case 18 in Table 2) illustrates the issue of The slides of the 4 false-positives were also reviewed. "equivocal" staining as seen a number of cases (asterisked in One case was undoubtedly MGMT-negative suggestive of Table 2). Please see Results for further commentary on this phenomenon. The case in row 4 (case 22 in Table 2) illustrates a situation where single-labelling (G) shows a high labelling index for MGMT due to a high content of non-tumourous cells (endothelial cells, lymphocytes and macrophages). Double-labelling (H) provides an easy "one-look" diagnosis of MGMT-negative (methylated). a methylated status but MLPA was unmethylated. The other 3 cases showed equivocal MGMT-immunostaining and we had chosen by convention to call these MGMT- negative (please see below). Equivocal MGMT-immunostaining Table 2 shows 7/40 cases (asterisked) with equivocal (focal or faint) MGMT-immunostaining of tumour cell nuclei on double-labelling. For illustrations of equivocal staining please see Figure 1 (E and F). These cases were difficult to classify as MGMT-negative (methylated) or MGMT-positive (unmethylated). We therefore decided on a convention by which such cases were interpreted as MGMT-negative (suggestive of a methylated status). Of the 7 such cases, 4 cases (3 glioblastomas and 1 oligo- dendroglial tumour) turned out to be correctly thus- called (i.e. they were methylated by MLPA). Three cases (all glioblastomas) were incorrectly called (i.e. they were unmethylated). Since there is no clear indication at present from these figures as to how the equivocal cases should be assessed (roughly equal numbers were methylated and unme- thylated) we suggest - until more data is available - treating these cases as MGMT-negative (suggestive of a methylated status) as we have here. It may be worth not- ing in the pathology report that such cases - in particular - Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 6 of 10 http://www.actaneurocomms.org/content/1/1/22 are liable to a change in status when molecular testing is through. This approach would seem most consistent with the default clinical position whereby patients with glio- blastomas in which the methylation status is unknown are managed as if the tumour were methylated. We are gath- ering further data on the issue of equivocal staining which, as indicated, accounts for about a fifth of our assessments. Comparison with single-labelling As mentioned, Figure 1 shows how quantitative assess- ment of double-labelling is quick and easy compared to single-labelling. We wondered how the automated as- sessment of the labelling index for MGMT on single- labelling performed against the qualitative assessment on double-labelling. For this we set a convenient cut-off of 50% for the labelling index (close to the actual median of 47.28 for the 40 cases in our series). Cases with a la- Figure 2 Chart of MGMT single-labelling, MGMT double- labelling and MLPA status. The chart shows the labelling index for belling index of up to 50% were considered methylated MGMT from the single-labelled slides on the Y-axis for the 40 cases (i.e. a positive test result) and cases with a labelling plotted against the results of double-labelling and MLPA status. The index greater than 50% were considered unmethylated 4 cases that were false-positive by double-labelling are seen in the (i.e. a negative test result) and this was compared with second column from the left. All of these cases have a labelling MLPA using the 0.25 cut-off of Jeuken et al. [10] as pre- index of less than 50% by single-labelling suggestive of a methylated status. The results of double-labelling and single- viously detailed. Please note that our choice of a median labelling are therefore concordant and combined analysis would not cut-off for the single-labelled MGMT labelling index have avoided calling these cases (falsely) positive. The 5 cases that may at first sight appear to be a departure from previous were false-negative by double-labelling are seen in the third column studies (e.g. Preusser et al. [6]). Generally these other from the left. Of these, 4 have a labelling index of greater than 50% studies have used assessment "by eye” and mentally by single-labelling suggestive of an unmethylated status. The results of double-labelling and single-labelling of these 4 cases are subtracted tumour-only elements whereas our data is therefore concordant and combined analysis would not have truly quantitative and applied to both tumourous and avoided calling these (falsely) negative. Only one of the 5 false- non-tumourous elements since the machine cannot sep- negative cases has a labelling index of less than 50% by single- arate the two. We feel that the median cut-off is appro- labelling suggestive of a methylated status. In this case the labelling priate in our context. index might have been helpful in avoiding calling this (falsely) negative on double-labelling. Overall however the extra effort For all cases (n = 40) for the single-labelling MGMT la- involved in measuring the labelling index by automated quantitative belling index versus MLPA sensitivity was 76%, specificity image analysis on the single-labelled slides may not be worthwhile, 55.33%, PPV 73.08% and NPV 57.14%. For the glioblast- particularly in labs not set up for image analysis. omas (n = 30) sensitivity was 60%, specificity 53.33%, PPV 56.25% and NPV 57.14%. Compared to double-labelling these results for quantitative single-labelling are not as (for endothelium), CD45 (for lymphocytes) and CD68 (for good and there are also other disadvantages compared to macrophages). The end result was a histology slide in double-labelling (please see Discussion). which we could easily distinguish between tumourous and We then wondered whether the single-labelling and non-tumourous components and thereby quickly come to double-labelling could be combined in some way to give a conclusion as to whether the tumour cell nuclei were a better result and the analysis is shown in Figure 2. MGMT-positive or MGMT-negative (and thence to a conclusion as to their likely methylation status). Discussion We found good agreement between observers (kappa We have developed a novel protocol for the analysis of 0.76) and within observer (kappa 0.84). Furthermore, MGMT-immunostaining using double-labelling. The aim double-labelling showed good specificity (80%), sensitiv- of our approach is to simplify the assessment of MGMT- ity (73.33%), positive predictive value (83.33%) and immunostaining by creating a visual separation of the negative predictive value (68.75%) compared to MLPA tumourous and non-tumourous elements. We did this by (the gold-standard). using the usual brown chromogen (DAB-peroxidase) for Compared to qualitative assessment of single-labelling nuclear MGMT staining and a red chromogen (Fast Red- MGMT-immunostaining, we found double-labelling to Alkaline Phosphatase) for a “cocktail" of non-tumourous be much quicker and easier to assess (Figure 1). Further- cytoplasmic elements. The cocktail is composed of CD34 more, double-labelling outperformed even quantitative Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 7 of 10 http://www.actaneurocomms.org/content/1/1/22 computerised image analysis of MGMT single-labelling raised the question of whether or not a strict correlation in terms of sensitivity, specificity, PPV and NPV. Further- should really be expected between MGMT staining and more, we did not find that much benefit would accrue methylation status. For example, Kreth and colleagues from combining the results of double-labelling with [12] found that patients with discordant results for quantitative single-labelling. Since quantitation is some- MGMT expression (determined by mRNA expression) what time-consuming we would therefore not necessarily and methylation status did worse than concordant coun- recommend using both double-labelling with quantita- terparts. A molecular basis for MGMT expression inde- tive single-labelling on a routine basis: we consider pendent of methylation may apply in some situations [13]. double-labelling alone to be sufficient for everyday Our double-labelling technique offers a possible new ap- purposes. proach to investigate such questions. We are aware that the double-labelling as with any test is not perfect and that certain cautions are required in Conclusions interpretation. The cocktail of three reagents (CD34, We have developed a novel method for double-labelling CD45 and CD68) should stain a good proportion of for MGMT, which is quick and easy to apply and might non-tumourous elements but not necessarily all. For ex- readily be subsumed into routine reporting for glioblast- ample, there may also be non-neoplastic astrocytes and omas (and other tumours). There is good inter-observer oligodendrocytes which are not identified by the cock- and intra-observer agreement and, compared to a mo- tail. For this reason we would recommend that interpret- lecular genetic gold-standard, the method yields good ation of the double-labelling be focussed on areas of sensitivity, specificity, positive predictive value and nega- tumour proper and caution exercised in areas where tive predictive value as a predictor of methylation status. tumour is infiltrating largely normal tissues. We are also aware that CD34 occasionally stains glioblastoma Methods tumour cells and in such situations tumour cells will be The study was carried out in compliance with local re- stained red by the red chromagen and will thus be ex- search ethics committee (LREC) guidelines as a service cluded from analysis. However, we feel the CD34 stain- development. ing by glioblastomas cells is usually just focal so that it This was a retrospective study. Forty formalin-fixed should be possible in most cases to find interpretable paraffin-embedded (FFPE) samples were obtained from areas in the slide. patients diagnosed with gliomas (10 oligodendroglial tu- In our own current practice, we carry out double- mours and 30 glioblastomas) from the archives of the labelling for MGMT and "cocktail" (CD34, CD45 and Cellular Pathology department at St George’s Hospital, CD68) to come to an initial conclusion as to the likely Tooting. The oligodendroglial tumours were from 2005 methylation status of the tumour. Our report contains a to 2010 and the glioblastomas were all from 2011. A caveat that this result may be overturned by MLPA. haematoxylin and eosin (H&E) stained section from each Nevertheless, because of the quicker turnaround time of sample was reviewed to ensure the specimen comprised immunohistochemistry compared to molecular genetic sufficient tumour for diagnosis and further testing. testing, the clinicians find this helpful with their initial management pending the definitive result from MLPA. Immunohistochemistry At present, all cases are followed up with MLPA for de- Immunohistochemistry (IHC) staining was carried out finitive diagnosis of methylation status. It may be pos- on 4 μm sections heated for 30 min at 60°C using Bond sible in time, when more information has been gathered III fully automated staining system with their Bond Poly- about double-labelling, to phase out some of the MLPA mer Refine detection system and associated reagents testing. supplied by Leica Microsystems, Newcastle-Upon-Tyne, In future studies we would like to compare the results UK. of double-labelling for MGMT and a “cocktail” of non- Antigen retrieval and dilution was carried out according tumourous elements to clinical outcomes. Quantitation of to antibody: CD34 clone QBEND10 (Novocastra, Leica MGMT expression in tumour cells in double-labelled im- Microsystems, UK) 1:100, CD45 clone 2B11&PD7/26 ages will require special thresholding protocols but should (DakoCytomation, Ely, UK) 1:500, MGMT clone 3.1 provide useful comparison with the methylation ratios (Millipore, Thermo Fisher, UK) 1:100 with Epitope obtained from MLPA. Although MLPA is a relatively new Retrieval Solution 1 (pH6) at 100°C for 30 min, CD68 technique for assessment of methylation status, in this re- clone 514H12 (Novocastra, Leica Microsystems, UK) gard it has the advantage of providing a quantitative result. 1:100 with Epitope Retrieval Solution 2 (pH9) at 100°C for The double-labelling-cocktail method may also be useful 20 min. All primary antibodies were applied to the section in the study of heterogeneity of MGMT expression in for 15 min except MGMT that was applied for 30 min. brain tumours. A number of recent studies [11–18] have The MGMT antibody was optimized using tonsil as a Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 8 of 10 http://www.actaneurocomms.org/content/1/1/22 positive control. Endothelial cell staining also acted as an Validation and scoring internal positive control. Quantitation of the labelling index for single-labelled Double-labelling utilized a sequential IHC staining MGMT (number of stained nuclei divided by total number method, incorporating a “cocktail” of CD34, CD45 and of nuclei expressed as percentage) was carried out in the CD68 as the first antibody visualized with the Bond software program imageJ version 1.46e from the National Polymer Red Refine detection system (supplied by Leica Institute of Health (Rasband, W.S., http://rsbweb.nih. Microsystems, Newcastle-upon-Tyne, UK) and MGMT gov/ij/). Three photomicrographs were taken by the clone 3.1 as the second antibody. neuropathologist (LB) as representative of the tumour, avoiding non-informative areas such as cortex and necrosis Methylation-specific multiplex ligand-dependent probe and without knowledge of the methylation status. We use amplification (MS-MLPA) the ImmunoRatio plug-in, which has been validated for the Ten x 10 μm sections were used for DNA extraction assessment of the labelling index of nuclear markers in and subsequent MS-MLPA testing. The area of tumour breast cancer [19]. In our material we found good correl- was manually micro-dissected to enrich the sample. ation of the labelling index for MGMT single-labelling DNA extraction was performed using an in-house obtained using ImageJ and ImmunoRatio compared to a method using heat for de-waxing and Chelex-100 direct cell count (Pearson correlation coefficient r = 0 .90) and sodium acetate for DNA purification. The probe and a semiquantitative manual assessment by group of mix was prepared by MRC Holland (Amsterdam, observers (r = 0.80) in 11 oligodendroglial tumours [20]. The Netherlands) and the procedure was carried out Please note that one of these cases was omitted from our according to the guidelines supplied. MS-MLPA was final study due to insufficient tissue. performed on the Beckman Coulter CEQ 8000. DNA Scoring of the double-labelling was carried out from yields were quantified using a NanoDrop (Thermo-Sci- photomicrographs. Slides were photographed “blind” entific, USA). (i.e. without knowledge of the methylation status) by A methylation ratio was produced by using an average the neuropathologist (LB) and the resulting 120 images methylation from 4 analysed probes. In fact 6 probes are (three representative images from each of 40 cases) provided in the kit but the manufacturers have recently were evaluated by LB and by two laboratory scientists recommended that only 4 are used because 2 of the EB and KE. The scheme followed was that images with probes are not particularly informative. A result of either mainly negative staining in tumour cell nuclei scored 0, methylated or unmethylated was obtained for the MS- images with equivocal staining scored 1 and images MLPA test. A ratio of less than or equal to 0.25 was used with mainly positive staining scored 2. The final score as a cut-off for unmethylated cases in accordance with for each observer was the modal average of their scores Jeuken et al. [10]. It was also possible, according to for the three images. One observer (LB) carried out a Jeuken et al. [10] to subcategorise the results as follows: second set of scores after 72 hours. Inter-observer and 0 to 0.25 unmethylated, 0.26 to 0.50 mildly methylated, intra-observer kappa statistics were calculated using the 0.51 to 0.75 moderately methylated and 0.76 to 1.00 ex- attribute agreement analysis functioninthe statistical tensively methylated. The analysis was performed using program Minitab 16. Photomicrographs were used to the software program Coffalyser v7 (MRC Holland, standardise the comparison bearing in mind the differ- Netherlands). ent skill-sets of the assessors (please note however that LB works directly from the double-labelled glass slides Photography in everyday diagnosis). The slides were viewed on a Nikon Eclipse 80i microscope Specificity, sensitivity, positive predictive value and with a Nikon DS Ri1 12 megapixel camera attached. Im- negative predictive value were calculated according to ages were captured with NIS-Elements BR 3.2 software on standard formulae in comparison with the gold-standard a Dell precision T7500 PC. All of the images were cap- MLPA after cross-tabulating the results in Minitab 16. tured under standard conditions of illumination. Tiff For this, the tripartite double-labelling scores were images (1280 ×1024 pixels) were taken at an objective lens converted to a binary scoring system (0 and 1). Original magnification of 40×. At the outset of the session, part of scores of 0 and 1 were mapped to 1 in the new system and the slide with no tissue (blankfield) was viewed in the original scores of 2 were mapped to 0. This "reverse" scor- microscope and a white balance carried out. The level of ing was adopted in order to correspond to the scoring of illumination was monitored as being between 204 and 206 the MLPA (i.e. unmethylated = 0, methylated = 1). After for the blankfield (between photographs the illumination due consideration, we adopted a convention by which MGMT-equivocal immunostaining was interpreted as level was checked as being within the same range). Photo- graphs were taken with a fixed exposure time and after MGMT-negative (i.e. presumptive of a methylated status; using the inbuilt focusing device. please see Results). Burke et al. Acta Neuropathologica Communications 2013, 1:22 Page 9 of 10 http://www.actaneurocomms.org/content/1/1/22 We wondered whether the "cocktail" (CD34, CD45 and (MGMT) immunohistochemistry in glioblastoma multiforme: observer variability and lack of association with patient survival impede its use as CD68) used in the double-labelling gave a comparable re- clinical biomarker. Brain Pathol 2008, 18:520–532. sult to the component antigens used singly. Using a 7. Nakasu S, Fukami T, Baba K, Matsuda M: Immunohistochemical study thresholding method in image J we measured the area for O6-methylguanine-DNA methyltransferase in the non-neoplastic and neoplastic components of gliomas. JNeurooncol 2004, fraction of staining in 10 oligodendroglial tumours for the 70:333–340. cocktail and for CD34, CD45 and CD68 stained singly. 8. Stupp R, Hegi ME: Methylguanine methyltransferase testing in There was good correlation between the area fraction for glioblastoma: when and how? J Clin Oncol 2007, 25:1459–1460. 9. Watanabe R, Nakasu Y, Tashiro H, Mitsuya K, Ito I, Nakasu S, Nakajima T: the cocktail (range 3 to 8%) and the sum of the area frac- O6-methylguanine DNA methyltransferase expression in tumor cells tions for CD34, CD45 and CD68 stained singly (range 3 to predicts outcome of radiotherapy plus concomitant and adjuvant 9%; Spearman's rho = 0 .76). The area fraction of the cock- temozolomide therapy in patients with primary glioblastoma. Brain Tumor Pathol 2011, 28:127–135. tail was somewhat lower than the combined area fractions 10. Jeuken JW, Cornelissen SJ, Vriezen M, Dekkers MM, Errami A, Sijben A, of the single labels consistent with a degree of overlap Boots-Sprenger SH, Wesseling P: MS-MLPA: an attractive alternative in antigenicity of target cells (e.g. CD68-positive macro- laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas. Lab Invest 2007, phages may also stain with CD45). 87:1055–1065. 11. Brell M, Tortosa A, Verger E, Gil JM, Vinolas N, Villa S, Acebes JJ, Caral L, Pujol Competing interests T, Ferrer I, et al: Prognostic significance of O6-methylguanine-DNA The authors declare that they have no competing interests. methyltransferase determined by promoter hypermethylation and immunohistochemical expression in anaplastic gliomas. Clin Cancer Res Authors’ contributions 2005, 11:5167–5174. EB to study design and immunohistochemistry. MG to study design and 12. Kreth S, Thon N, Eigenbrod S, Lutz J, Ledderose C, Egensperger R, Tonn JC, molecular genetics. KE to study design and immunohistochemistry. FB to Kretzschmar HA, Hinske LC, Kreth FW: O-methylguanine-DNA molecular genetics. RT to study design and molecular genetics. MC to methyltransferase (MGMT) mRNA expression predicts outcome in clinical aspects. LRB to study design, image analysis, statistics and drafting of malignant glioma independent of MGMT promoter methylation. manuscript. All authors read and approved the final manuscript. PLoS One 2011, 6:e17156. 13. Kreth S, Limbeck E, Hinske LC, Schutz SV, Thon N, Hoefig K, Egensperger R, Acknowledgments Kreth FW: In human glioblastomas transcript elongation by alternative Thanks are due to Prof. Anthony Bell and Mr. Henry Marsh for funding polyadenylation and miRNA targeting is a potent mechanism of MGMT and Dr. Frank Saran for helpful discussions. silencing. Acta Neuropathol 2013, 125:671–681. 14. Uno M, Oba-Shinjo SM, Camargo AA, Moura RP, Aguiar PH, Cabrera HN, Author details Begnami M, Rosemberg S, Teixeira MJ, Marie SK: Correlation of MGMT Cellular Pathology, St George’s Hospital, Blackshaw Road, London SW17 promoter methylation status with gene and protein expression levels in 0QT, UK. Molecular Genetics, St George’s Hospital, Blackshaw Road, London glioblastoma. Clinics (Sao Paulo) 2011, 66:1747–1755. SW17 0QT, UK. Neurosurgery, St George’s Hospital, Blackshaw Road, London 15. Pollack IF, Hamilton RL, Sobol RW, Burnham J, Yates AJ, Holmes EJ, Zhou T, SW17 0QT, UK. Finlay JL: O6-methylguanine-DNA methyltransferase expression strongly correlates with outcome in childhood malignant gliomas: results from Received: 10 April 2013 Accepted: 28 May 2013 the CCG-945 Cohort. J Clin Oncol 2006, 24:3431–3437. Published: 10 June 2013 16. Lalezari S, Chou AP, Tran A, Solis OE, Khanlou N, Chen W, Li S, Carrillo JA, Chowdhury R, Selfridge J, et al: Combined analysis of O6-methylguanine References -DNA methyltransferase protein expression and promoter methylation 1. Gorlia T, van den Bent MJ, Hegi ME, Mirimanoff RO, Weller M, Cairncross JG, provides optimized prognostication of glioblastoma outcome. 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Karayan-Tapon L, Quillien V, Guilhot J, Wager M, Fromont G, Saikali S, Etcheverry A, Hamlat A, Loussouarn D, Campion L, et al: Prognostic value of O6-methylguanine-DNA methyltransferase status in glioblastoma patients, assessed by five different methods. J Neurooncol 2010, 97:311–322. doi:10.1186/2051-5960-1-22 Cite this article as: Burke et al.: Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours. Acta Neuropathologica Communications 2013 1:22. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Acta Neuropathologica CommunicationsSpringer Journals

Published: Jun 10, 2013

References