The next generation of glioma biomarkers: MGMT methylation, BRAF fusions and IDH1 mutations.


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For some, glioma biomarkers have been expected to solve common diagnostic problems in routine neuropathology service caused by insufficient material, technical shortcomings or lack of experience. Further, biomarkers should predict patient outcome and direct optimal therapy for the individual patient. Unfortunately, current biomarkers still fall somewhat short of these grand expectations. While there has been some progress, it has generally been slow and in small steps. In this review, the newest set of glioma biomarkers: O(6) -methylguanine-DNA methyltransferase (MGMT) methylation, BRAF fusion and IDH1 mutation are discussed. MGMT methylation is well established as a prognostic/predictive marker for glioblastoma; however, technical questions regarding testing remain, it is not currently utilized widely in guiding patient management, and it has proven to be of no assistance in diagnostics. In contrast, BRAF fusion and IDH1 mutation analyses promise to be very helpful for classifying and grading gliomas, while their potential predictive value has yet to be established.

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The next generation of glioma biomarkers: MGMT methylation, BRAF fusions and IDH1 mutations.

  1. 1. Brain Pathology ISSN 1015-6305MINI-SYMPOSIUM: Molecular Diagnostics in Neuro-Oncology bpa_454 74..87The Next Generation of Glioma Biomarkers: MGMTMethylation, BRAF Fusions and IDH1 MutationsAndreas von Deimling; Andrey Korshunov; Christian HartmannDepartment of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, and Clinical Cooperation Unit Neuropathology, GermanCancer Research Center, Heidelberg, GermanyKeywords Abstractbiomarker, BRAF, glioma, IDH1, MGMT. For some, glioma biomarkers have been expected to solve common diagnostic problems inCorresponding author: routine neuropathology service caused by insufficient material, technical shortcomings orProfessor Dr. Andreas von Deimling, MD, lack of experience. Further, biomarkers should predict patient outcome and direct optimalDepartment of Neuropathology, Institute of therapy for the individual patient. Unfortunately, current biomarkers still fall somewhatPathology, Ruprecht-Karls-Universität short of these grand expectations. While there has been some progress, it has generallyHeidelberg, Clinical Cooperation Unit been slow and in small steps. In this review, the newest set of glioma biomarkers:Neuropathology, German Cancer Research O6-methylguanine-DNA methyltransferase (MGMT) methylation, BRAF fusion and IDH1Center, Im Neuenheimer Feld 220/221, mutation are discussed. MGMT methylation is well established as a prognostic/predictiveD-69120 Heidelberg, Germany (E-mail: marker for glioblastoma; however, technical questions regarding testing remain, it is currently utilized widely in guiding patient management, and it has proven to be of no assistance in diagnostics. In contrast, BRAF fusion and IDH1 mutation analyses promise toReceived 11 October 2010; accepted 11 be very helpful for classifying and grading gliomas, while their potential predictive valueOctober 2010. has yet to be established.doi:10.1111/j.1750-3639.2010.00454.x promoter hypermethylation in gliomas varies widely. In clinicalMGMT PROMOTER studies, it has ranged from 35% to 73% in GBM (8–10, 17, 18, 21,HYPERMETHYLATION 23, 38–40, 74, 90, 96–98, 101). In diffusely infiltrating anaplastic gliomas [World Health Organization (WHO) grade III], it has beenBrief history found in 50%–84% (7, 91, 99), while 43%–93% of the WHO gradeThe gene encoding the O6-methylguanine-DNA methyltransferase II counterparts are reportedly positive (24, 51, 52). The substantial(MGMT) has become one of the most, if not the most studied range of reported MGMT promoter hypermethylation frequenciesmolecular marker in neurooncology since the first description of is probably at least partially due to technical challenges (seean association between MGMT promoter hypermethylation and below).response to alkylating drugs a decade ago (23). However, the inter-est in this gene was particularly enhanced in 2005 by the publica- Mechanisms of action, tumor inactivation andtion of Hegi et al showing in a prospective phase III trial, that chemosensitivityglioblastoma (GBM) patients with methylated MGMT promoterstatus demonstrated a significant survival advantage with temozo- The MGMT gene located on 10q26 has five exons and a CpG-richlomide treatment (39). Nonetheless, the dealkylating function of island of 763 bp with 98 CpG sites encompassing the first exon andMGMT had already been described in the early 1980s (69, 72), large parts of the promoter. A minimal promoter and an enhancerwith its cDNA being cloned 10 years later (87). In 1999, promoter region are located within the CpG island (Figure 1) (65). In normalhypermethylation was found to be the main inactivating mecha- tissue, most CpG sites within the island are unmethylated. Innism of this gene in a broad variety of human cancers (22). tumors, the cytosine in CpG sites often carries a methyl group, thereby increasing the affinity of proteins like methyl-CpG-binding protein 2 and methyl-CpG-binding domain protein 2 to the DNA.Distribution of MGMT promoter These proteins subsequently alter the chromatin structure andhypermethylation in tumor entities prevent binding of transcription factors, thereby silencing expres-MGMT promoter hypermethylation has been identified in a sion of MGMT (65).wide range of human cancers, including lung-, head and neck-, MGMT is a suicide DNA repair protein that normally catalyzespancreatic-, renal- and bladder carcinomas, as well as lymphomas, the transfer of a methyl group from the O6-position of a guanineleukemias and melanoma (22). The reported frequency of MGMT DNA nucleotide to a cysteine residue at its own position 145. This74 Brain Pathology 21 (2011) 74–87 © 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  2. 2. von Deimling et al The Biomarkers MGMT, BRAF and IDH1alkylation of MGMT is a one-way process, with alkylated MGMT The role of MGMT testing in patients with WHO grade II or IIIultimately targeted for degradation (30). In gliomas, this is relevant diffuse gliomas is even less clear at this time. In anaplastic gliomas,because application of alkylating chemotherapeutic drugs like MGMT promoter hypermethylation is associated with longer pro-temozolomide cause, among other actions, the binding of an gression free survival for patients treated either by radiochemo-alkyl group to the O6-position of guanine, thereby inducing DNA- therapy or radiation alone (90, 99). A predictive role of MGMTmismatching, DNA-double-stand breakage and ultimately apopto- promoter hypermethylation for response to temozolomide treat-sis in proliferating cells. Thus, MGMT protein counteracts the ment was documented for grade II gliomas (24, 51). However, anormally lethal effect of temozolomide by repairing DNA damage. positive prognostic effect of MGMT promoter hypermethylationWhen a tumor has a hypermethylated MGMT promoter, the was not found for patients with WHO grade II astrocytomas (A II)hypothesis is that MGMT expression is reduced and the cytotoxic (52) and oligodendrogliomas (O II) (94), who received no alkylat-effects of alkylating drugs are then enhanced. ing chemotherapy. Yet, this widely accepted and intuitive concept that MGMT pro-moter hypermethylation acts as a chemosensitizer recently becamechallenged by the observation that patients suffering from anaplas- METHODS FOR MGMT ANALYSIStic gliomas with hypermethylated MGMT similarly exhibited a MGMT promoter hypermethylation assayssurvival benefit when treated with radiotherapy alone (90, 99).Therefore, it remains unclear whether MGMT also plays a role in Technical limitations of all available methods used in a routinerepairing radiotherapy-induced DNA damage, if other DNA repair setting prohibit analysis of all 98 CpG sites. Generally, eachgenes are silenced by promoter hypermethylation in addition to method focuses on only a couple of CpG sites based on the assump-MGMT, or if the survival advantages are better explained by other tion that they reflect the methylation status of the whole CpGprognostically favorable genetic alterations like the 1p/19q codele- island, in turn predicting patient response to alkylating drugs. Yet,tions (6, 63, 90) and IDH1 mutations (80) that often coexist with the methylation pattern is not always homogeneous and methodsMGMT promoter hypermethylation. targeting different CpG sites may yield conflicting results (25). Only a few reports are available that allow conclusions regarding methylation of the majority of the CpG sites in gliomas. By clonalClinical relevance sequencing, Nakagawachi et al determined the methylation patternIn one of the most important series on therapy of patients with of all CpG sites in glioma cells that did not express MGMT. Theynewly diagnosed GBM in the last decade and its follow-up study, it identified an upstream highly methylated region (UHMR) and awas shown that those patients with a hypermethylated MGMT pro- downstream highly methylated region (DHMR) and a region inmoter demonstrated survival rates of 49% and 14% at 2 and 5 years between with varying methylation (65). According to their data, allrespectively, when treated with concomitant and adjuvant temozo- CpG sites of the methylation-specific polymerase chain reactionlomide and radiotherapy. In contrast, estimated 2- and 5-year sur- (MS-PCR) and the commercially available pyrosequencing assayvival rates were only 24% and 5% respectively, in similar patients (see below) are located within the DHMR. Everhard et al analyzedthat were initially treated with radiotherapy alone. GBM patients the methylation profile of 52 CpG sites by pyrosequencing in GBMwhose tumors lacked MGMT hypermethylation demonstrated 2- and compared the results with MGMT mRNA expression. In thisand 5-year survival rates of 15% and 8% when they received com- way, they were able to identify six single CpG sites and two CpGbined radiochemotherapy, which dropped to only 2% and 0% when regions that best correlated with overall expression. Only a partialtreated with radiotherapy alone (39, 85, 86). As such, temozolo- overlap between these CpG sites and the CpG sites tested by themide response was most dramatic in the methylation-positive routine assays was observed (25) (Figure 1).group, yet there was some advantage even in the non-methylated Most clinical studies have used MS-PCR as first introduced bycohort. Given this data, the fact that temozolomide is a well- Esteller et al (22) to compare MGMT status with survival andtolerated oral drug, and that few highly efficacious alternatives are therapeutic response. As such, MS-PCR has become the standardcurrently available, most neuro-oncologists still opt to treat their method for MGMT testing to date. Five to nine CpG sites arepatients with this drug first, regardless of MGMT status. Neverthe- usually covered by MS-PCR, in which DNA bisulfite treatmentless, multiple studies have subsequently confirmed the observation converts unmethylated cytosine to uracil. Next, PCR amplificationthat MGMT promoter hypermethylation is one of the strongest is done with two different primer pairs that align to regions encom-prognostic factors for patients with newly diagnosed GBM and that passing those specific CpG sites. One primer pair is specific forit is a potent predictor for response to treatment with alkylating methylated, “non-uracilized” DNA, while the other is designed fordrugs (23, 31, 38, 40, 97), even in elderly patients (9). Furthermore, DNA in which cytosine residues have been converted to uracil. InMGMT promoter hypermethylation was identified as the only this way, converted and unconverted CpG sites can be distin-molecular marker that was enriched in so-called long-term survi- guished from each other. In addition to the MGMT promoter, avors of GBM (OS > 36 months) (54). However, prolonged survival completely unmethylated fragment of DNA (eg, COL2A1 pro-can be seen without MGMT promoter hypermethylation, indicating moter) and a completely methylated sample are often used as nega-the existence of other factors conferring this favorable prognosis tive and positive controls, respectively. Finally, PCR products are(54, 64). So, while testing currently provides powerful prognostic visualized on an agarose gel. But while this method does notinformation, its role in guiding patient management is more require expensive equipment and is widely established, the bisulfitetenuous. Presumably however, as additional and perhaps more tar- reaction requires a great deal of technical expertise and is rathergeted therapeutic options become available, MGMT testing may labor-intensive. Also, MS-PCR is frequently used with anbecome all the more important for patient management. increased number of PCR cycles and may result in false positiveBrain Pathology 21 (2011) 74–87 75© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  4. 4. von Deimling et al The Biomarkers MGMT, BRAF and IDH1Figure 1. Genomic area covering the promoter, CpG island and exon 1 to (35). Minimal promoter region, enhancer region, upstream highlyof O6-methylguanine-DNA methyltransferase (MGMT). Genomic data methylated region and downstream highly methylated region accordingbased on University of California Santa Cruz (UCSC) Genome Browser to (65). CpG sites with best correlation between methylation status andFebruary 2009 assembly (, chromosome 10 MGMT expression according to (25). Methylation-specific PCR (MS-genomic contig GL000100.1 (GenBank)—a web-based service offered PCR) primers according to (23, 39, 99). Pyrosequencing region accordingby the NIH, MGMT sequence: NM_002412.3 (GenBank), CpG island to (62). HhaI cleaving sites used for methylation-specific multiplex liga-predicted by UCSC Genome Bioinformatics position chr10: tion dependent probe amplification (MS-MLPA) according to (47). Real-131 264 949–131 265 710. Whole promoter DNA sequence according time-MS-PC (RT-MS-PCR) region according to (92).᭣amplification. Depending on numerous factors, PCR efficiency pyrosequencing, quantitative MS-MLPA data require an algorithmvaries, and therefore, the reproducibility is not as high as one would to calculate a qualitative +/- threshold. In our hands MS-MLPAlike for a clinical assay. emerged as a less reliable method when formalin-fixed paraffin- An improvement on MS-PCR is real-time MS-PCR which embedded tissue (FFPE) was analyzed.allows a higher quantity of standardization, greater throughputand the definition of a cut-off (92). In this variant, a methylation-specific probe has an attached fluorophore and quencher, sitting MGMT protein-based assaysbetween primer sites. After the primers hybridize and initiate PCR, Immunohistochemistry (IHC) would seem to be the most directthe probe is degraded by an exonuclease that acts during the exten- and convenient assay to determine amount of actual MGMTsion phase of PCR. This results in separation of the fluorophore protein. Although some studies have reported an associationfrom the quencher, with fluorescence being quantifiable. Results between IHC-based MGMT expression and response to alkylatingcan be expressed as the ratio of methylation-specific amplification drugs in gliomas (3, 11, 15, 46, 58), the technique is fraught withof the tumor MGMT gene to the COL2A1 reference gene, that is the problems. In glial tumors, various non-neoplastic cells includingmethylation index (MI). Based on prior work (90), an MI less than lymphocytes, microglia, blood vessel cells and reactive astrocytes4 is negative, an MI between 4 and 16 has a low level of methylation all express MGMT, thereby hampering targeted IHC-based evalua-with uncertain clinical significance, and an MI greater than 16 is tion of tumor cells (26). A high interobserver variability has thusconsidered methylated. been reported and the cut-off levels in the literature for positive Another attractive alternative is methylation-specific pyrose- staining have ranged from >10% to >50% positive cells (76).quencing (62).This method also requires DNA bisulfite treatment as Finally, many researchers have failed to identify an associationthe initial step, followed by sequencing of a short DNA fragment between IHC results and either MGMT methylation status based oncovering usually five CpG sites using a luciferase-based detection MS-PCR or patient survival/chemosensitivity (32, 55, 76, 77).mechanism. Primers can be individually designed, but a commercial Detection of MGMT mRNA expression is an alternative to IHC butkit is available. The method allows analysis of highly fragmented currently no clinical study is available that has utilized this method.DNA and is therefore robust and reproducible in formalin-fixed, Measurement of enzymatic MGMT activity could potentiallyparaffin-embedded clinical tissues. However, methylation-specific provide a more accurate assessment at the mechanistic level, but nopyrosequencing requires expensive equipment and the costs per study has demonstrated a value to this approach in a clinical settingsample are high if only a few cases are analyzed per run.An unsolved to date. Both methods suffer from a dependence on fresh frozenissue is the quantitative character of the results: each of the five tissue and otherwise elaborate, error-prone methodology. Both alsotested CpG sites are assigned a value between 0 and 100% which suffer the same risk of false-negative “dilution” by non-neoplasticneeds to be finally condensed to a qualitative +/- result (Figure 2). MGMT-producing cells.No clinical study has yet been produced that allows a valid definitionof an algorithm to calculate such a threshold. An example of anambiguous result is depicted in Figure 2C. Another method suitable for routine diagnostics that generates CONSEQUENCESquantitative methylation data is methylation-specific multiplex liga- Impact on diagnostic aspectstion dependent probe amplification (MS-MLPA) (47). MS-MLPAwas successfully used to analyze the tumor samples of the large Due to demand by neuro-oncologists for MGMT methylation statusEuropean Organization for Research and Treatment of Cancer on their patients’ tumors, some neuropathology departments(EORTC) 26951 study for MGMT promoter hypermethylation (90). already offer this test. However, in different labs, various methodsMS-MLPA does not require a DNA consuming bisulfite treatment of testing across diverse CpG sites are applied and even if MS-PCRstep. After probe annealing and ligation, digestion with the restric- is chosen, the same primers and conditions are not always used,tion enzyme HhaI only cuts DNA at unmethylated CpG sites. making it difficult to assess inter-laboratory concordance rates. AsFinally, probes are amplified by PCR and visualized by fragment a result, divergent results are sometimes generated if the sameanalysis together with a non-HhaI digested control sample. The tumor is analyzed in multiple laboratories. To avoid doubts regard-short probe annealing sequence should allow analysis of highly ing the quality of MGMT testing, a methodological standardizationfragmented DNA. However, MS-MLPA requires HhaI restriction combined with round-robin testing organized by national neuropa-sites within a CpG island and only one of the three suitable MGMT thology and molecular pathology societies is recommended. Com-CpG sites are located within the region that is commonly analyzed mercially available MGMT test kits may enhance inter-laboratoryby MS-PCR or pyrosequencing (Figure 1). Furthermore, similar to reproducibility. An alternative and less desirable approach wouldBrain Pathology 21 (2011) 74–87 77© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  5. 5. The Biomarkers MGMT, BRAF and IDH1 von Deimling et al Figure 2. Bar plot showing different results for MGMT promoter hypermethylation analysis by pyrosequencing according to (62). T: amount of thymidine in %. C: amount of cytosine in %. CpG 1—5: the different CpG sites. Control: a cytosine not followed by guanine that always becomes converted to 100% uracil/thymidine when DNA bisulfite treatment was successful. A. Nonambiguous pattern indicating methylation of all five CpG sites between 17% and 53%. B. Nonambiguous pattern indicating no methylation of all five CpG sites. C. Methylation of only CpG site no. 2 with to delegate MGMT promoter hypermethylation analysis to exter- nervous system (CNS) tumors. Multiple studies (see above) clearlynal commercial companies, who are less likely to have skill in indicate that GBM can be separated into two biologically differentglioma tissue evaluation and would not interpret the data in the groups based on MGMT status, potentially justifying incorporationoverall context of other clinicopathological features. of this variable in the next WHO classification. However, molecular A growing debate in the neuropathology community revolves assays are still not available worldwide and the WHO classificationaround whether or not MGMT status should be incorporated into remains primarily a morphology-based system for tumor stratifica-the next revision of the WHO classification scheme for central tion, aimed at the broadest audience possible.78 Brain Pathology 21 (2011) 74–87 © 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  6. 6. von Deimling et al The Biomarkers MGMT, BRAF and IDH1Impact on clinical practice Distribution of KIAA1549:BRAF fusion in tumor entitiesPatients suffering from GBM do appear to benefit from concomi-tant and adjuvant temozolomide therapy when MGMT promoter is The KIAA1549:BRAF fusion in PA I has been found across all agehypermethylated (39). However, as already mentioned, the lack of groups and in various tumor locations, including cerebellum, cere-good alternative treatment options for patients with unmethylated bral hemispheres, hypothalamus, optic nerve and brain stem,GBMs and the small documented number of such patients that still although some studies revealed that BRAF duplication/fusion is arespond to such therapy argues for the commonly utilized approach more frequent event in cerebellar PA I (28, 48, 49, 83). In contrast,of trying temozolomide treatment first, independent of the MGMT BRAF gene fusion is a rare event in diffusely infiltrating gliomas,status. Thus, routine MGMT analysis does not appear essential for instead often containing BRAF point mutations, whereas, forpatient management at this time, although MGMT testing in new example, BRAFV600E is found in approximately 25% of grade II–IVglioma clinical trials has become a conditio sine qua non so pediatric astrocytomas (53, 81). Occurrence and distribution ofadequate comparisons with the current standard of care can be KIAA1549:BRAF fusions in other brain tumor subtypes are stillmade. unknown. Mechanism of actionConclusions Genomic sequencing has revealed a few breakpoint variants:Currently, MGMT is one of the most requested molecular assays in KIAA1549 exon16–BRAF exon 9, KIAA1549 exon 15–BRAF exonclinical neuro-oncology. Due to lack of therapeutic alternatives, 9, KIAA1549 exon19–BRAF exon 9, KIAA1549 exon18–BRAFMGMT evaluation is only currently essential for clinical trials, exon 10 and KIAA1549 exon 16–BRAF exon 11 (28, 48, 49, 83,although that is likely to change once effective alternative therapies 88). The common thread through all these breakpoint variants is thebesides temozolomide are identified. A range of new methodolo- formation of an oncogenic BRAF-KIAA1549 fusion incorporatinggies have become available for MGMT testing that potentially the BRAF kinase domain but lacking the amino-localized auto-allow higher levels of sensitivity, specificity, robustness, and repro- inhibitory domain. This truncated BRAF, is constitutively activeducibility. However, clinical studies that critically compare these (48, 83, 88). While such activity likely is tumorigenic, it is interest-new assays to the current “gold standard” of MS-PCR are urgently ing to note that constitutive activation of BRAF can lead toneeded. Furthermore, systematic analyses to identify which CpG oncogene-induced senescence in slowly growing benign tumors.sites best reflect treatment outcome and patient survival are still An alternate mechanism of MAPK pathway activation in PA Ilacking. constitutes tandem duplication at 3p25 in approximately 2% of cases leading to an in-frame oncogenic fusion between SRGAP3 and RAF1, the latter of which shares high sequence homology withBRAF FUSIONS BRAF (28, 49, 56). In general, RAF gene fusion variants have been found in 80% of PA I and thus constitutes a hallmark aberration inBrief history this tumor entity.RAF kinases are part of the mitogen-activated protein kinase(MAPK) cascade, a pathway that ultimately leads to the regulation Clinical relevanceof a wide range of substrates, including transcription factors andother protein kinases that control cell proliferation, differentiation Prognostic significance of the KIAA1549:BRAF fusion, if any, isand apoptosis. Oncogenic activation of BRAF has been well docu- still unclear, although a few studies have found no differences inmented in many tumors (67). However, this usually results from survival between tumors with and without BRAF duplication/fusionpoint mutation rather than gene rearrangement, with a hotspot at (28, 42, 45, 48). Nevertheless, the frequent BRAF alteration in PA Iresidue 600 (BRAFV600E). An activating rearrangement of BRAF in may serve as a novel therapeutic target for pharmacological inhibi-a primary tumor was first reported in four papillary thyroid carci- tion of the MAPK pathway, particularly in tumors that are difficultnomas, where an AKAP9:BRAF fusion was described (16). Trans- to fully excise surgically (75, 78, 88). Previous in vitro studies havelocations involving BRAF have also been reported in large congeni- revealed that stable silencing of BRAF through lentiviral transduc-tal melanocytic nevi (67). Among astrocytic neoplasms, sporadic tion with inhibition of Map/Erk kinase (MEK)1/2 blocked prolif-and hereditary (NF1 associated) pilocytic astrocytomas (PA I) eration and arrested growth of glioma cells (73). In addition, analy-share constitutive activation of the MAPK pathway. A few studies sis of the KIAA1549:BRAF fusion might be very helpful forhave characterized the proto-oncogene BRAF as a particularly differential diagnosis between PA I and A II, especially in combina-important factor in the pathogenesis of PA I in childhood (73, 88). tion with mutational analyses of the IDH1/2 genes (53, 81).Most notably, duplication of the BRAF locus at 7q34 with consecu-tive up-regulation of BRAF expression and MAPK target genessuch as CCND1, was found in more than 50% of these tumors (73). METHODS FOR KIAA1549:BRAF FUSIONSeveral subsequent studies confirmed similar or even higher fre- ANALYSISquencies of BRAF tandem duplications in PA I (2, 28, 42, 45, 48, Fusion detection in genomic DNA49, 56, 83). Moreover, a few studies showed that tandem duplica-tion at 7q34 leads to a fusion between KIAA1549 and BRAF in For the mapping of breakpoints on genomic level, long-distanceapproximately 70% of these tumors (48, 49, 56, 83). inverse PCR (LDI-PCR) is applied (60). LDI-PCR is based on theBrain Pathology 21 (2011) 74–87 79© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  7. 7. The Biomarkers MGMT, BRAF and IDH1 von Deimling et al Conclusions DNA- and RNA-based methods for detection of KIAA1549:BRAF fusion are well-standardized and may be universally applied but snap frozen tissue samples produce the most optimal and reproduc- ible results. Unfortunately, most of these methods require special equipment and are not generally applicable for every day diagnos- tic practice. In terms of routine application, DNA-based markers for FISH are particularly attractive because of their robustness and applicability to FFPE samples. Nevertheless, some obstacles for universal application of this method exist—such as difficulties in standardization of the optimal cut-off levels for fusion detection, absence of reliable criteria to distinguish real gene fusion from randomly overlapping signals and finally, absence of commercially available probe sets. IDH1 MUTATIONS Brief history IDH1 is no doubt the “new kid in town”, receiving considerable attention since the discovery of its relation with human gliomas. Mutations in IDH1 emerged as a surprising finding from aFigure 3. Detection of KIAA1549/BRAF fusion by fluorescence in situ sequencing project addressing the genome of human GBM withhybridization (FISH) analysis. Nucleus of pilocytic astrocytoma carrying 18/149 of these tumors exhibiting a heterozygous point mutation intandem duplication of BRAF (red signals) and KIAA1549 (green signals) codon 132 (70). Interestingly, these IDH1 mutations were associ-resulting in a fusion of these loci (yellow signal). ated with young patient age and the secondary glioblastoma (sGBM) pattern, tumors that evolved from previously confirmed lower grade gliomas. This observation drew attention to A II and anaplastic astrocytoma WHO grade III (A III), both of which werehydrolysis of tumor DNA using distinct combinations of restriction found to carry IDH1 mutations in the majority of cases (1, 33, 44,enzymes, self-ligation of the resulting DNA fragments and a subse- 68, 80, 84, 91, 95, 97, 99, 100). Even more provocative was thequent PCR reaction using a specific set of oligonucleotides. The finding that IDH1 mutations were just as common in oligodendro-analysis is followed by direct-sequencing with corresponding glial tumors. Prior to this, much effort had been put in separatingprimers, confirming the fusion. astrocytic from oligodendroglial tumors on a molecular level, with the general consensus being that TP53 mutations are more associ- ated with diffuse astrocytomas while combined 1p/19q deletionsFusion detection in cDNA predominantly occurred in oligodendroglial tumors. Therefore, theTo detect gene fusion, 5′ rapid amplification of cDNA ends shared trait of IDH1 mutations in both astrocytic and oligodendro-(RACE) method is usually applied (48). RACE results in the pro- glial gliomas suggests a possible origin of both entities from aduction of a cDNA copy of the RNA sequence of interest, produced common precursor cell type. Apart from A II, A III, O II, anaplasticthrough reverse transcriptase PCR (RT-PCR) assay. The amplified oligodendroglioma WHO grade III (O III), oligoastrocytoma WHOcDNA copies are then sequenced and, if a fusion is present, should grade II (OA II), anaplastic oligoastrocytoma WHO grade III (OAmap to a unique mRNA that contains exons from both fusion III) and sGBM, IDH1 mutations have been described only in aboutpartners (Figure 4). 10% of acute myeloid leukemias (59, 93). With the exception of rare individual cases, all other neoplasms studied thus far are essentially negative for IDH1 mutations (4, 50, 79).FISH (fluorescence in situ hybridization) Distribution of IDH1 mutations in tumor entitiesFor interphase FISH analysis of KIAA1549:BRAF fusion, the sim-plest strategy is to use two fluorochrome-conjugated probes: a Mutations in the cytosolic NADP-dependent isocitrate dehydroge-FITC-labeled clone RP11-355D18 corresponding to KIAA1549 nase (IDH1) gene have recently been detected in a fraction rangingand a digoxigenin-labeled clone 726N20 mapping to BRAF (53). between 50% and 80% of astrocytomas, oligodendrogliomas andThe BRAF-KIAA1549 gene fusion is defined in the cases showing oligoastrocytomas (1, 41, 44, 84, 95, 100). There is no differencenuclei with a single fusion red-green or yellow signal in addition to regarding IDH1 mutation frequency between WHO grade II andthe normal pair of split red and green signals (Figure 3). Signals are WHO grade III tumors, with sGBM also exhibiting a comparablescored in at least 100 non-overlapping, intact nuclei, and BRAF mutation rate. This is contrasted by the distinct rarity of IDH1fusion is typically detected in 20%–50% of nuclei in positive cases. mutations in primary or de novo GBMs, arising in the absence of aNon-neoplastic control tissues do not reveal this pattern. lower-grade precursor. Interestingly, the rates of IDH1 mutations in80 Brain Pathology 21 (2011) 74–87 © 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  8. 8. von Deimling et al The Biomarkers MGMT, BRAF and IDH1 KIAA1549:BRAF 15_11T C G A G T C C C G T C T AC C A A G T G T T T T AC T G C A G G G AT T G T T G G C C G T C T G G G C T G G G C T G T A G A A G G KIAA1549:BRAF 16_9 C G G C C A A C A A T C C C T G C A G T G A C T T G A T T A G A G A C C A A G G A T T T C G T KIAA1549:BRAF 15_9C C C T T C G T C CAC A A C T C A G C C T A CA T C G G A T GC C CA G A C T T G A T T A G A G A C C A A G G A T T T C G T G G T GFigure 4. Detection of KIAA1549/BRAF fusion by 5′ rapid amplification of cDNA ends (RACE) analysis. Sequence traces consequently demonstrating afusion between KIAA1549 exon 15: BRAF exon 11, KIAA1549 exon 16: BRAF exon 9 and KIAA1549 exon 15: BRAF exon 9.PA I and ependymal tumors, as well as in less common glial tumor This contrasts with the situation in acute myeloid leukemiaentities are very low, providing diagnostic utility (1). (AML), the only other tumor entity so far known to carry IDH1 IDH1 mutations affect codon 132 in the vast majority of the mutations in a significant portion of tumors, where the distributioncases and have been detected in only one of the parental alleles, of IDH1 mutations is wider (34, 59, 93). Interestingly, mutations inwith the remaining gene copy apparently being wild type. We mitochondrial NADP-dependent isocitrate dehydrogenase (IDH2)sequenced the codon 132 region of IDH1 in more than 3000 gene also occur predominantly in astrocytomas, oligodendroglio-primary human brain tumors and detected (as of August 2010) mas, oligoastrocytomas and AML. IDH2 mutation are encountered1212 mutations. Major portions of these data have been reported in in roughly 3% in gliomas (37), but are more frequent in AML (5,prior studies (1, 37, 97, 99), but the distribution of mutation types is 71, 89).summarized in Table 1. In gliomas, approximately 93% of themutations are of the R132H type, followed by R132C exchanges in4%, R132S and R132G mutations in approximately 1.5% each, and Mechanism of actionsingle R132L mutations. Wild-type IDH1 decarboxylates isocitrate to a-ketoglutarate (aKG), thereby reducing NADP to NADPH (29). Several concepts for the tumorigenic potential of mutant IDH1 protein have beenTable 1. Types of IDH1 mutations detected in 1212 brain tumors promulgated. First, the mutation at codon 132 affects the binding(Heidelberg). Abbreviation: N (%) = number of tumors with mutation and site for isocitrate. Hence, it is not surprising that the decarboxylat-corresponding percentage of mutation type among all mutations. ing activity of mutant IDH1 protein is significantly reduced orNucleotide change Amino acid change N (%) obliterated (44, 100). Activity drops below 50% because of a domi- nant effect of the mutation via inhibition of heterodimeric IDH1G395A R132H 1130 (93.2) complexes (102). Thus, the amount of NADPH equivalents neces-C394T R132C 45 (3.7) sary for cellular protection from oxidative stress might be reducedC394A R132S 16 (1.3) resulting in an increase of reactive oxygen species with subsequentC394G R132G 14 (1.2) pro-oncogenic DNA damage (57). Another hypothesis suggestsG395T R132L 6 (0.5) that IDH1 mutations cause reduced production of aKG. BecauseC394G, G395T R132V 1 (0.1) aKG has prolylhydroxylase activity, it normally promotes degrada-Brain Pathology 21 (2011) 74–87 81© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  9. 9. The Biomarkers MGMT, BRAF and IDH1 von Deimling et altion of HIF1-a. Therefore, IDH1 mutations may promote tumor Pyrosequencinggrowth and angiogenesis by reducing inhibition of HIF1-a (102). However, the hypotheses described above do not entirely explain Recent studies have detected IDH1 mutations by pyrosequencinga peculiarity of IDH1 mutations, its nearly exclusively heterozy- (27, 82). This approach appears as robust as traditional sequencinggous nature, wherein only one of the two IDH1 alleles is affected and allows for rapid analysis and high throughput. A major advan-and the other one is perfectly intact, in stark contrast to the two-hit tage of pyrosequencing is the quantitative nature of the readings,mechanism classically encountered in tumor suppressor genes. allowing mutation detection in tissues intermingled with non-Heterozygous IDH1/2 mutations are more consistent with a gain of tumorous cells. It has been estimated that a concentration offunction, rather than a loss of function. More recent work has approximately 7% mutant alleles is sufficient for detection. Assuggested just such a function, in which the mutant enzyme mentioned above in the context of MGMT analysis, though, a keyreduces aKG to 2-hydroxyglutarate (2-HG), in the process con- disadvantage of pyrosequencing is the considerable investment insuming NADPH. In support of this, gliomas carrying IDH1/2 machinery and consumables and the reduced cost efficiency ifmutations have tissue levels of 2-HG that are 1–2 full orders of looking at single samples per run.magnitude higher than comparable wild-type tumors (19). Simi-larly, the serum of AML patients with IDH1 or IDH2 mutationsalso has markedly elevated 2-HG (33). It bears mentioning, though, dCAPSthat a clear-cut tumorigenic effect of 2-HG has yet to be elucidated. An alternative to DNA sequencing is the so-called “derived cleaved amplified polymorphic sequence” (dCAPS) analysis (66). ThisClinical relevance method relies on the application of mismatched primers, which upon PCR amplification will generate restriction endonucleaseBoth the original GBM sequencing work and subsequent studies sites that will vary depending on whether the mutation is present ordescribing IDH1 mutations have shown better outcomes in these not. Consecutive digestion with the appropriate endonucleases willtumors than grade-matched gliomas lacking this alteration. Analy- yield DNA fragments of different sizes which can be separated onsis of patients enrolled in prospective studies or cohorts have agarose gels. We have developed a battery of primers that allow thealso demonstrated a favorable association of IDH1 mutation with detection of both wild-type IDH1 and all known R132 mutants,overall survival in A III (99), O III (91) and GBM (97) patients. including R132H, R132C, R132G, R132L and R132S (61). WhileFrom a diagnostic perspective, other studies have established that similar DNA quality is needed for this as is needed for sequencing,assays aimed at detecting IDH1 mutation are fairly sensitive for the this approach does not require expensive sequencing facilities. Andpresence of a diffuse glioma (vs. reactive gliosis) and are highly although only predefined sequence alterations are detectable withspecific when other primary CNS tumors are considered in the this assay, the aforementioned list appears to cover the vast major-differential (discussed further below). ity of mutant IDH1.METHODS FOR IDH1 ANALYSIS Melting curve analysisCurrent methods of IDH1 analyses usually address either thesequence of the gene or the structure of the protein. However, it Yet another approach to detect IDH1 and IDH2 mutations isalso might be possible to focus on analysis of the mutant enzyme melting curve analysis performed on real-time PCR products. Aproduct, 2-HG. One such method is gas chromatography-mass suitable protocol has recently been published, utilizing two fluores-spectrometry. Because (so far) all mutations in R132 on IDH1 and cent probes, one of which serves as sensor probe either spanningR172 on IDH2 have been shown to result in the production of codon 132 of IDH1 or codon 172 of IDH2 and a light cycler (43).2-HG, this approach might prove to be highly efficient, yet has the Having even a single base pair mismatch between the fluorescentobvious limitation of needing a solid sample of unfixed tissue. probe and the PCR product will result in a lower melting tempera- ture. PCR products from a typical mutated glioma would therefore have two melting peaks, a wild-type sequence showing a higherDirect sequencing melting point and a lower one showing a lower melting point. Sequencing can then be targeted to those cases with two peaksNearly all of the published studies rely on sequence analyses from to verify the fluorescent melting curve analysis (FMCA). Thistumor DNA. With development of rapid sequencing facilities, both method, which is already clinically employed in the detection ofworkload and time required is rapidly decreasing. However, it KRAS and BRAF point mutations in other tumors, appears to be lessshould be kept in mind that the success of sequencing analysis vulnerable to non-neoplastic DNA “dilution” such that the detec-relies heavily on suitability of the material. Contaminating DNA tion threshold is roughly 10% mutant alleles.from adjacent brain tissue, lymphocytic infiltrates, microglial cellsand endothelia can dilute mutant DNA below detection thresholdsleading to false negative readings. On the other hand, background IHCsignals might also reach threshold levels resulting in false positiveresults. Classic Sanger sequencing requires at least 20% mutant The development of an IDH1 R132H mutation-specific antibodyalleles for reliable detection of the mutant. Thus, thorough adjunct (DIANOVA, clone H09) suitable for FFPE tissue immediatelyhistological analysis of tumor tissue prior to DNA extraction is of made IDH1 analysis in routine specimens accessible and relativelycourse mandatory. simple even for modestly-sized pathology laboratories (13, 14). A82 Brain Pathology 21 (2011) 74–87 © 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  10. 10. von Deimling et al The Biomarkers MGMT, BRAF and IDH1Figure 5. Binding of antibody H09 to tumorcells in infiltrating edge of oligodendroglioma.caveat is that clone H09 is highly specific to R132H and doesnot recognize wild-type sequence or the C, S, L or V amino acid CONSEQUENCESsubstitutions in codon 132. Thus, approximately 93% of brain Impact on diagnostic aspectstumor-associated IDH1 mutations in codon 132 are readily detect-able, while the remaining 7% are missed, as are all IDH2 muta- Knowledge of the IDH1 status is of both diagnostic and clinicaltions. In our hands, however, we found the detection rate of R132H relevance. On diagnostic grounds, IDH1 status greatly assistsmutations by IHC to be slightly higher than that recovered by classification of gliomas. For example, it clearly separates oligo-sequencing. This is due to the ability to detect single tumor cells dendroglial tumors from several entities that are sometimes diffi-(Figure 5), which cannot be accomplished in a practical way by cult to distinguish, including central (or extraventricular) neuro-sequencing. Our routine procedure for detection of IDH1 mutation cytoma, tanycytic ependymoma, PA I with oligodendroglial-likeis therefore an initial IHC screen, followed by DNA sequencing of cytology and dysembryoplastic neuroepithelial tumor (DNT), allcases negative or equivocal with the H09 antibody. In order to of which are characterized by the absence of IDH1 mutationscapture as many possible relevant tumors as possible, those cases (12). Because IDH1 mutations do not occur in reactive gliosis,are also sequenced for IDH2 mutations. conditions producing reactive gliosis can often be separated fromTable 2. IDH1 mutations in gliomas from the Diagnosis N Mut (%)Heidelberg series. Astrocytoma WHO grade II (A II) 299 225 (75.3) Anaplastic astrocytoma WHO grade III (A III) 339 213 (62.8) Secondary glioblastoma WHO grade IV (secGBM) 16 12 (75) Oligodendroglioma WHO grade II (O II) 196 160 (81.6) Anaplastic oligodendroglioma WHO grade III (O III) 227 158 (69.6) Oligoastrocytoma WHO grade II (OA II) 125 99 (79.2) Anaplastic oligoastrocytoma WHO grade III (OA III) 260 184 (70.8) Primary Glioblastoma WHO grade IV (prGBM) 518 42 (8.1) Giant cell glioblastoma WHO grade IV (gcGBM) 10 2 (20) Pediatric Glioblastoma WHO grade IV (pedGBM) 13 1 (7.7) Gliosarcoma WHO grade IV (GS) 10 0 Ependymoma WHO grade II (E II) 17 0 Anaplastic ependymoma WHO grade III (E III) 11 0 Pilocytic astrocytoma WHO grade I (PA I) 114 2 (1.7) Subependymal giant cell astrocytoma WHO grade I (SEGA I) 12 0 Pleomorphic xanthoastrocytoma WHO grade II (PXA II) 9 0 Ganglioglioma WHO grade I (GG I) 22 0 Dysembryoplastic neuroepithelial tumor WHO grade I (DNT) 21 0 Central neurocytoma WHO grade II 35 0Brain Pathology 21 (2011) 74–87 83© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology
  11. 11. The Biomarkers MGMT, BRAF and IDH1 von Deimling et althe frequently mutated diffuse gliomas. The potential of antibody enzyme isoforms 1 and 2 mutations in acute myeloid leukemia: aH09 to detect single cells is sometimes of great assistance in study by the Acute Leukemia French Association group. J Clindetection of tumor in very small samples or in specimens origi- Oncol 28:3717–3723.nating from the infiltrating tumor edge. Further, considering the 6. Brandes AA, Nicolardi L, Tosoni A, Gardiman M, Iuzzolino P, Ghimenton C et al (2006) Survival following adjuvant PCV orfrequency of R132H among IDH1/2-mutated gliomas, screening temozolomide for anaplastic astrocytoma. Neuro Oncol 8:253–260.with H09 IHC is expected to reduce the need for sequencing by 7. Brandes AA, Tosoni A, Cavallo G, Reni M, Franceschi E, Bonaldi Lapproximately 90%. An overview of the distribution of IDH1 et al (2006) Correlations between O6-methylguanine DNAmutations in human brain tumors is given in Table 2. methyltransferase promoter methylation status, 1p and 19q deletions, and response to temozolomide in anaplastic and recurrent oligodendroglioma: a prospective GICNO study. J Clin OncolImpact on clinical aspects 24:4746–4753.Our recent analyses on patients enrolled in the NOA-04 study (99) 8. Brandes AA, Franceschi E, Tosoni A, Blatt V, Pession A, Tallini G et al (2008) MGMT promoter methylation status can predict theand the German Glioma Network (97) revealed that IDH1 status incidence and outcome of pseudoprogression after concomitantwas of higher prognostic relevance than WHO diagnosis within a radiochemotherapy in newly diagnosed glioblastoma patients. J Clinset of tumors consisting of A III and GBM. In this series, the order Oncol 26:2192–2197.of most favorable to least was: A III with mutation; GBM with 9. Brandes AA, Franceschi E, Tosoni A, Benevento F, Scopece L,mutation; A III without mutation; and GBM without mutation (36). Mazzocchi V et al (2009) Temozolomide concomitant and adjuvantWe believe that these findings may have imminent importance for to radiotherapy in elderly patients with glioblastoma: correlationthe classification of A III and GBM and therefore, also on further with MGMT promoter methylation status. Cancer 115:3512– of these patients. There may also be a favorable prog- 10. Brandes AA, Tosoni A, Franceschi E, Sotti G, Frezza G, Amista Pnostic effect of IDH1 mutations in A II, OA II and O II (20, 80). et al (2009) Recurrence pattern after temozolomide concomitant with and adjuvant to radiotherapy in newly diagnosed patients with glioblastoma: correlation With MGMT promoter methylation status.Conclusions J Clin Oncol 27:1275–1279. 11. Capper D, Mittelbronn M, Meyermann R, Schittenhelm J (2008)The recognition of IDH1 mutations in gliomas has already greatly Pitfalls in the assessment of MGMT expression and in its correlationextended our understanding of these tumors, in particular under- with survival in diffuse astrocytomas: proposal of a feasiblescoring the probable role of metabolism in gliomagenesis. The immunohistochemical approach. Acta Neuropathol 115:249–259.presence of IDH1 mutations in both diffuse astrocytic and oligo- 12. Capper D, Reuss D, Schittenhelm J, Hartmann C, Bremer J, Sahm Fdendroglial gliomas highlights the likely common origin of these et al Mutation specific IDH1 antibody differentiatesentities, which have been mostly set apart from each other by oligodendrogliomas and oligoastrocytomas from other brain tumors with oligodendroglioma-like morphology. Acta Neuropathol Berlinmolecular markers for the last two decades. Diagnostic challenges (in press).may be better met by knowledge of the IDH1 status. The high 13. Capper D, Zentgraf H, Balss J, Hartmann C, von Deimling A (2009)relevance of IDH1 status on clinical outcome of patients with A III Monoclonal antibody specific for IDH1 R132H mutation. Actaand GBM is likely to prompt revisions of the current WHO classi- Neuropathol Berlin 118:599–601.fication of brain tumors. We expect that on grounds of the strong 14. Capper D, Weißert S, Balss J, Habel A, Meyer J, Jäger D et al (2010)prognostic effect of IDH1 status on survival, future clinical studies Characterization of R132H mutation sSpecific IDH1 antibodyon diffuse gliomas and GBM will uniformly include this analysis. binding in brain tumors. Brain Pathol 20:245–254.These implications are of considerable interest to neuropatholo- 15. 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