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. 2010 Nov 3;21(3):308–320. doi: 10.1111/j.1750-3639.2010.00447.x

The Nature and Timing of Specific Copy Number Changes in the Course of Molecular Progression in Diffuse Gliomas: Further Elucidation of Their Genetic “Life Story”

Judith WM Jeuken 1,✉, Angelique Sijben 1,2,†, Fonnet E Bleeker 3, Sandra HE Boots‐Sprenger 1, Jos Rijntjes 1, Johanna MM Gijtenbeek 4, Wolf Mueller 5, Pieter Wesseling 1,6
PMCID: PMC8094293  PMID: 21029244

Abstract

Up till now, typing and grading of diffuse gliomas is based on histopathological features. However, more objective tools are needed to improve reliable assessment of their biological behavior. We evaluated 331 diffuse gliomas for copy number changes involving 1p, 19q, CDKN2A, PTEN and EGFR(vIII) by Multiplex Ligation‐dependent Probe Amplification (MLPA®, Amsterdam, The Netherlands). Specifically based on the co‐occurrence of these aberrations we built a model for the timing of the different events and their exact nature (hemi‐ → homozygous loss; low‐level gain → (high‐copy) amplification) in the course of molecular progression. The mutation status of IDH1 and TP53 was also evaluated and shown to correlate with the level of molecular progression. The relevance of the proposed model was confirmed by analysis of 36 sets of gliomas and their 39 recurrence(s) whereas survival analysis for anaplastic gliomas confirmed the actual prognostic relevance of detecting molecular malignancy. Moreover, based on our results, molecular diagnostic analysis of 1p/19q can be further improved as different aberrations were identified, some of them being indicative for advanced molecular malignancy rather than for favorable tumor behavior. In conclusion, identification of molecular malignancy as proposed will aid in establishing a risk profile for individual patients and thereby in therapeutic decision making.

Keywords: EGFR amplification and rearrangements, loss of 1p and 19q, PTEN and CDKN2A loss, molecular pathology, TP53 and IDH1 mutations

INTRODUCTION

Based on their histology, diffuse gliomas can be classified as pure astrocytic, pure oligodendroglial or mixed oligoastrocytic neoplasms (As, Os and OAs, respectively) 30, 32, 34, 35. Gliomas are subsequently graded for malignancy according to the most recent World Health Organization (WHO) guidelines as low‐grade (WHO grade II), anaplastic (WHO grade III) or glioblastoma (GBM) multiforme [WHO grade IV; A‐IV = GBM or OA‐IV = GBM with oligodendroglial differentiation (GBMO)](34). Although the histopathological diagnosis provides information on the expected survival and determines the choice of treatment, criteria for typing and grading are not unequivocal 7, 9, 30, 32. Although nearly all low‐grade diffuse gliomas eventually progress to high‐grade malignancy, time to progression varies considerably (35) and unfortunately there is currently no valid parameter that unambiguously predicts in individual patients how rapidly malignant progression will occur. An additional and more objective tool to determine malignant progression in gliomas is therefore warranted. In this respect, molecular analysis is a very promising tool as molecular changes largely determine tumor behavior.

Over the last decades, it has become increasingly clear that molecular genetic markers are helpful in recognizing more uniform subgroups of gliomas with regard to prognosis and response to therapy and thereby can help to improve the management of individual glioma patients (40). Losses of chromosome arms 1p and 19q (which usually present as co‐deletions affecting both complete chromosome arms) are reported to occur as an early event in the oncogenesis of Os and OAs and to predict longer survival and better response to (chemo)therapy 3, 8, 11, 20, 25, 29, 33, 39, 41, 44, 46. Although these aberrations are only occasionally detected in As, they are relatively frequent in malignant As of patients with an unexpectedly long survival, these latter tumors possibly representing misdiagnosed OAs 18, 31. Other early events in glioma oncogenesis encompass IDH1 mutations and TP53 mutations which have been reported in, respectively, 60%–90% and >60% of the A‐IIs, 50%–100% and 30% of the OA‐IIs, and 70%–85% and <5% of the O‐IIs 1, 4, 10, 15, 34, 38, 49, 50. In fact, IDH1 is now considered as an initiating event in glioma oncogenesis, preceding TP53 mutations and loss of 1p and 19q. Furthermore genetic changes affecting, for example, PTEN, CDKN2A and EGFR have been identified mainly in high grade malignant As and are therefore considered to be involved in glioma progression 30, 34, 45. The exact timing and interrelation between these aberrations during molecular (malignant) progression, however, has not yet been investigated systematically. Moreover, usually no distinction is made between the different levels of copy number changes, that is, hemizygous vs. homozygous loss, or low‐level gain, amplification or high‐copy (HC) amplification. Meanwhile, such information may contribute to an accurate (molecular) identification of tumor progression and malignancy. While serial analysis of a large set of primary gliomas and their recurrences will provide such information, this approach is usually hampered by the fact that biopsy or resection of the recurrent tumor is in many patients not performed. Alternatively, parallel analysis of multiple aberrations in the complete spectrum of diffuse gliomas and evaluation of their co‐occurrence will provide insights in the sequence of events occurring during molecular (malignant) progression.

In the current study we first used Multiplex Ligation‐dependent Probe Amplification (MLPA) to investigate a spectrum of 331 diffuse gliomas for copy number changes that are most frequently detected in these tumors, that is, involving chromosome arm 1p and/or 19q, CDKN2A, PTEN and EGFR (including EGFR rearrangements, EGFRΔ). In contrast to commonly performed loss of heterozygosity (LOH)‐analysis, MLPA analysis allows for distinguishing between hemizygous and homozygous losses, as well as between low‐level gains, amplifications and HC‐amplifications. Thereby, this analysis increases our knowledge on the molecular underpinnings of oncogenesis and (malignant) progression of gliomas. Specifically, the evaluation of the co‐occurrence and the interrelation of the aberrations identified allowed us to build a model for the timing of these events during molecular progression of diffuse gliomas. IDH1 and TP53 mutation analysis was performed and correlated with malignant progression at the molecular level. For further evaluation of the model, we not only performed parallel analysis of 36 primary tumors and their 39 recurrences but also investigated its prognostic significance in a group of patients with anaplastic (WHO grade III) gliomas.

MATERIALS AND METHODS

Tumor samples

Diffuse glioma samples for this study were retrieved from the neuro‐oncology archive at the Department of Pathology of the Radboud University Nijmegen Medical Centre and the Academic Medical Center, Amsterdam, the Netherlands. The use of brain tumor tissue after completing histopathological diagnosis for research purposes has been approved by the regional ethics committee. The investigated tumors (n = 331) were classified and graded according to the WHO‐2007 classification as diffuse astrocytoma (A‐II; n = 20), anaplastic astrocytoma (A‐III; n = 7), GBM (n = 209), oligodendroglioma (O‐II; n = 21), anaplastic oligodendroglioma (O‐III; n = 26), oligoastrocytoma (OA‐II; n = 13), anaplastic oligoastrocytoma (OA‐III; n = 8) or GBMO (n = 27) (34). Following the WHO‐2007 classification, anaplastic oligoastrocytic tumors with necrosis that were previously (and according to the WHO‐2000 classification) diagnosed as OA‐III were now designated as GBMO. In this initial group of gliomas only a single glioma sample of each patient was included.

Overall, 313 cases were evaluated using MLPA assay P088 (27) (1p/19q; described below), whereas 329 were evaluated using MLPA assay P105 26, 28 (PTEN, CDKN2A and EGFR rearrangements including EGFRvIII). Furthermore, the majority of the samples (n = 238) were sequenced for hotspot IDH1 mutations in exon 4 and/or mutations in exon 5–8 of TP53 as described previously 4, 10, 37. Overall, 16 and 12 A‐IIs, 4 and 3 A‐IIIs, 142 and 101 GBMs, 17 and 16 O‐IIs, 19 and 17 O‐IIIs, 11 and 9 OA‐IIs, 7 and 6 OA‐IIIs, and 21 and 17 GBMOs were analyzed for IDH1 and TP53 mutations, respectively.

To genetically validate the model proposed for molecular progression of gliomas, an additional 39 diffuse glioma specimens were analyzed in 36 patients of which both the original (n = 36) and recurrent tumor tissue (n = 39) was available (10 A‐IIs, 11 GBMs, 7 O‐IIs, 3 O‐IIIs, 7 OA‐IIs and 1 OA‐III) of which 35 were also analyzed for TP53 and IDH1. Furthermore, as a proof of principle, a pilot study evaluating the prognostic meaning of detecting molecular malignancy as proposed was conducted for anaplastic gliomas (n = 41). Overall survival was measured after surgery for the analyzed biopsy and MedCalc statistical software (Mariakerke, Belgium) was used for analysis.

MLPA

DNA was isolated from routinely processed, formalin fixed, paraffin embedded tumor samples as well as from snap frozen tumor tissue using the DNeasy Tissue Kit (Qiagen, Venlo, the Netherlands) as described previously (27). MLPA analysis was used to detect copy number changes of multiple loci simultaneously ( http://www.mlpa.com) (43) and all assays used were prepared by MRC‐Holland (Amsterdam, the Netherlands). MLPA assay P088 (lot nr 0804, 0305, 0706 or 0608) was used to detect complete or partial losses involving chromosome 1p (15–16 probes depending on lot nr) and 19q (8 probes), whereas MLPA assay P105 (lot nr 0306, 0407 or 1008) was used to detect copy number changes in the genes CDKN2A (5 probes), PTEN (10–11 probes) and EGFR (11 probes), and identification of EGFR rearrangements (EGFRΔ), for example, EGFRvIII. Copy number analysis for TP53 (8 probes included in P105) was not performed as TP53 LOH in gliomas is associated with mitotic recombination resulting in a copy number neutral event (47). As described previously, we fully validated the sensitivity and specificity of these MLPA assays 27, 28. Part of the samples analyzed in the present study was also included in these previous studies.

MLPA was performed as described by the manufacturer with minor modifications and data analysis was performed in Excel (Microsoft Office, Microsoft Corporation, Redwood, WA, USA), both described in more detail previously (27). MLPA copy number detection thresholds were set at 1.2 and 0.8 for the detection of low‐level gains and hemizygous losses, respectively (27). Furthermore, ratios below 0.4 were considered homozygous losses, ratios above 2.0 as amplifications and those exceeding 10 as high copy number amplifications (HC‐amplification). As described previously, we identified EGFRvIII by assessing the average ratio for exon 2–7 probes and comparing this with the average ratio of probes for exons 1, 8, 13, 17 and 22 (EGFRvIII ratio). EGFRvIII ratios below 0.8 were considered to harbor the EGFRvIII deletion variant (28). Additionally, we inspected the individual probe ratios in order to confirm the presence of EGFRvIII and/or to identify other EGFRΔ as indicated by a clear increase or decrease of the ratios identified by repeated experiments and confirmed using MLPA assay P315 evaluating all EGFR exons. For chromosome 1p and 19q losses, a distinction was made between complete and partial losses, the latter were defined as a ratio <0.8 for at least 3 adjacent probes but not of all probes for these chromosome arms.

RESULTS

Copy number changes involving CDKN2A, PTEN, EGFR, 1p and 19q

An overview of the distribution of copy number changes of these genes and chromosome arms in the different glioma types and grades is shown in Figure 1. Both CDKN2A and PTEN losses were detected with increasing frequency in gliomas of a higher malignancy grade. Interestingly, while in low‐grade gliomas the majority of CDKN2A losses consisted of hemizygous losses, homozygous losses prevailed in grade IV lesions. Homozygous PTEN losses also increased with malignancy grade, but are less common compared with CDKN2A. Additional copies of EGFR (n = 183) were mainly detected in GBM(O) whereas the remaining cases (<10%) were diagnosed as anaplastic glioma or occasionally as A‐II. Rearrangements of EGFR (EGFRΔ, n = 39) mainly encompassed EGFRvIII (n = 28) and were detected only in gliomas with additional EGFR copy numbers, most frequently co‐occurring with (HC‐)amplifications (37/39 cases) suggesting that gene rearrangement takes place during gene amplification as a late event in glioma oncogenesis.

Figure 1.

Figure 1

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Distribution of copy number changes in diffuse gliomas. The distribution is shown for the different malignancy grades (II, III or IV) of astrocytic tumors (As), oligoastrocytic tumors (OAs) and oligodendroglial tumors (Os). The cases were evaluated for IDH1 and TP53 mutations (wt = wild type, mut = mutant) and copy number changes involving 1p/19q, CDKN2A, PTEN and EGFR status using Multiplex Ligation‐dependent Probe Amplification (− = loss, + = gain, comp or part = complete or partial chromosome arm lost), each represented in a specific color (orange, purple, blue, red and green, respectively). An increase in color saturation indicates a more malignant character of the aberration [IDH1/TP53: mut/wt < mut/mut < wt/mut; 1p/19q: complete co‐deletion < isolated (complete or partial) loss or a combined partial loss involving 1p/19q of which at least one is a partial loss < gain involving 19(q); CDKN2A and PTEN: hemizygous < homozygous loss; EGFR: low‐level gain < amplification < high‐copy (HC) amplification]. Red bars on the green EGFR column represent the occurrence of EGFRΔ (72% of these representing EGFRvIII). Number of cases investigated for, respectively, IDH1, TP53, 1p/19q and CDKN2A/PTEN/EGFR analysis encompassed: 16, 12, 19 and 20 A‐IIs; 4, 3, 7 and 7 A‐IIIs; 142, 101, 195 and 207 glioblastomas (GBMs); 11, 9, 13 and 13 OA‐IIs; 7, 6, 8 and 8 OA‐IIIs, 21, 17, 25, 27 GBMs with oligodendroglial differentiation (GBMOs); 17, 16, 20 and 21 O‐IIs; 19, 17, 26 and 26 O‐IIIs.

More than half of the gliomas investigated contained no losses of either 1p or 19q (∼60%). Complete 1p or 19q losses were detected in approximately 20% of the gliomas in our series whereas partial losses were detected less frequently (<15%). As expected, complete 1p and 19q losses were mainly present in Os (up to 80%), usually presenting as a co‐deletion affecting both complete chromosome arms (Figure 1), and less frequently in OAs. Gliomas containing a partial 1p or 19q loss may still show an additional loss on the other chromosome arm (either complete or partial), this type of combined partial 1p/19q loss was considered as distinct from the complete co‐deletion. Finally, some gliomas may contain an isolated loss (complete or partial) involving either 1p or 19q, this was detected more frequently in OAs and As than Os. Gains involving 1p or 19q (complete or partial) were less frequent and were identified in only 5% and 6% of the Os, 11% and 6% of the OAs, and 6% and 22% of the As, respectively. Most gliomas showing a gain involving 19q (n = 55) were high‐grade malignant (93%), showed gain of the adjacent 19p region as well (75%) and lacked 1p loss (82%) (Figure 1).

IDH1 and TP53 mutation analysis

IDH1 and TP53 mutations were evaluated in a random subset of the cases screened for copy number changes and frequencies detected are summarized in Figure 1. IDH1 mutations are commonly detected in grade II and III gliomas (65%–90% in different categories) and less frequently in GBM and GBMO (15% and 25% of the cases, respectively) whereas TP53 mutations are most frequently detected in OA‐II/III (approximately 75%), less frequently in A‐II/III/IV and O‐IIs (25%–40%) and occasionally in O‐IIIs and GBMOs (6%–12%). In grade II/III gliomas the majority of the TP53 mutations occur in the presence of an IDH1 mutation (As > Os > OAs) whereas in grade IV gliomas only half (GBMO) or less than half (GBM) of the TP53 mutations occur in the presence of an IDH1 mutation. Interestingly, of the cases with a 1p/19q co‐deletion that were sequenced for both IDH1 and TP53, 74% harbored an IDH1 mutation in the presence of a TP53 wild type (25/34).

A model for molecular progression in gliomas

Evaluating the co‐occurrence of the detected copy number changes (simplified overviews in 2, 3) and their interrelation, we build a model incorporating information on the nature and timing of these events (Figure 4). Our results suggest that for both PTEN and CDKN2A, the initial hemizygous loss can evolve into homozygous loss during molecular progression. Furthermore, as hemizygous loss of PTEN (152 cases) can be detected next to a hemizygous or homozygous CDKN2A loss (23% and 51%, respectively) whereas homozygous PTEN losses (13 cases) are mainly associated with homozygous CDKN2A losses (70%), we propose that CDKN2A is generally lost prior to PTEN alterations. As gliomas without EGFR copy number changes may already harbor CDKN2A losses (34% of the cases, one‐third of these already being homozygously deleted) and/or PTEN losses (25% of the cases, only 1.5% being a homozygous deletion), these losses seem to occur most often prior to EGFR copy number changes. The detected correlation between increasing copy numbers of EGFR during molecular progression clearly correlated with the other copy number changes investigated; EGFR gains coincide with deletions involving CDKN2A and PTEN in approximately 60% of the cases each, whereas cases with (HC‐) amplifications of EGFR most frequently show co‐occurrence of homo‐ or hemizygous loss of CDKN2A (18% and 65%) or PTEN (75% and 8%). Concordantly, EGFRΔ (39 cases) usually (37/39) coincides with loss of CDKN2A (33, of which 27 homozygous) and/or PTEN (35, of which 5 homozygous). No differences were detected in this distribution among cases showing EGFRvIII (72% of EGFRΔ) and those with other EGFR rearrangements. Finally, as HC‐amplification of EGFR, either with or without EGFR rearrangements, is associated with a hemizygous rather than with a homozygous loss of PTEN, the latter may signify an end‐stage of molecular progression of diffuse gliomas.

Figure 2.

Figure 2

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Co‐occurrence of 1p/19q loss, EGFR gain/amplification, CDKN2A loss and PTEN loss in a spectrum of diffuse gliomas. The (co‐)occurrence of EGFR (green), CDKN2A (blue) and PTEN (red) is shown for gliomas grade II, III and IV, separately. In this figure, next to information on the copy number changes in the group as a whole (all cases), the frequency of such changes is given in subgroups as defined by their 1p/19q status (see Results section for definitions of the different categories of 1p/19q copy number changes). The number of cases investigated and the number of cases containing a specific aberration is shown. The size of the circles reflects the relative number of cases showing the corresponding aberration whereas the overlap of circles reflects their co‐occurrence (frequency also indicated by percentages in the different segments of the circles).

Figure 3.

Figure 3

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Co‐occurrence of IDH1 and TP53 mutation status, EGFR gain/amplification, CDKN2A loss and PTEN loss in a spectrum of diffuse gliomas. The (co‐)occurrence of EGFR (green), CDKN2A (blue) and PTEN (red) is shown for gliomas grade II, III and IV, separately. The frequencies are shown for subgroups as defined by their IDH1/TP53 status (wt = wild type, mut = mutant). Frequencies were not shown for groups smaller than five cases (gliomas‐II and gliomas‐III IDH wt/TP53 mut). The number of cases investigated and the number of cases containing a specific aberration is shown. The size of the circles reflects the relative number of cases showing the corresponding aberration whereas the overlap of circles reflects their co‐occurrence (frequency also indicated by percentages in the different segments of the circles).

Figure 4.

Figure 4

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Simplified scheme of the proposed timing and nature of chromosomal copy number changes during molecular progression of diffuse gliomas. A. IDH1 and TP53 mutations as well as a combined and complete hemizygous loss of chromosome arms 1p and 19q (1p/19q co‐deletion) are early events in glioma oncogenesis, IDH1 now being considered as an event preceding TP53 mutations and 1p/19q co‐deletion. B. Evaluating copy number changes of chromosome 1p and 19q, CDKN2A, PTEN and EGFR, as well as EGFR rearrangements including EGFRvIII, in a spectrum of 331 diffuse gliomas encompassing the different histopathological subtypes provides clues about the frequency and “timing” of molecular events in the course of tumor progression. The correlation between the degree of molecular progression and the identified early events is indicated by the order in which the latter are shown whereas the correlation with histopathology is indicated as well. For example, high‐grade cases with an IDH1‐mut/TP53‐wt/1p/19q co‐deletion cases are common among (pure) oligodendroglial cases and can additionally harbor losses of CDKN2A and/or PTEN, occasionally an EGFR low‐level gain whereas EGFR (HC‐)amplification or EGFR gene rearrangements are (relatively) rare in this group. Vice versa, IDH‐wt/TP53‐wt/no. 1p/19q co‐deletion cases generally have an astrocytic phenotype and often harbor multiple other aberrations including isolated (complete or partial) loss of 1p or 19q, or a combined partial loss (the partial loss involving at least one chromosome arm), 19q gains and EGFR amplification and rearrangements, reflecting the highest degree of molecular progression. Of note: in this model homozygous deletion of PTEN is a very late event, however, biallelic activation of PTEN can occur more early through, for example, a hemizygous loss in combination with an inactivating mutation as is frequently detected for PTEN. Abbreviations: mut = mutant; wt = wild type.

A complete co‐deletion of 1p/19q occurred as an early event in Os and part of the OAs (Figure 1). Interestingly, some of these tumors additionally harbor a CDKN2A loss (hemizygous > homozygous loss), PTEN loss or EGFR gain. Frequencies as well as co‐occurrence increases with malignancy grade which suggests that these aberrations are also involved in the malignant molecular progression of gliomas with a complete 1p/19q co‐deletion (Figure 2). Importantly, gliomas showing “other” 1p/19q losses either as an isolated loss (complete or partial; “isolated”, n = 50) or a combined loss on 1p and 19q at least one being a partial loss (“combined partial”, n = 21) were associated with malignant (oligo‐)astrocytic tumors (Figure 1). Compared with cases with a complete 1p/19q co‐deletion, such “other” 1p/19q losses containing cases more frequently showed a CDKN2A loss (homozygous > hemizygous), a PTEN loss or an increase of EGFR (gains > (HC‐)amplifications > EGFRΔ), often occurring in combination (Figure 2). These “other” 1p/19q losses thus signify molecular progression of gliomas. Finally, gliomas with a gain involving chromosome 19q commonly (82%) showed multiple additional changes such as homo‐ or hemizygous CDKN2A loss (47% and 27%), hemizygous PTEN loss (71%), and gains or (HC‐)amplification of EGFR (29% and 50%) or even the presence of EGFRΔ (18%). This co‐occurrence suggests that a gain of 19q (either complete or partial; approximately 1:2) signifies advanced molecular progression, that is, molecular malignancy in diffuse gliomas.

The relation between the IDH1/TP53 mutation status and the frequencies and co‐occurrence of the copy number changes is summarized in Figure 3. Whereas in grade II and III cases the majority (70%) contained an IDH1 mutation, either in the presence of TP53 wild type or a TP53 mutation (II: 40% and 31%; III: 52% and 20%, respectively), grade IV gliomas mainly had wild‐type IDH1 (82%) in the presence of a wild‐type TP53 (61%). Frequencies of copy number changes as well as their co‐occurrence increases with malignancy grade whereas in grade III and IV gliomas an increase was also detected from IDH1mutant/TP53 wild type, IDH1mutant/TP53 mutant, IDH1wild type/TP53 mutant to IDH1wild type/TP53 wild type cases (3, 4). Interestingly, evaluating molecular progression in tumors with a specific IDH1/TP53 status, we noticed that cases with an IDH1 mutation and TP53 wild type (n = 32) most frequently were grade II (44%) or III (40%) and only occasionally grade IV (16%) and infrequently showed additional copy number changes. This may suggest that in these cases molecular progression occurs less easily and/or remains less pronounced. In cases with both an IDH1 mutation and a TP53 mutation (n = 32) half of the cases are grade IV, the majority (82%) containing progression associated aberrations, however, only occasionally involving CDKN2A, PTEN and EGFR together. Malignant progression is even more pronounced in cases with an IDH1 wild type, 89% of the IDH1 wild type/TP53 mutant cases (n = 27) and 84% of the IDH1 wild type/TP53 wild type cases (n = 86) being grade IV, the latter showing the most pronounced degree of molecular progression, that is, 63% containing all three aberrations as opposed to 38% in IDH1 wild type/TP53 mutant cases. As aberrations more or less frequently (co‐)occur, the proposed model is applicable to the different IDH1/TP53 types of gliomas which may show molecular malignancy more or less pronounced.

Validity of the model for molecular progression in gliomas

The validity of the proposed model for molecular glioma progression was evaluated by parallel analysis of primary tumors (n = 36) and their recurrences (n = 39) as summarized in Table 1. Aberrations associated with molecular progression emerged in 28 recurrences and was accompanied by an increased histopathological malignancy grade in half of the cases. Interestingly, however, in the 23 recurrences without histopathological evidence for increased malignancy (including 11 cases that already showed the highest WHO grade in the primary tumor), 14 (including 2 O‐IIIs and 3 GBMs) acquired copy number changes indicating further molecular progression.

Table 1.

Copy number changes involved in molecular progression as detected in primary gliomas and their recurrence(s). Abbreviations: WHO = World Health Organization; A = astrocytoma; OA = oligoastrocytoma; O = oligodendroglioma; GBM = glioblastoma; M = Male; F = Female.

Set Patient Tumor‐ID WHO Interval IDH1 TP53 1p 19q CDKN2A PTEN EGFR EGFRvIII
 1) M,35 N872* A‐II mut +
N873 A‐II 17 mut −part − − +
 2) F,22 N871 A‐II mut mut
N050* OA‐II 59 −part −part −
 3) F,49 N260* A‐II mut mut − −part
N123 OA‐II 61 mut mut −part
+part −− +
 4) M,39 N897 A‐II mut mut
N175* OA‐II 48 mut mut −
 5) F,28 N281* OA‐II mut mut −
N375 OA‐II 24 mut mut −
 6) F,22 N174* OA‐II −
N310 OA‐II 14 −
 7) M,37 N866* O‐II − −
N449 O‐II 35 mut wt − − −− +
 8) F,34 N868 O‐II mut wt − −
N676* O‐II 35 mut wt − −
 9) F,37 N858* O‐II mut mut − −
N859 O‐II 11 mut mut − − − −
10) M,37 N472* O‐III mut wt − − +
N869 O‐III 32 mut wt − − − +
11) M,60 N107* O‐III mut wt − −
N293 O‐III 47 mut wt − − +
12) F,44 N880 GBM wt wt + −− −− +++ +
N881* GBM 14 wt wt + −− −− +++# +
13) M,47 N852* GBM wt wt + − − +++
N853 GBM 10 wt wt − − +++
14) M,52 N885* GBM wt wt + −− −− ++
N886 GBM 21 wt wt + −− − +++
15) F,50 N878* GBM wt wt −part − ++
N879† GBM 17 wt wt −part +
16) M,27 N870 GBM mut mut −part −part −
N436* GBM 109 −
17) F,39 A001* GBM mut mut
A016 GBM mut mut −
18) M,40 A105* GBM wt wt −− − +
A106 GBM wt wt −− − +
19) F,39 A107* GBM wt wt +part −− − +++
A108 GBM wt wt +part −− − +++
20) M,26 A115* GBM wt mut
A002 GBM wt mut
21) M,66 N306 A‐II wt wt
N124* OA‐III 60 wt wt −part
+part
22) F,53 N898 O‐II wt wt
N899* O‐III 6 wt wt +
23) M,34 N047* O‐II mut wt − −
N560 OA‐III 113 mut wt − −
24) M,26 N036 O‐II mut mut −part
N162* OA‐III 28 mut mut −part −
25) M,30 N079* O‐II mut wt − −
N867 O‐III 70 mut wt − − −
26) M,53 N856 OA‐II mut wt − −
N678* O‐III 59 mut wt − − −
27) F,23 N035 O‐II mut wt −part −part
N182* OA‐III 29 mut wt −part −part +
28) M,39 N168 O‐II mut wt − −
N862* OA‐III 86 mut wt − − −
29) M,54 N854 A‐II mut mut −
N855* A‐III 34 mut mut −part −− −
30) M,57 N850 A‐II mut wt −
N851* GBM 10 mut wt −part −part −−
31) M,48 N167* OA‐II mut wt − −
N857 GBM 56 mut wt# − − −− −
32) M,21 N887 A‐II mut
N888* GBM 27 mut −part
33) F,30 N863 A‐II mut mut
N864* GBM 38 mut mut −− −
34) F,39 N861 A‐II mut mut −
N860 OA‐II 90 mut mut − −
N901* OA‐III 3 mut mut − −−
35) F,26 N211* A‐II mut wt +
N874 A‐II 18 mut wt +
N423 GBM 23 − +
36) F,34 N902 OA‐II mut mut
N462 OA‐II 93 mut mut −
N865* O‐III 14 mut mut −−
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*

Tumors that were included in the first set of gliomas analyzed.

†

The presence of preexistent normal brain tissue is mentioned in the pathology report. Age at surgery in years and sex (F for female, M for male) are indicated for each set. IDH1 and TP53 status is indicated as mutant (mut) or wild type (wt), an additional # indicating a case in which exon 6 could not reliably be analyzed. Copy number changes are semi‐quantitatively scored as hemizygous (−) or homozygous loss (−−), a partial hemizygous loss (−part), a low‐level gain (+), a partial low‐level gain (+part), amplification (++), or high‐copy amplification (+++), whereas an additional # indicates a clear increase in EGFR copy number, even though both are classified as +++. The interval is indicated in months. For set 17–20 the exact interval is unknown, but they represent recurrences instead of re‐operation samples.

Moreover, evaluation of the actual changes in copy number detected between the first biopsies of gliomas and their matching recurrences corroborated the model build for molecular progression, that is, molecular malignancy. Events considered to be an early aberration in glioma oncogenesis like IDH1 mutation, TP53 mutation and a co‐deletion of the complete 1p/19q were always identified in both the original tumor and its recurrence. In contrast, changes in copy number involving CDKN2A, PTEN, EGFR and other 1p/19q alterations generally emerged in the matched recurrences. Hemizygous and homozygous loss of CDKN2A (8 and 7) and PTEN (6 and 1) were newly identified in the recurrence, whereas for both genes a transition from a hemizygous into a homozygous loss was also detected (2 and 1 cases, respectively). EGFR copy number changes were newly identified in the recurrences (n = 5) and a further increase in copy number was also detected in some cases (n = 2). Finally, isolated and/or partial 1p/19q losses (n = 10) or 19q gains (n = 2) were newly identified in recurrent tumors confirming their involvement in glioma progression. In a few cases (n = 5), previously identified aberrations were absent (case 3, 13, 15 and 16) or less severe (case 14 and 15) which may result from tumor heterogeneity or a decreased tumor load in the specimen (the latter being histologically proven in the recurrence of case 15).

For a “proof of principle” testing of the potential clinical relevance of detecting molecular malignancy according to the model proposed, we choose to analyze anaplastic gliomas (n = 41), also because in these WHO grade III gliomas survival is often highly variable. Evaluating the individual aberrations, a significant correlation was detected between the different types of copy number changes identified and survival (Figure 5A–D). Moreover, an overall picture of the severity of molecular progression was created by calculating the sum of points given to the specific aberrations (eg, normal = 0, hemizygous loss = 1, homozygous loss = 2, see Figure 5E). In this group molecular malignancy as calculated using our model significantly correlated with overall survival (Figure 5F).

Figure 5.

Figure 5

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Survival analysis of patients with anaplastic gliomas. Overall, 41 patients were analyzed and the prognostic value on survival was evaluated for the different types of CDKN2A, PTEN, EGFR and 1p/19q copy number aberrations. Different types of copy number changes of individual genes already significantly correlate with survival (A–D), survival given in days on the x‐axis (see Results section for definition of the different types of 1p/19q copy number changes). To evaluate the prognostic value of the model, we propose for assessment of malignant molecular progression, points were assigned to each aberration as indicated (E) and the resulting scores of the tumors showed a strongly significant correlation with survival (F).

DISCUSSION

Quite a number of studies investigated (individual) aberrations in different types and grades of gliomas, and in fact this information is incorporated in the current WHO‐2007 classification 6, 12, 13, 42, 45, except for the more recently identified IDH1 mutations 1, 4, 10, 15, 38, 49, 50. The frequencies described in those earlier studies are in concordance with those detected in the current study. Combinations of aberrations are less frequently studied 14, 36, 45 and especially evaluation of the interrelations between the aberrations involved in such a co‐occurrence allowed us to build a model for the timing and nature of copy number changes in the course of molecular (malignant) progression for the spectrum of diffuse gliomas (Figure 4). Not only can certain aberrations acquire a more malignant character during malignant progression (eg, hemizygous vs. homozygous loss; gain vs. (HC‐)amplification), but also the co‐occurrence of such aberrations can increase. It is important to realize that the functional consequences of the gains/losses depicted in our model for molecular progression of gliomas (Figure 4) may also occur as a result of (combinations of) other (epi)genetic aberrations; for instance, complete loss of function of PTEN is described to frequently occur in gliomas via a combination of hemizygous loss and mutation of the remaining PTEN gene (34). Our subsequent parallel analysis of primary tumors and their recurrence(s) as well as previous reports 14, 23, 36 corroborate the proposed scheme. As discussed in more detail below, such a model can be of additional clinical value next to the histopathological classification by allowing identification of so far unexpected (aggressive) tumor behavior.

Clinical relevance of identifying molecular malignancy

The impact of assessment of molecular malignancy in diffuse gliomas is underlined by our observation that signs of molecular progression in the form of genetic aberrations as evaluated and described n the current paper may already be present prior to histopathological features of high‐grade malignancy (detected in one‐third of the grade II lesions). Moreover, we detected molecular progression in 28 of the 39 recurrent cases, whereas in half of these cases this did not coincide with increased histopathological malignancy suggesting that these tumors may actually be further progressed than shown by their histopathological features (n = 9). In fact, when already showing the highest histopathological malignancy grade, progression into an even more malignant tumor can occur at the molecular level (n = 5).

The complete 1p/19q co‐deletion, which is considered to occur as an early event in the oncogenesis of (oligodendro)glial tumors (Os > OAs) and is indicative for prolonged survival and therapy response, has been suggested to be mutually exclusive with PTEN and EGFR alterations 17, 19, 21, 22, 23, 24. However, we clearly show that these aberrations can co‐occur, underlining the relevance of the model proposed for molecular progression for this specific genetic subtype of (oligodendro)gliomas 19, 24, 25, 27. Previous studies also reported that loss of CDKN2A or PTEN and gain of EGFR next to a 1p/19q co‐deletion is indicative for decreased survival and duration of response to therapy (34).

Interestingly, the prognostic relevance of detecting molecular progression is also indicated by the inverse correlation between the extent of molecular malignancy (O‐IIIs < OA‐IIIs < A‐IIIs; GBMO < GBM) and historical overall survival for these glioma categories (O‐IIIs > OA‐IIIs > A‐IIIs; GBMO > GBM) 2, 3, 5, 11, 19, 20, 44, 46. Similarly, molecular malignancy in high‐grade gliomas with the clinically favorable IDH1 mutation is less pronounced than in gliomas with an IDH1 wild type. This may suggest that, rather than the presence of the IDH1 mutation itself, the absence of molecular progression explains the prolonged survival.

Although detailed exploration of the correlation between (a combination of) specific aberrations and different clinical aspects is beyond the scope of the current study, we investigated the prognostic value of assessing molecular malignancy as a proof of principle in a group of anaplastic gliomas. Unequivocal grading (ie, delineation from WHO grade II lesions on the one hand and grade IV lesions on the other) can be difficult and consequently, the survival of patients with an anaplastic glioma is highly variable. The significant correlation between particular copy number changes and prognosis became even more significant and effective when molecular malignancy was evaluated in a comprehensive way as described in our model. The exact clinical impact of this analysis, however, needs to be further investigated for the complete spectrum of gliomas as well as related to clinical aspects such as treatment protocols.

Among GBMs, secondary GBMs are known to carry a somewhat better prognosis than de novo GBMs. Although we do not have the clinical information available yet for the cases investigated, our results (Figure 3) suggest that IDH1 mutation analysis allows for identification of the most favorable GBMs (ie, lowest degree of molecular malignancy). The fact that those mutations commonly occur in cases of a lower malignancy grade support the idea that a GBM with an IDH1 mutation most likely progressed from such a lower grade lesion (ie, secondary GBMs). TP53 mutations have previously been suggested as indicative for secondary GBMs (34). The fact that TP53 mutant cases also showing an IDH1 mutation display a lower degree of molecular progression than TP53 mutant cases harboring IDH1 wild type may explain the variation in survival among (secondary) GBMs and underline that IDH1 mutations might be a better marker for the identification of secondary GBMs than TP53. Moreover, TP53 mutant/IDH1 wild type cases are only occasionally low‐grade (3/25) supporting the idea that such GBMs actually occur de novo. Using this approach, approximately one‐fifth of the GBMs investigated in the present study is expected to represent secondary GBMs.

Overall, molecular analysis as proposed may thus allow for identification of molecular malignancy, an aspect that remains undetected by histopathological evaluation alone. For example, absence of molecular progression may be of clinical relevance in predicting prolonged survival provided that tumor‐load and sampling of the analyzed fragment were adequate. Vice versa, when samples that histopathologically not yet show malignant progression but do show pronounced molecular malignancy, patients can be expected to show decreased survival.

Clinical importance of detecting the different types of 1p/19q aberrations

Identification of 1p loss is advocated as a valuable molecular diagnostic tool in diffuse gliomas as this, especially when combined with a loss of 19q, has been described to be indicative for prolonged survival and therapy response 16, 19, 20, 48. The techniques used and the locus/loci investigated, however, differ among institutions. As clearly shown in our present study and suggested previously, different types of 1p/19q copy number changes exist with opposite clinical implications. As opposed to the most commonly detected complete co‐deletion of 1p/19q, “other” 1p/19q copy number changes exist that encompass an isolated loss on 1p or 19q (either partial or complete), a combined partial 1p/19q loss (at least one of these losses involving only part of the chromosome arm), as well as a 19(q) gain. Our present data clearly show that these “other” 1p/19q copy number changes occur during molecular malignant progression of gliomas and are thus indicative for molecular malignancy and more aggressive tumor behavior. As commonly used techniques do not allow this distinction, including “other” 1p/19q aberrations most likely will (falsely) decrease the clinical impact of 1p/19q analysis. This is underlined by our observation that these “other” 1p/19q aberrations are mainly detected in OAs and As, whereas 1p/19q analysis has been reported to be especially clinically relevant for pure Os as opposed to OAs and As.

From a molecular diagnostic point of view, this observation raises some important issues. First, evaluation of both 1p and 19q by testing only a limited set of loci, as, for example, in 1p36 and 19q13.3 LOH or FISH (fluorescent in situ hybridization) analysis, may not allow for a distinction between complete 1p/19q co‐deletion and combined partial 1p/19q losses. Furthermore, analysis of (multiple) 1p loci spanning the chromosome arm (allowing detection of partial vs. complete 1p losses) without 19q evaluation does not allow for a distinction of the complete 1p/19q co‐deletion and an isolated complete 1p loss or a combination of complete 1p loss and partial 19q loss. Finally, the widely used LOH analysis is based on the identification of an allelic imbalance which can result from either an allelic loss or gain and does therefore not allow for an accurate distinction between a 19(q) gain or loss. Based on the current results, our recommendation for a more accurate molecular diagnostic analysis would be to evaluate multiple loci spanning both 1p and 19q, the spacing and number of loci investigated reflecting the sensitivity and specificity of the assay. Furthermore, parallel analysis of molecular markers indicating molecular malignancy as described in this study is expected to improve assessment of the biological behavior of the tumor even further.

Future perspectives

The identification of molecular progression as we propose here shows a clear correlation with the historical overall survival data. Moreover, the actual prognostic value was confirmed for anaplastic gliomas as a proof of principle. The exact prognostic relevance for the complete spectrum of diffuse gliomas requires extensive further evaluation of clinical records. The proposed model can be extended by including information on other (epi)genetic aberrations, especially of those genes already reported to be involved glioma progression and indicative of poor prognosis (eg, RB1, MDM2 and CDK4). Ultimately, such efforts should result in a model that allows for the establishment of a molecular risk profile that carries important prognostic information and is helpful for therapeutic decision making for individual glioma patients.

ACKNOWLEDGMENTS

This project was sponsored by the Dutch Cancer Society (KWF: KUN‐2008‐4214) and STOPhersentumoren.nl.. We thank Marjolijn Klomp, Martijn Kramer, Lonneke Wigman and Marieke Dekkers for their valuable contribution to the MLPA analysis.

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