Abstract
Background
Astrocytoma of the isocitrate dehydrogenase (IDH) wild-type gene is described as a provisional entity within the new World Health Organization (WHO) classification. Some groups believe that IDH wild-type lower-grade gliomas, when interrogated for other biomarkers, will mostly turn out to be glioblastoma. We hypothesize that not all IDH wild-type lower-grade gliomas have very poor outcomes and the group could be substratified prognostically.
Methods
Seven hundred and eighteen adult WHO grades II and III patients with gliomas from our hospitals were re-reviewed and tested for IDH1/2 mutations. One hundred and sixty-six patients with IDH wild-type cases were identified for further studies, and EGFR and MYB amplifications, mutations of histone H3F3A, TERT promoter (TERTp), and BRAF were examined.
Results
EGFR amplification, BRAF, and H3F3A mutations were observed in 13.8%, 6.9%, and 9.5% of patients, respectively, in a mutually exclusive pattern in IDH wild-type lower-grade gliomas. TERTp mutations were detected in 26.8% of cases. Favorable outcome was observed in patients with young age, oligodendroglial phenotype, and grade II histology. Independent adverse prognostic values of older age, nontotal resection, grade III histology, EGFR amplification, and H3F3A mutation were confirmed by multivariable analysis. Tumors were further classified into “molecularly” high grade (harboring EGFR, H3F3A, or TERTp) (median overall survival = 1.23 y) and lower grade (lacking all of the 3) (median overall survival = 7.63 y) with independent prognostic relevance. The most favorable survival was noted in molecularly lower-grade gliomas with MYB amplification.
Conclusion
Adult IDH wild-type lower-grade gliomas are prognostically heterogeneous and do not have uniformly poor prognosis. Clinical information and additional markers, including MYB, EGFR, TERTp, and H3F3A, should be examined to delineate discrete favorable and unfavorable prognostic groups.
Keywords: glioma, IDH wild-type, molecular grade, MYB amplification, prognosis
Importance of the study.
IDH mutation has been applied as a major classifier in the 2016 WHO classification of diffuse gliomas. Wild-type tumors have worse prognosis and thus are regarded as unrecognized glioblastomas. Based on EGFR amplification, TERTp, and H3F3A mutations, we stratified IDH wild-type grades II/III gliomas into “molecularly” high grade (with any of the 3 biomarkers) and lower grade (lacking all) with clinical relevance. Molecularly high-grade tumors exhibited glioblastoma-equivalent outcome, suggesting the need for more aggressive clinical intervention. Molecularly lower-grade tumors showed a better prognosis compatible with a clinically low-grade diagnosis despite a genetic background of wild-type IDH. The most favorable outcome was observed in molecularly lower-grade tumors with MYB amplification, providing evidence that not all IDH wild-type gliomas have uniformly poor prognoses. Our findings highlight the prognostic heterogeneity of IDH wild-type lower-grade gliomas and contribute to the refinement of substratification of this newly established WHO entity.
Grading of gliomas traditionally relies on morphology and immunohistochemical evaluation.1,2 Maximum safe resection followed by chemo- or radiotherapy remains the standard treatment.3,4 In the new classification by the World Health Organization (WHO), mutations in isocitrate dehydrogenase (IDH) 1 and 2 are key genetic events, not only in glioblastomas, but also in adult lower-grade gliomas. Mutations can be found in more than 70% of WHO grades II and III gliomas and predict a more favorable prognosis.5 On the contrary, IDH wild-type patients usually have dismal clinical outcomes even when the tumors are histologically grade II or III.5–7 Moreover, a recent study of IDH wild-type lower-grade gliomas suggested that the majority of IDH wild-type low-grade gliomas were actually unrecognized glioblastomas.8
Our previous studies have shown that IDH status alone does not supersede WHO histological grades for prognostication in lower-grade gliomas, and IDH wild-type lower-grade gliomas do not have a universally poor survival.9,10 In this study, we screened the IDH mutation status in 718 grades II and III diffuse gliomas, and 166 IDH wild-type tumors were so identified and further studied. Also examined were mutation status of the telomerase reverse transcriptase promoter gene (TERTp), BRAF, and H3F3A and amplification status of the epidermal growth factor receptor gene (EGFR) as well as v-Myb avian myeloblastosis viral oncogene homolog (MYB).
Materials and Methods
Patients, Tumor Tissues, and Clinical Data
A total of 718 adult patients with lower-grade gliomas (WHO grade II and grade III in the 2007 WHO classification) diagnosed between 1994 and 2014 in Prince of Wales Hospital (Hong Kong) and Huashan Hospital (Shanghai) were reviewed according to the 2016 WHO classification of tumors of the central nervous system by 2 experienced neuropathologists (H-K.N. and H.C). As one of the authors (H-K.N.) was part of the WHO Consensus Panel, we were able to work from the new classification before it was published. Glioma tissues were retrieved from the archive of the Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, and the Department of Neuropathology, Huashan Hospital. The New Territories East Cluster–Chinese University of Hong Kong ethics committee and the Shanghai Huashan Hospital ethics committee approved this study.
Fluorescence In Situ Hybridization for EGFR and MYB Amplification
Detailed steps and probes for EGFR amplification were performed just as described previously.10,11 Fluorescence signals in at least 100 non-overlapping signals were counted. EGFR amplification was defined as more than 5% of counted nuclei showing target (red) to reference (green) signal >2.10,12 Commercially available probe was applied for the MYB amplification detection (Cytocell). Detailed steps for MYB amplification were the same as EGFR amplification following the protocol provided by the manufacturer. At least 100 non-overlapping signals were counted and MYB amplification was defined as more than 5% of cells showing clusters or a ratio of target (red) to reference (green) signal >2. A representative photograph of MYB fluorescence in situ hybridization is shown in Supplementary Figure S1.
Mutational Analysis for IDH, TERTp, BRAF, and H3F3A
Direct sequencing of IDH (IDH1 and IDH2), TERTp, BRAF , and H3F3A was performed as described previously.9,13 Only IDH1 and IDH2 wild-type cases were included in this project. Hotspot mutations at codon 132 of IDH1, codon 172 of IDH2, codon 600 of BRAF, and codons 27 and 34 of H3F3A were scanned. Every mutant case was confirmed by independent PCR amplification and sequencing performed twice.
The Cancer Genome Atlas IDH Wild-Type Lower-Grade Gliomas
The list of The Cancer Genome Atlas (TCGA) IDH wild-type lower-grade gliomas, as well as mutational status of TERT, H3F3A, and BRAF of each case, was adopted from the supplemental data sheet of 2 published papers of TCGA group.14,15EGFR and MYB amplification statuses were examined by analyzing the copy number variation data downloaded from the website of TCGA using Integrative Genomics Viewer software (Broad Institute).
Statistical Analysis
Statistical analysis was conducted using IBM SPSS software v20. Overall survival (OS) was defined as the time between operation and death or the last follow-up. Chi-square was used to define the relationship between the molecular markers and clinical parameters. Survival curves were drawn by the Kaplan–Meier method, and survival between different groups was compared by the log-rank test. Survival comparison was performed separately for each clinical and molecular parameter with no adjustment for multiple comparisons. Clinical and molecular parameters with significant P-value or strong trend of prognostic association were included in multivariable analysis. Multivariable analysis of survival was performed by the Cox proportional hazards model. Proportional hazard assumption was tested using log[-log] plots. P < 0.05 (two sided) was considered statistically significant.
Results
Clinical Characteristics of IDH Wild-Type Lower-Grade Gliomas
We examined 718 adult lower-grade gliomas for IDH mutation and identified 166 (23.1%) IDH wild-type lower-grade tumors (WHO grades II and III) (Supplementary Table S1). Among the IDH wild-type lower-grade glioma cohort, 58 cases were diffuse astrocytoma, IDH wild-type (WHO grade II), 9 were oligodendroglioma not otherwise specified (NOS) (WHO grade II), 14 were oligoastrocytoma NOS (WHO grade II), 64 were anaplastic astrocytoma, IDH wild-type (WHO grade III), 9 were anaplastic oligoastrocytoma NOS (WHO grade III), and 12 were anaplastic oligodendroglioma NOS (WHO grade III).
Comparison of the clinical and pathologic characters of IDH wild-type and mutant tumors is presented in Supplementary Table S2. Briefly, patients with IDH wild-type gliomas were older (mean age, 44.6 vs 41.8 y, P = 0.02) and the majority of them had astrocytomas (73.5% vs 48.7%, P < 0.00001). IDH wild-type tumors also demonstrated grade III histology more frequently than IDH mutant tumors (51.2% vs 31%, P < 0.00001). It was also noteworthy that IDH wild-type tumors had a lower total resection rate compared with IDH mutant tumors (55.1% vs 70.8%, P < 0.001).
Clinical characteristics of IDH wild-type lower-grade tumors are presented in Table 1. Mean and median ages of patients were 44.6 and 46.0 years, respectively (range, 18‒76 y). Male to female ratio was 1.30:1. Tumors involving midline structures like the ventricular system, thalamus, and spinal cord were defined as midline located tumors. Thirty-nine tumors were midline located and 127 were nonmidline located. Resection status of 118 patients was available, with total resection achieved in 65. Adjuvant therapy data were available in 125 of 166 cases, with 56 patients receiving both radio- and chemotherapy, 8 receiving chemotherapy, and 29 receiving radiotherapy only; 32 patients did not receive any adjuvant therapy.
Table 1.
Clinical and molecular characteristics of IDH wild-type lower-grade gliomas
| All Tumors (n = 166) | TERTp mut (n = 41) | BRAF-V600E (n = 10) | EGFR amp (n = 20) | H3F3A-K27M (n = 12) | H3F3A-G34R (n = 2) | MYB amp (n = 33) | |
|---|---|---|---|---|---|---|---|
| Age, mean/median/range | 44.6/46.0/ 18–76 | 51/54/ 18–73 | 40.9/41.5/ 18–71 | 46.8/47.5/ 20–68 | 39.8/39/ 18–58 | 30.5/30.5/ 18–43 | 40.8/43/ 18–65 |
| Age group | |||||||
| ≤45 | 81 (48.8%) | 14 (34.1%) | 6 (60%) | 7 (35%) | 7 (58.3%) | 2 (100%) | 17 (51.5%) |
| >45 | 85 (51.2%) | 27 (65.9%) | 4 (40%) | 13 (65%) | 5 (41.7%) | – | 16 (48.5%) |
| Gender | |||||||
| Female | 72 (43.4%) | 15 (36.6%) | 4 (40%) | 8 (40%) | 6 (50%) | 2 (100%) | 10 (30.3%) |
| Male | 94 (56.6%) | 26 (63.4%) | 6 (60%) | 12 (60%) | 6 (50%) | – | 23 (69.7%) |
| Histologic Grade | |||||||
| II | 81 (48.8%) | 16 (39%) | 6 (60%) | 9 (45%) | 6 (50%) | 1 (50%) | 16 (48.5%) |
| III | 85 (51.2%) | 25 (61%) | 4 (40%) | 11 (55%) | 6 (50%) | 1 (50%) | 17 (51.5%) |
| Histologic Type | |||||||
| Astrocytoma | 122 (73.5%) | 33 (80.5%) | 8 (80%) | 15 (75%) | 10 (83.3%) | 2 (100%) | 23 (69.7%) |
| Oligodendroglioma | 21 (12.7%) | 5 (12.2%) | 1 (10%) | 3 (15%) | – | – | 5 (15.2%) |
| Oligoastrocytoma | 23 (13.9%) | 3 (7.3%) | 1 (10%) | 2 (10%) | 2 (16.7%) | – | 5 (15.2%) |
| Tumor Location | |||||||
| Nonmidline | 127 (76.5%) | 32 (78%) | 6 (60%) | 16 (80%) | 5 (41.7%) | 2 (100%) | 24 (72.7%) |
| Midline | 39 (22.9%) | 9 (22%) | 4 (40%) | 4 (20%) | 7 (58.3%) | – | 9 (27.3%) |
| Operation | |||||||
| No total excision | 53 (31.9%) | 13 (31.7%) | 3 (30%) | 7 (35%) | 5 (41.7%) | 1 (50%) | 9 (27.3%) |
| Total excision | 65 (39.2%) | 16 (39%) | 5 (50%) | 6 (30%) | 3 (25%) | 1 (50%) | 15 (45.5%) |
| Not available | 48 (28.9%) | 12 (29.3%) | 2 (20%) | 7 (35%) | 4 (33.3%) | 9 (27.3%) | |
| Adjuvant Therapy | |||||||
| No adjuvant therapy | 32 (19.3%) | 7 (17.1%) | 1 (10%) | 3 (15%) | 3 (25%) | – | 7 (21.2%) |
| Chemotherapy only | 8 (4.8%) | – | – | 2 (10%) | 1 (8.3%) | – | 1 (3%) |
| Radiotherapy only | 29 (17.5%) | 10 (24.4%) | 2 (20%) | 5 (25%) | 2 (16.7%) | 1 (50%) | 4 (12.1%) |
| Radiotherapy and chemotherapy | 56 (33.7%) | 17 (41.5%) | 5 (50%) | 7 (35%) | 3 (25%) | – | 12 (36.4%) |
| Not available | 41 (24.7%) | 7 (17.1%) | 2 (20%) | 3 (15%) | 3 (25%) | 1 (50%) | 9 (27.3%) |
n, number of cases; mut, mutant; amp, amplification.
Follow-up data for 142 patients with IDH wild-type lower-grade gliomas were available. Mean and median OS were 3.4 and 1.9 years, respectively (range, 0.01‒14.5 y). Median OS of patients with IDH wild-type grade II gliomas was 8.4 years and that of patients with IDH wild-type grade III gliomas was 1.32 years (P < 0.001). Young age was associated with better prognosis (P < 0.001), and such prognostic impact was more pronounced in grade II tumors (P < 0.0001) than in grade III tumors (P = 0.072). Statistical survival difference existed between different histologic types, with median OS of 7.63 years in oligodendrogliomas, 3.1 years in oligoastrocytomas, and 1.92 years in astrocytomas (P = 0.016). Stratified by histologic grade, tumor phenotype exhibited prognostic difference in only grade III tumors (P = 0.021), not in grade II tumors. Totally resected tumors tended to have better clinical outcome compared with nontotal resection (median OS 2.86 vs 1.55 y, P = 0.066). The favorable prognostic value of total resection in IDH wild-type gliomas was more prominent in grade III tumors (P = 0.002) than in grade II tumors (P = 0.057). Nonmidline tumors, with a median OS of 2.83 years, also showed a trend to longer survival compared with midline located tumors (median OS 1.55 y, P = 0.06). Collectively, single variate analysis showed that clinico-pathologic parameters including younger age, grade II histology, and oligodendroglial histology are predictors for better clinical outcome in IDH wild-type lower-grade gliomas (Table 2).
Table 2.
Univariate analysis of IDH wild-type lower-grade gliomas
| All IDH Wild-Type Tumors | Grade II | Grade III | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Variables | n | Median OS, y | HR (95% CI) | P | n | Median OS, y | P | n | Median OS, y | P |
| Age a | 142 | 1.05 (1.03–1.06)* | <0.001 | 72 | 1.06 (1.03–1.08)b | <0.0001 | 70 | 1.02 (1.00–1.04)b | 0.072 | |
| Gender | ||||||||||
| Male | 83 | 1.78 | 1 | 36 | 8.4 | 47 | 1.23 | |||
| Female | 59 | 3.42 | 0.71 (0.46–1.08) | 0.707 | 36 | 6.93 | 0.61 | 23 | 1.62 | 0.07 |
| Histologic Type | ||||||||||
| Oligodendroglioma | 15 | 7.63 | 1 | 8 | 7.63 | 7 | 3.5 | |||
| Oligoastrocytoma | 19 | 3.1 | 2.24 (0.80–6.46) | 13 | NR | 6 | 0.5 | |||
| Astrocytoma | 108 | 1.92 | 3.06 (1.24–7.58)* | 0.016 | 51 | 6.9 | 0.256 | 57 | 1.18 | 0.021 |
| Histologic Grade | ||||||||||
| II | 72 | 8.4 | 1 | |||||||
| III | 70 | 1.32 | 4.10 (2.58–6.54)* | <0.001 | ||||||
| Total Resection | ||||||||||
| Yes | 62 | 2.86 | 1 | 27 | NR | 35 | 1.78 | |||
| No | 52 | 1.55 | 1.54 (0.97–2.43) | 0.066 | 32 | 3.9 | 0.057 | 20 | 0.4 | 0.002 |
| Tumor Location | ||||||||||
| Nonmidline | 107 | 2.83 | 1 | 57 | 8.4 | 51 | 1.32 | |||
| Midline | 34 | 1.55 | 1.56 (0.99–2.47) | 0.06 | 15 | 4.4 | 0.228 | 19 | 1.09 | 0.387 |
| EGFR | ||||||||||
| Non-amp | 107 | 2.67 | 1 | 51 | NR | 57 | 1.62 | |||
| Amp | 18 | 1.03 | 2.31 (1.30–4.10) * | 0.004 | 8 | 0.82 | <0.0001 | 10 | 1.03 | 0.63 |
| TERT promoter | ||||||||||
| wt | 98 | 3 | 1 | 53 | 10.65 | 45 | 1.55 | |||
| mut | 35 | 1.32 | 2.44 (1.53–3.87)* | <0.001 | 14 | 1.76 | 0.0003 | 21 | 1.05 | 0.18 |
| H3F3A | ||||||||||
| wt | 116 | 2.24 | 1 | 54 | 8.4 | 62 | 1.32 | |||
| K27M | 10 | 0.5 | 2.25 (1.16–4.36)* | 0.016 | 5 | 2.83 | 0.007 | 5 | 0.5 | 0.03 |
| BRAF | ||||||||||
| wt | 114 | 2.43 | 1 | 55 | 8.4 | 59 | 1.52 | |||
| V600E | 10 | 10.65 | 0.46 (0.17–1.28) | 0.139 | 6 | NR | 0.355 | 4 | 1.85 | 0.657 |
| MYB | ||||||||||
| Non-amp | 83 | 2.57 | 1 | 45 | 8.4 | 38 | 1.17 | |||
| Amp | 29 | 2.58 | 0.88 (0.50–1.53) | 0.64 | 15 | NR | 0.518 | 14 | 1.55 | 0.54 |
aAge is a continuous variable; bhazard ratio and 95% CI shown for age; n, number of cases with data available; wt, wild-type; mut, mutant; amp, amplified; *denotes P < 0.05.
Molecular Characteristics of IDH Wild-Type Lower-Grade Gliomas
We examined mutations of TERTp, H3F3A, and BRAF, as well as EGFR and MYB amplifications in the IDH wild-type lower-grade glioma cohort. Among samples with analyzable data, we detected 41 TERTp mutant tumors, 10 BRAF mutant tumors, 14 H3F3A mutant tumors, 20 EGFR amplified tumors, and 33 MYB amplified tumors. The clinico-pathologic and molecular information of each IDH wild-type lower-grade glioma patient is illustrated in Fig. 1 and Supplementary Figure S2. Clinical characteristics of tumors harboring each of the molecular aberrations are detailed in Table 1. Representative medical images of IDH wild-type lower-grade gliomas harboring the molecular markers are in Fig. 1. Single variate analysis of molecular markers is shown in Table 2.
Fig. 1.
Grid figure summarizing frequency and distribution of the clinical as well as molecular markers of IDH wild-type lower-grade gliomas. Representative radiologic images of IDH wild-type lower-grade gliomas harboring each of the molecular markers are shown. Cases without survival data are not included in this figure.
TERTp Mutation
TERTp mutation can be identified in 26.8% (41/153) of IDH wild-type tumors, including thirty-two C228T and nine C250T mutations. Patients with TERTp mutated gliomas were older than those with wild-type tumors (mean age 51 y vs 42 y, P = 0.001). Median OS of patients with TERTp mutant tumors was 1.32 years compared with 3 years for those with TERTp wild-type tumors (P < 0.001) (Fig. 2A). There were no significant associations between TERTp mutation and histologic grade in IDH wild-type gliomas. Stratified by histologic grade, TERTp mutation predicted worse OS in grade II tumors (P = 0.0003) but not in grade III tumors (Table 2). Among patients with TERTp mutant tumors, longer OS was observed in those receiving total resection (P = 0.039) of grade II histology (P = 0.016) (Supplementary Table S3).
Fig. 2.
Kaplan–Meier survival analysis of TERT mutation, EGFR amplification, H3F3A K27M mutation, BRAF mutation, MYB amplification, and different molecularly graded subgroups. Survival analysis of different biomarkers revealed that (A) TERTp mutation, (B) EGFR amplification, and (C) H3F3A K27M mutation were associated with shorter OS compared with their wild-type or non-amplified counterparts (P < 0.001, P = 0.003, and P = 0.009, respectively). (D) BRAF V600E mutant tumors tended to have better prognosis compared with BRAF wild-type tumors (P = 0.13). (E) There was no statistical survival difference between the MYB amplified and non-amplified tumors (P = 0.64). (F) Molecularly lower-grade tumors (IDH wild-type lower-grade gliomas lacking EGFR amplification, H3F3A mutation, and TERT promoter mutation) had longer OS compared with molecularly high-grade tumors (IDH wild-type lower-grade gliomas harboring either EGFR amplification, H3F3A K27M, or TERT promoter mutation) (P < 0.001). (G) Kaplan–Meier analysis of prognostic value revealed that MYB amplification appeared to be a favorable prognosticator in molecularly lower-grade gliomas. (H) The prognostic split was more obvious within grade II gliomas. Abbreviations: amp, amplified; non-amp, non-amplified.
EGFR Amplification
EGFR amplification was identified in 13.8% (20/145) of tumors, including 9 grade II and 11 grade III. The mean ages of patients with EGFR amplified tumors and non-amplified tumors were 46.8 years and 44.4 years, respectively. Patients with EGFR amplified lower-grade gliomas had significantly shorter OS than those with EGFR non-amplified tumors (median OS 1.03 y vs 2.67 y, P = 0.003) (Fig. 2B). The prognostic difference was only observed in grade II gliomas (P < 0.001) but not in grade III tumors (Table 2). Among patients with EGFR amplified tumors, older age (P = 0.047), astrocytic histology (P = 0.008), and midline tumor location (P < 0.001) were associated with shorter OS (Supplementary Table S3).
H3F3A Mutations
Mutational frequency of H3F3A in IDH wild-type lower-grade gliomas was 9.5% (14/148), which included 12 cases of K27M mutation and 2 cases of G34R mutation. Half of the K27M and G34R tumors, respectively, were of grade II histology. The mean ages of patients with K27M and G34R tumors were 39.8 years and 30.5 years, respectively, compared with mean age of 45.7 years for patients with wild-type H3F3A (P = 0.167). Seven of 12 K27M gliomas were in midline structures (P = 0.009) and both G34R gliomas were of hemispheric locations. Prognostically, K27M tumors exhibited shorter OS than the wild-type tumors (median OS 0.5 y vs 2.24 y, P = 0.016) (Fig. 2C). The prognostic impact of K27M was independent of histologic grade (Table 2). Follow-up data were only available in one patient with a G34R tumor, who had OS of 3.56 years. Among the K27M mutant tumors, none of the clinico-pathologic variables was prognostic.
BRAF Mutation
BRAF V600E mutation was detectable in 6.9% (10/144) of IDH wild-type lower-grade gliomas, including 6 grade II and 4 grade III tumors. The mean age of patients with BRAF mutant tumors was 40.9 years. These patients tended to have better OS than those with wild-type BRAF (10.65 vs 2.43 y, P = 0.13) (Fig. 2D). The trend of better prognosis was observed in only grade II tumors, not in grade III tumors. We did not identify any clinical parameter of prognostic value among BRAF mutant gliomas.
MYB Amplification
MYB amplification was identified in 25.8% (33/128) of IDH wild-type lower-grade gliomas, including 16 grade II tumors and 17 grade III tumors. The mean ages of patients with MYB amplified gliomas and non-amplified gliomas were 40.8 years and 45.6 years, respectively. Though there was no significant difference in OS between the MYB amplified and non-amplified tumors, it was noteworthy that the survival curve starts to separate clearly after the point of 2.5 years (Fig. 2E), suggesting that MYB might predict better survival outcomes in some “long survivors.” Among patients with MYB amplified gliomas, younger age (P = 0.016) and grade II histology (P = 0.01) were associated with longer OS (Supplementary Table S3).
Correlation Between Molecular Markers and Multivariable Survival Analysis
Further analysis of the molecular distribution revealed that TERTp mutation tended to coexist with EGFR amplification, since 13 of 20 EGFR amplified tumors were TERT mutant (P < 0.001). Eight of 9 BRAF mutants and 11 of 12 H3F3A mutant tumors were TERTp wild type. EGFR amplification status of 6 BRAF mutant cases was available and only 1 of them was EGFR amplified. EGFR amplification was mutually exclusive with H3F3A K27M mutation, since all 12 H3F3A mutant cases were EGFR non-amplified. No BRAF mutant cases harbored H3F3A mutation. No statistically significant relationship was found between the MYB amplification and any other molecular markers. However, it should be well noted that 8 of the 9 patients with H3F3A mutants, whose MYB amplification status was available, were MYB non-amplified. Frequency and distribution of all the clinical and molecular markers are detailed in Table 1, Fig. 1, and Supplementary Fig. S2.
Multivariable analysis including the significant parameters in univariate analysis is presented in Table 3, which confirms the prognostic value of age (hazard ratio [HR] = 1.03, P = 0.02), grade III histology (HR = 9.32, P < 0.001), nontotal resection (HR = 2.42, P = 0.002), EGFR amplification (HR = 3.77, P = 0.037), and H3F3A K27M mutation (HR = 4.30, P = 0.005) in IDH wild-type lower-grade gliomas.
Table 3.
Multivariable analysis of IDH wild-type lower-grade gliomas
| Variables | HR (95% CI) | P |
|---|---|---|
| Age a | 1.03 (1.005–1.06)* | 0.02 |
| Histologic Type | ||
| Oligodendroglioma | 1 | |
| Oligoastrocytoma | 1.08 (0.24–4.93) | 0.923 |
| Astrocytoma | 0.90 (0.26–3.07) | 0.86 |
| Histologic Grade | ||
| II | 1 | |
| III | 9.32 (3.94–22.0)* | <0.001 |
| Total Resection | ||
| Yes | 1 | |
| No | 2.42 (1.31–4.48)* | 0.005 |
| Tumor Location | ||
| Nonmidline | 1 | |
| Midline | 1.21 (0.57–2.60) | 0.621 |
| H3F3A | ||
| wt | 1 | |
| K27M | 4.30 (1.56–11.8)* | 0.005 |
| EGFR | ||
| Non-amp | 1 | |
| Amp | 3.77 (1.08–13.1)* | 0.037 |
| TERT promoter | ||
| wt | 1 | |
| mut | 1.53 (0.73–3.18) | 0.259 |
| EGFR by TERT promoter interaction | 0.79 (0.17–3.64) | 0.766 |
aAge is a continuous variable; wt, wild-type; mut, mutant; amp, amplified; *denotes P < 0.05.
Molecular Grading of IDH Wild-Type Lower-Grade Gliomas
Given that (i) the impact of EGFR amplification and H3F3A and TERTp mutations on the clinical outcome of the patients and their histopathologic implications in existing literature (ie, EGFR amplification represented a classic marker of glioblastoma),16,17 (ii) H3F3A K27M mutation has been adopted as a classification marker of grade IV gliomas in the 2016 WHO classification,1 and (iii) the IDH wild-type/TERTp mutant genetic profile characterized ~70%‒80% of glioblastomas,18,19 we subclassified the IDH wild-type lower-grade gliomas into “molecularly high grade” and “molecularly lower grade.” IDH wild-type lower-grade gliomas harboring EGFR amplification, H3F3A mutation, or TERTp mutation were classified as molecularly high-grade tumors. IDH wild-type lower-grade gliomas lacking all these 3 biomarkers were classified as molecularly lower-grade tumors, thus making 59 IDH wild-type tumors graded as molecularly high grade and 75 tumors graded as molecularly lower grade, with median patient OS of 1.23 and 7.63 years, respectively (Fig. 2F, P < 0.001). There was no correlation between molecular grading and histologic grading, since both molecularly high-grade and lower-grade tumors were equally distributed over different histologic grades (P = 0.65; Supplementary Figure S3). Therefore, the clinical value of the molecular grading was independent of histologic grade. Since the prognosis of IDH wild-type gliomas with MYB amplification was very heterogeneous, with OS ranging from 0.1 year to 13.6 years, the prognostic value of MYB amplification was further interrogated in the molecular grade subsets. Further analysis of prognostic value in different molecular groups revealed that MYB amplification appeared to be a favorable prognosticator in molecularly lower-grade gliomas (P < 0.001) (Fig. 2G). The prognostic split was more obvious within grade II gliomas (Fig. 2H). Further multivariable analysis of IDH wild-type lower-grade gliomas stratified by molecular grade and MYB amplification confirmed the independent prognostic value of histologic grade and tumor resection status. Compared with molecularly high-grade tumors, molecularly lower-grade MYB amplified tumors had the best survival (HR = 0.23, P = 0.008), followed by molecularly lower-grade MYB non-amplified tumors (HR = 0.26, P < 0.001). The detailed multivariable analysis is presented in Table 4.
Table 4.
Multivariable analysis of IDH wild-type lower-grade gliomas stratified by “molecular grade” and MYB amplification
| HR | (95% CI) | P | |
|---|---|---|---|
| Agea | 1.02 | (0.99–1.05) | 0.25 |
| Histologic Type | |||
| Oligodendroglioma | 1 | ||
| Oligoastrocytoma | 1.51 | (0.33–6.97) | 0.59 |
| Astrocytoma | 0.77 | (0.20–2.90) | 0.7 |
| Histologic Grade | |||
| II | 1 | ||
| III | 12 | (4.68–30.8)* | <0.001 |
| Total Resection | |||
| Yes | 1 | ||
| No | 2.51 | (1.32–4.79)* | 0.004 |
| Tumor Location | |||
| Nonmidline | 1 | ||
| Midline | 1.76 | (0.88–3.53) | 0.21 |
| Effect of MYB in Molecular Subgroup | |||
| Molecularly high gradeb | 1 | ||
| Molecularly lower grade,cMYB non-amp | 0.26 | (0.13–0.5)* | <0.001 |
| Molecularly lower grade,cMYB amp | 0.23 | (0.08–0.68)* | 0.008 |
aAge is a continuous variable; amp, amplified; non-amp, non-amp lified; *denotes P < 0.05. bIDH wild-type lower-grade gliomas harboring either EGFR amplification, H3F3A-K27M, or TERT promoter mutation. cIDH wild-type lower-grade gliomas lacking EGFR amplification, H3F3A mutation, and TERT promoter mutation.
Prognostic Relevance of Molecular Grade in TCGA Validation Cohort
We further validated the clinical relevance of the molecular grading by analyzing 86 cases of IDH wild-type lower-grade gliomas in the database of TCGA (Supplementary Table S4). Among the IDH wild-type lower-grade gliomas with available molecular data, TERTp mutation, EGFR amplification, and H3F3A mutation were identifiable in 66.1% (37/56), 40% (34/85), and 3.6% (3/84) of tumors, respectively. Stratification according to our molecular grading criteria identified 13 molecularly high-grade and 57 molecularly lower-grade gliomas. Patients with molecularly lower-grade gliomas showed a trend of better prognosis compared with molecularly high-grade gliomas (median OS 11.1 y vs 1.5 y, P = 0.17; Supplementary Figure S4). Multivariable analysis also revealed a trend of poor outcome for patients with IDH wild-type molecularly high-grade gliomas (Supplementary Table S5). Combining the cohort of TCGA and our cohort revealed the independent prognostic value of molecular high grade in IDH wild-type lower-grade gliomas (HR = 2.08, P = 0.0013) (Supplementary Table S5).
Discussion
Numerous studies have found that IDH wild-type gliomas have distinct characteristics compared with IDH mutant tumors, both biologically and clinically.8,14,20–22 The 2016 WHO classification uses IDH mutation status for classification not only of glioblastoma but also of lower-grade astrocytomas and oligodendrogliomas.1 While it is clear that IDH mutations segregate glioblastomas into good and poor prognostic groups, their role in prognostication in the adult lower-grade gliomas remains uncertain. The vast majority of oligodendrogliomas will be IDH mutated,23 and the WHO has used IDH mutation and 1p/19q codeletion to define oligodendrogliomas. However, whether IDH wild-type astrocytoma should remain as a distinct entity is still controversial and the WHO classification has retained the entity but described it as provisional, pending further studies.
We investigated several important molecular markers in a large cohort of IDH wild-type lower-grade gliomas which can potentially improve the clinical stratification of the newly established WHO entity. EGFR amplification was found in more than 40% of primary glioblastoma and predicted a worse clinical outcome, thus is regarded as a classic genetic marker of glioblastomas.16,17TERTp mutation was frequently detected in glioblastoma and was reported as an independent poor prognostic factor in both IDH mutant and wild-type glioblastomas.18,24 Labussiere and colleagues also identified the coexistence of TERTp mutation and EGFR amplification, as well as the poor prognostic value of EGFR amplification in TERTp wild-type glioblastomas.18 These 2 molecular markers conferred a similarly aggressive tumor behavior in the lower-grade lesion, as shown in our current study. The median OS of patients with EGFR amplified lower-grade gliomas in our cohort was 1.03 years and that of patients with TERTp mutant lower-grade gliomas was 1.32 years, both of which were significantly shorter than the wild-type counterparts. Reuss et al in a recent paper studied a series of IDH wild-type lower-grade gliomas and found that most of them could be diagnosed as the molecular equivalents of conventional glioblastomas, and the median survival of patients was 19.4 months.8 The authors also identified a subgroup of IDH wild-type lower-grade gliomas harboring the H3F3A K27M mutation, which were also of poor prognosis. Our findings were similar in that H3F3A mutation predicted a poor prognosis in IDH wild-type lower-grade gliomas, with median patient OS of 6 months. Combining the 3 biomarkers, IDH wild-type lower-grade gliomas were classified into molecular high grade and lower grade. Further survival analysis based on this segregation showed the distinct clinical outcome in these 2 groups. In other words, IDH wild-type lower-grade tumors harboring TERTp mutation, EGFR amplification, or H3F3A mutation have poor clinical outcomes compared with other IDH wild-type tumors without any of these genetic events. The median OS of patients with molecularly lower-grade tumors, even in the genetic background of wild-type IDH, was 7.63 years. The 5-year OS of patients with IDH wild-type molecularly lower-grade gliomas was even up to 81% (data not shown), highlighting the prognostic heterogeneity of lower-grade gliomas with wild-type IDH and the need for further classification. Notably, our proposed molecular grading could also prognostically stratify the IDH wild-type lower-grade gliomas in the cohort of TCGA into 2 groups with distinct clinical outcomes. The subset of molecularly high-grade tumors presented with more aggressive clinical behavior (median OS in our cohort = 1.2 y; median OS in TCGA cohort = 1.5 y) and might draw special attention both in clinical practice as well as in patient selection and stratification in upcoming clinical trials.
Amplification of the MYB gene can be identified in around 30% of BRCA1 mutant breast tumors, but is rare in 2% of sporadic breast cancers.25MYB amplification can also be identified in 10% of pancreatic cancers,26 as well as in 2 colorectal cancer cell lines27 and 2 glioblastoma cell lines.28 Further survival analysis revealed that expression of B-MYB was associated with an increased risk of death in neuroblastomas,29 which also predicted worse survival probability in neuroblastomas without MYCN amplification. Whole genome sequencing on pediatric low-grade gliomas has found genetic alterations involving MYB and BRAF as among the most common genetic events in these tumors.30 Partial duplication of MYB was identified in 28% of pediatric grade II diffuse astrocytomas.31 Rearrangements of MYB were recurrent with FGFR1 and mutually exclusive in 53% of pediatric grade II diffuse low-grade gliomas.32 Tatevossian et al also identified novel MYB amplifications, which upregulate the protein expression, in 2 of 14 pediatric low-grade gliomas.33 However, the impact of this genetic event on the clinical outcome of the patients remained unclear. In our cohort, we found that MYB amplification can be identified in 25.8% of adult IDH wild-type grade II and grade III tumors, a higher frequency than EGFR amplification and H3F3A mutation or BRAF mutation. Patients with MYB amplified lower-grade gliomas tended to be younger than those without the amplification (mean age 40.8 y vs 45.6 y, P = 0.12). The mean age of patients with molecularly lower-grade, WHO grade II tumors harboring MYB amplification was 34 years and these patients enjoyed excellent prognosis (Fig. 2H). The only deceased patient in this subgroup was a 54-year-old man with recurrent oligoastrocytoma surviving for only 9.9 months, who had primary tumor 28 years ago. The other patients in this subgroup were all surviving at the time of last follow-up. These results suggested that there exists a subset of IDH wild-type low-grade gliomas in young adults which exhibits similar clinical outcome as low-grade gliomas of pediatric patients. We speculated that these molecularly lower-grade, IDH wild-type/MYB amplified grade II gliomas in young adults extended the age spectrum of the pediatric diffuse low-grade gliomas with MYB amplification.32,33
BRAF V600E mutation is a common gene abbreviation in pleomorphic xanthoastrocytoma, ganglioglioma, and extracellular pilocytic astrocytoma, with a frequency up to 66%.34 However, the frequency of BRAF mutation in adult diffuse gliomas is low, only 15% (5/33) according to the case report by Chi et al.35 Prognostic studies based on young adult glioblastoma and pediatric glioblastoma had identified BRAF mutation as a predictor for a better clinical outcome, which may result from total surgical resection.13,36 In their study based on 1122 diffuse grades II to IV gliomas, Ceccarelli et al also identified a subgroup of pilocytic astrocytoma-like tumors in IDH wild-type tumors (6% of the IDH wild-type tumors).15 This subgroup of tumors also had a better prognosis, which also displayed a low frequency of typical glioblastoma alterations like TERTp expression and EGFR amplification. Re-review of these cases rediagnosed 3/26 as pilocytic astrocytoma grade I. BRAF mutation was found in 6.9% (10/144) of cases in our cohort, similar to Ceccarelli et al. BRAF mutant tumors showed a trend for a better prognosis compared with wild-type tumors (median OS 10.65 vs 2.43 y, P = 0.14), even though that was not statistically significant in both single and multivariable analyses. This may be due to the small BRAF mutant sample size, since only 10 BRAF mutant cases were identified. No difference in the total resection rate or age distribution between the BRAF mutant and wild-type tumors (P > 0.05) was found. Although in this study, BRAF mutant tumors showed only a trend for good survival, this group still draws special attention because of its potential survival benefit from the BRAF inhibitor vemurafenib.37
Prediction of patient outcome based on histology as well as WHO grade has been challenged with the development of more precise molecular stratification of gliomas.6,10,38,39 Reuss et al also found that prediction power of the WHO grading existed between only grades II and III IDH wild-type tumors but not IDH mutant gliomas.39 This conclusion was corroborated in our study. Grade III histology remained as a strong adverse factor in both single and multivariable analyses in IDH wild-type gliomas. In grade II tumors, TERTp wild-type cases have a better clinical outcome than TERTp mutant ones (median OS 10.65 vs 1.76 y, P = 0.003). Grade II EGFR amplified cases had a worse outcome compared with non-amplified cases. Median OS of grade II EGFR amplified tumors was 0.82 year, while the median OS of WHO grade II EGFR non-amplified tumors was not reached (P < 0.001). However, these phenomena were not observed in grade III cases (Table 2).
In summary, we presented evidence showing that adult IDH wild-type lower-grade gliomas are prognostically heterogeneous and do not have uniformly poor prognosis. Additional biomarkers including TERT promoter mutation, EGFR amplification, H3F3A mutation, and MYB amplification could be utilized individually or in combination to further classify IDH wild-type lower-grade gliomas into favorable and unfavorable subgroups. The concept of “molecular grading” could be of potential use in clinical risk stratification of IDH wild-type lower-grade gliomas.
Supplementary Material
Supplementary material is available at Neuro-Oncology online.
Funding
This work was supported by Shanghai Municipal Commission of Health and Family Planning (201540145), 973 program (2015CB755503), Shanghai Sailing Program (16YF1415200), Health and Medical Research Fund of Hong Kong (02133146), and the S. K. Yee Medical Foundation (2151229).
Conflict of interest statement. The authors declare no potential conflicts of interest.
Supplementary Material
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