Lithuanian University of Health Sciences
HIGH-GRADE GLIOMA MUTATIONS
IN 15-25 YEARS OLD PATIENTS
By
Mathilde Desplanques
A thesis submitted in part fulfilment for the Degree of Master of Medicine
In the
Faculty of Medicine Department of Neurosurgery
Kaunas, 2019/2020
Supervisor: Adomas Bunevicius MD, PhD Consultant : Pascale Varlet MD, PhD
Table of contents
1. TITLE PAGE 1 2. TABLE OF CONTENTS 2 3. SUMMARY 3 4. ACKNOWLEDGE 4 5. CONFLICT OF INTEREST 4 6. ABBREVIATIONS 5 7. TERMS 5 8. INTRODUCTION 69. AIM AND OBJECTIVES 7
10. LITERATURE REVIEW 8 -12 10.1 IDH mutations 8
10.2 Diffuse astrocytoma 8
10.3 Diffuse oligodendroglioma 8- 9 10.4 IDH mutation in adults 9 -10 10.5 IDH mutation in children 10
10.6 H3F3A mutation 10
10.7 H3F3A mutation in children 10-11 10.8 H3F3A mutation in adults 11
10.9 BRAF mutation 11-12 10.10 MGMT promoter methylation 12
11. RESEARCH METHODOLOGY AND METHODS 13 - 14 12. RESULTS 15- 24 12.1Study sample 15-18 12.2 Molecular landscape in AYA HGG patients 18-21 12.3 Prognosis data 21-24 13. DISCUSSION OF RESULTS 25-27 14. CONCLUSIONS 28 15. LITERATURE LIST 29-37 16. ANNEXES
Summary
I, Mathilde Desplanques, wrote my thesis about « high-grade gliomas mutations in 15-25 years old patients ».
Aim: Numerous publications about pediatric and adult gliomas molecular alterations have already been published in the past, yet only a few focused on teenager's tumours mutations. However, studies exploring the importance of molecular signatures on the intermediate age group of adolescent-young adults (AYA) (15 to 25 years old) of HGG glioma patients remain limited. This study aimed to evaluate the mutational landscape in high-grade glioma (HGG) in this intermediate age group. Objectives: Pediatric and adult's high-grade gliomas harbouring significative distinct molecular alteration patterns, our objective was
1. to analyze which mutations are the most frequent in intermediate age group (15-25 years old HGG patients)
2. to compare mutational landscape of pediatric and adult gliomas
3. to evaluate if this intermediate age group patients should be treated considering their age or molecular profile
Methods: From Pubmed database we collected and analysed research studies focused on paediatrics and adults patients with HGGs published from August 2015 until August 2018, integrating meta-analysis by Mckay et al. that considered data published prior to 2015. We separated our research in 2 distinct database queries: the first, pediatric part using keywords «high-grade glioma ; mutation ; pediatric » and the second, adult part with keywords «high-grade glioma ; mutation ; adult ». The study inclusion criteria were: high-grade glioma patients, patient age between 15 and 25 years old and publications with enough clinical characteristics and molecular informations. This led us to select 13 publications under the pediatric glioma research (1–13) and 36 publications from adults glioma
research (1–3,8,10,14–44). We removed 5 publications from the adult's research that were already used from pediatric research (1–3,8,10) and remained with 31 adult publications (14–44). We then analyzed those 44 publications and gathered a cohort of 473 patients with HGG in the intermediate age group. Results : The distribution of our cohort was the following: 44,4% females and 55,6% males. Most of the tumours were located in cerebral hemispheres 66,4%, others were in the brainstem 22.5% and non-brainstem location 11.1%. 25.9 % of HGGs were IDH mutant, 18.4 % were H3K27M mutant, 11.1 % were H3G34R mutant and 16.9% were BRAF v600E mutant. The 27.7 % were either IDH wild type or not otherwise specified.
We found a mixed of « pediatric histone mutant » and « adult IDH mutant » molecular alterations in our AYA group. However, HGGs molecular alterations of AYA patients were more similar to pediatrics moleculars alterations.
Conclusions/Recommendations:
AYA gliomas represent a heterogeneous group of tumours, and their molecular signatures mostly resemble pediatric gliomas. It seems more important to treat those young adults based on their molecular characteristics and then refer them toneuro-oncologist specialising in pediatric oncology or adult neuro-oncology, more than treating them based on their age of diagnosis.
Acknowledge
I would like to thanks my consultant MD. PhD. Pascale Varlet and my supervisor MD. PhD. Adomas Bunevicius without whom I could not have done such a thesis. I would also like to thanks my parents Stephane and Valerie who have supported me each day as well as my sisters Eleonore and Marie.
Conflict of interest
Abbreviations/Terms
AA – anaplastic astrocytoma AOA – anaplastic oligoastrocytoma AOD – anaplastic oligodendroglioma
aPXA – anaplastic pleomorphic xantho-astrocytoma ATRX – alpha thalassemia X-linked mental retardation AYA – adolescent young adults
BRAF – proto-oncogene B-Raf gene
CDKN2A – cyclin-dependent kinase inhibitor 2A CNS – central nervous system
EGFR – epidermal growth factor receptor DIPD – diffuse intrinsic pontine glioma DMG – diffuse midline glioma
eGBM – epithelioid glioblastoma multiform GBM – glioblastoma multiforme
H3 – histone 3
H3G34R – mutation on glycine 34 of histone 3 H3K27M – mutation on lysine 27 of histone 3 HGG – high grade glioma
IDH – isocitrate dehydrogenase LGG – low grade glioma
MGMT – O6-methylguanine-DNA methyltransferase OD – oligodendroglioma
OS – overall survival
PDGFRA – platelet derived growth factor receptor alpha PXA –pleomorphic xantho-astrocytoma
PTEN - Phosphate and TENsin homolog gene TERT – telomerase transverse transcriptase TMZ – temozolomide
TP53 – tumour protein 53
WHO – world health organisation WT – wild type
Introduction
Primary brain tumours represent the most common solid tumours in children, adolescents and young adults (AYAs)(45). High-grade gliomas (HGG) (i.e. World Health Organization – WHO), including astrocytoma grade III and IV, anaplastic oligodendroglioma grade III and anaplastic pleomorphic xanthoastrocytoma grade III, have the highest mortality rate among patients under the age of 25 years (46), (47). Paediatric HGGs, because of their similar histopathological appearance to adult HGGs, are classified, graded, and treated according to the same system and rationale as adult HGG(48). However, recent molecular studies have demonstrated that paediatric HGGs present with different genomic alterations and oncogenic activation pathways than adult HGGs (10,49–51). Also, comparative genomic hybridization/transcriptome studies have identified different molecular subgroups in adult HGGs (52) that are rarely found in paediatric HGGs (11). For instance, isocitrate dehydrogenase (IDH) 1 and 2 mutations and 1p/19q co-deletions are very rare in children (<3%), as well as PTEN mutations and EGFR amplification (<10%) (53).
Contrarily, the H3K27M or H3G34R/V variants represent distinct clinicopathological subgroups in paediatrics, even defining diffuse midline gliomas, and H3K27M-mutant gliomas was introduced as a new tumoral entity in the latest WHO Classification of CNS tumours (54). This molecular subgroup is found in 35% of paediatric HGG with a predominance for diffuse intrinsic pontine gliomas (DIPG) (36.2%) and diffuse midline glioma (DMG) (12.4%)(10), and remains rare in adult HGG (<3%) and mutually exclusive from IDH mutant cases (55). There are only scarce available data about HGGs in AYAs (45) and no study has been specifically dedicated to this patient group. Available information can be only extrapolated from the subgroups of older patients in paediatric series or subgroups of younger patients in adult series. In this study, we sought to investigate the distribution and
clinicopathological and molecular characteristics of HGG in AYAs (15-25-year-olds), through analysis of publications published in the PubMed database from 2015 to 2018, by analysing HGG patients in the intermediate age group.
Aim and objectives
Aim: To evaluate which gene mutations are the most frequent in the intermediate adolescent age group of HGG patients.
Objectives:
1. to analyze which mutations are the most prominent in this intermediate age group (15-25 years old patients)
2. to compare respective molecular signatures in pediatric and adult glioma patients
3. to evaluate if this intermediate age group should be treated depending on their age or molecular profile
Literature review
Isocitrate dehydrogenase (IDH) mutation
In the IDH1 gene, the R132H mutation is the most common and occur at codon 132 with an exchange of arginine amino acid to histidine. This IDH1 mutation is known to be an early event in the
pathogenesis of glioma (56). IDH mutation is highly associated with various clinical and other molecular parameters, such as histopathological diagnosis, patient's age, 1p/19q codeletion, TP53 mutation, as well as prognosis and survival. IDH1 mutation occurs mainly in oligodendrogliomas (OD), WHO grade II and III astrocytomas and secondary GBM (57). Most IDH mutant tumours occur in young adult infiltrating gliomas rather than children and older patients, and IDH mutation is known as a favorable prognosis factor since tumours harbouring this mutation have slower growth rate than those without IDH mutation, so named IDH wild-type (IDH-WT) (56).
Diffuse astrocytoma
Diffuse astrocytomas are histologically diagnosed by their irregular, elongated hyperchromatic nuclei and their fibrillary degree. Grading of diffuse astrocytomas will depend on mitotic activity (WHO grade II-III) and necrosis degree, combined with microvasculature proliferation (WHO Grade IV) (56). IDH mutant infiltrating astrocytomas and secondary GBMs are associated with the ATRX mutation and TP53 mutation (mutually exclusive with 1p/19q codeletion) and primarily characterized by their IDH mutant status(58). TP53 mutation is frequent in IDH mutant anaplastic astrocytomas (AA) and IDH mutant GBMs and has been reported to be present in up to 80% of these cases(56). In previous studies, it was mentioned that grade II-III IDH mutant astrocytomas harbour ATRX mutation in 78% of cases and only 5% of IDH mutant astrocytoma harbour TERT mutation. Similarly, patients with IDH mutated GBM have higher frequency of ATRX, around 63%, compared to TERT mutation, which is less frequent and represented only 12% of GBM IDH mutated cases (32). Loss of ATRX is known to be a prognostic marker for astrocytic tumours (28). Patients with IDH mutated AA with ATRX loss have a better survival rate than patients with IDH mutant AA without ATRX alterations (57).
Diffuse oligodendroglioma (OD)
Oligodendrogliomas are histologically diagnosed by their round, regular nuclei with perinuclear halos. Criteria to differentiate oligodendroglioma will be based on mitosis activity (WHO grade II or III) as well as microvascular proliferation and necrosis (56).
30-40% of IDH mutated gliomas have 1p/19q codeletion (56) and also harbor TERT promoter
codeletion and TERT mutation are highly associated with oligodendrogliomas. TERT mutation is found in almost all OD. 94% oligodendrogliomas,that are IDH mutant, 1p/19q co-deleted tumours, had been reported to harbour TERT mutation (32) and only in 4% of IDH mutated glioma that lack 1p/19q codeletion (56). As previously reported the TERT and ATRX mutations are mutually exclusive in GBMs and astrocytomas, therefore TERT mutation is rare in IDH mutant GBMs and IDH mutant astrocytomas (56,57,60). This is why TERT mutation is rare in young adults GBMs harbouring IDH mutation with ATRX mutation frequently (11). 1p/19q codeletion is the most valuable diagnostic marker of this subgroup of diffuse glioma since this 1p/19q codeletion is specific for
oligodendroglioma. 1p/19q codeletion is known as a favourable prognosis marker for survival and chemotherapy treatment response (15), providing to patients with oligodendroglioma a better
prognosis than patients with IDH mutant tumours lacking 1p/19q codeletion (18,61). The survival rate of patients with TERT mutated tumours depends on IDH and MGMT status (8). However, it was reported that TERT mutations in IDH mutant GBMs confer a worse prognosis and survival (32). IDH mutated tumours in adults
IDH1 mutation occurs in 10% of GBMs. They mostly occur in WHO grade II-III infiltrating gliomas of adults, and secondary GBMs (62)(60)(56). Studies have shown that IDH status is the most
important prognostic marker as well as a diagnostic marker in adult diffuse gliomas (58).
IDH1 mutations are often seen in young adults (20-40 years old) but are rare in children and patients over 55 years old (10,11,60,62). 16.8% of young adults with GBM harbour IDH mutation (11). Young adults IDH mutated HGG most commonly occur in cerebral hemispheres (63).
ATRX loss appears almost exclusively in the IDH mutant tumours (57)(63) and coexists with TP53 mutations (20). IDH mutant astrocytomas harbour approximately 80% TP53 mutation (56). ATRX mutation is found in 57% of secondary GBMs(56) and in 33.2-71% of WHO grade III gliomas (63). TERT and ATRX alterations are frequent in IDH mutant gliomas but are rarely seen together (11). Only 6% of IDH mutated GBM and only 2% of WHO grade II-III IDH mutated astrocytoma have been reported to harbour both mutations simultaneously (32). Therefore TERT mutation is rare in young adult GBMs who mostly have IDH mutated GBMs with ATRX alteration (11). IDH mutant oligodendrogliomas have a better prognosis than IDH mutant astrocytomas (60). IDH mutant
glioblastomas have a better prognosis than IDH wild type GBMs (60). IDH mutant GBMs have better survival than AA wild type. This would suggest that IDH status is the main prognosis marker
independent on histological grading (64).
IDH mutant GBMs never show EGFR amplification (60) because EGFR and IDH mutation are mutually exclusive (65). MGMT promoter methylation is frequent in IDH mutated tumours (10) (9)
which confer patients with IDH mutated tumors a better prognosis than their IDH wild type counterpart.
IDH mutated tumours in children
IDH mutation is rare in pediatric tumours and had been reported to be present in less than 10% of pediatric GBMs but typically absent in youngest children (56). Previous studies mentioned that they were no IDH mutations in HGGs of pediatric patients younger than 14 years old. ATRX alteration can be seen in IDH mutant pediatric HGGs but IDH mutations are rarely seen in children (5), therefore ATRX mutation found in only 20% of pediatric GBMs since they are mostly IDH wild type GBMs (56). Pediatric gliomas mostly harbour H3F3A and ATRX mutation but rarely IDH mutations (56). Both alterations, the ATRX and H3F3A mutations can coexist in pediatric HGGs (58).
However, studies of oligodendrogliomas in children show that 18% of tumors harbour IDH mutation and 25% harbour 1p/19q codeletion, which is different from their adult counterparts (56). TERT occurs in only 2% of DIPG (66). TERT mutation increases with age regardless of IDH status of gliomas (34).
Pediatric GBMs are mostly IDH wild type and those IDH wild type GBMs are mostly co-occurring with PDGFRA alteration (18%), CDKN2A deletion (49%), TP53 mutation (35%) and EGFR amplification (40-50%) (56).
H3F3A mutation
H3.3 is a histone protein coded by the H3F3A gene and appears to be mutated in a subset of HGGs. H3.3 histone mutations include H3K27M, H3G34R and H3K36 mutations. However, only H3K27M and H3G34R mutations are specific for gliomas. Those two last H3.3 mutations are highly specific to GBMs and mostly occur in pediatric glioma (67). H3K27M mutation is the substitution of lysine to methionine at codon 27 in the H3F3A gene while H3G34R mutation is the substitution of glycine to arginine amino acid at codon 34 of H3F3A gene (68)
H3F3A mutated tumours in children
H3.3 mutation is frequently found in pediatric HGG and represent 40% of pediatric’s GBMs (56). First, the H3G34R mutation accounts for around 15% of H3 mutated tumours (69) and is common in pediatric HGGs. Nearly all mutated tumors are located in hemispheres (69). The H3G34R mutated tumours have a median age of 18 years old (56). H3G34R mutation is not found in DIPG tumours in children (69). H3G34R mutation co-occurs with ATRX and p53 mutations (10,53,69). ATRX and p53 mutations found in almost all H3G34R mutant tumours (67). MGMT promoter methylation is frequent in H3G34R mutated tumours (9,10) therefore patients with H3G34R mutated tumours have a better
prognosis and good response to chemotherapy treatment than patients with H3K27M mutated tumours or H3/IDH wild type tumo(70).
Then, the H3K27M mutations are found in the youngest age group (1,10,69) and account for 30% of pediatric HGGs tumours. H3K27M mutated tumours are now classified as grade IV diffuse glioma since the new WHO 2016 classification (71).
H3K27M mutant tumours mostly appear in the midline (pons, medulla, thalamus, spinal cord) (3,63,69) and especially in DIPG (diffuse intrinsic pontine glioma).
Up to 78% DIPG are H3K27M mutant tumours, while only 36% of non-brainstem gliomas harbour H3K27M or H3G34R mutation and less than 25% of non-brainstem glioma harbour H3K27M
mutation (69). H3K27M mutant tumours have a median age of 10.5 years old (56). ATRX mutation in H3K27M mutated tumours are less frequent than ATRX mutation in H3G34R mutated tumours
(10)(69)(63). Yet, it was reported that 30% of GBMs with H3K27M mutation also harbour ATRX mutation (69). Also, H3K27M mutation often harbours TP53 in 60% of cases (69). MGMT promoter methylation rarely appears in H3K27M tumours (70). The H3K27M mutated tumors are very
aggressive and survival is less than a year for children (13). H3F3A mutated tumours in adults
H3.3 mutations are rare and are found in only 5% of adult GBM (60).
Some studies mentioned that H3.3 mutation in young adult glioblastoma is up to 18.7% (11).
H3 mutation in young adults GBMs can also overlap with ATRX (58,60) however it is less frequent than in pediatric GBMs (56). H3K27M mutated tumours are very aggressive and survival rate in patients with those tumours is around 20 months for adult (13).
BRAF mutated tumours
Firstly described in 2004, BRAF mutation in gliomas is most specific to low grade gliomas (LGGs). The most common alteration in BRAF gene is V600E mutation (72). BRAF V600E is present in a wide range of brain tumours, including ganglioglioma (~30%), pleomorphic xanthoastrocytoma (~40%), and epithelioid GBM (~50%) (58), (21). Around 2% of all GBM harbour BRAFv600E mutation or rearrangements (62).
BRAF mutation in GBMs is a sign of secondary progression from a lower grade glial tumor (73). BRAF mutation is diagnostically useful for confirmation of the neoplastic process but does not help for identification of tumour type (60). BRAFv600E, IDH1, H3G34R mutations and EGFR
adult glioma study, loss of BRAF was more charasteristic for pediatrics gliomas , while retained BRAF was more dominant in adults gliomas (5).
BRAFv600E mutation have previously been reported to be present in 20% of pediatrics GBMs, and 3% of adults GBMs (74). Also around 15% of young adult GBMs harbour BRAF mutation. It seems that BRAFv600E mutation frequency decreases with age (75).
GBMs with BRAF mutation have frequent CDKN2A deletion and affect younger age patients (11). 80% of BRAF mutated GBMs harboured CDKN2A deletion while only 26.4% of BRAF wild type GBMs harbour CDKN2A deletion (11). BRAFv600E mutation is rare in adults GBMs, it is however very common in epithelioid GBM (eGBM)(74). Others reported that eGBM harboring BRAFv600E mutation was up to 93%, 71% harboring TERT promoter mutation and 79% - CDKN2A/B deletion (38). It has been hypothesized that epithelioid glioblastoma is a malignant form of PXA (60). PXA and eGBM have similar histological and molecular alterations. TERT is used to differentiate PXA from GBM. Both eGBM and PXA (aPXA) harbour CDKN2A/B deletion and BRAF mutation. However, PXA harbour BRAFv600E mutation in 50-78% of cases and CDKN2A/B deletion in 60-83% of cases, while TERT promoter mutation was only present in 4% of PXAs and 23% aPXAs (38).
MGMT Methylation
O (6)- methylguanine – DNA methyltransferase (MGMT) promoter methylation status is not used as a diagnostic marker but it is highly useful to determine temozolomide (TMZ) treatment effectiveness, and patient survival and prognosis.
MGMT promoter methylation is a prognostic marker for WHO grade III IDH mutant gliomas but also predict the effectiveness of alkylating chemotherapy in patients with IDH wild type gliomas (57). Combined therapy of temozolomide (TMZ) and radiotherapy shows an impact on survival and progression-free compared to monotherapy and is standard of care of GBMs (76), (65). Patients with MGMT promoter methylated tumors have better survival when combined therapy with TMZ and radiotherapy are used compared to those only treated with radiotherapy (57). Previous studies
mentioned that the median OS for MGMT methylated glioma was 18.2 months versus 12.2 months for tumours that didn't have MGMT methylation (64). Currently, there is no effective alternative
Research methodology and methods
We identified research papers reporting molecular phenotypes of HGGs from the PubMed database that were published in a period from August, 2015 until August, 2018. We also integrated
metananalysis by Mckay et al. who also reported molecular data from HGGs published before 2015. We separated our literature search into 2 distinct analyses : one of pediatric patients and another of adult patients as described in Figure 1.
First, we focused on the pediatric cohort: we used the keywords « High-grade glioma » ; « mutation » ; and « pediatric » and added filters: « full text », « 2015/10/01- 2018/10/31 », and age specificity « 13-18/19-24/ 19+ and 19-44 years old ». This literature search identified a total of 164 publications. We then excluded all retrospective studies, review papers, radiological publication, treatment publications, low-grade glioma publications, and those which were previously used by Mackay et al. analysis, or were not related to our topic or not harbouring enough molecular data for our study. We ended up with 13 publications from this « pediatric high-grade glioma mutation » research (1–13)
In the same manner, we used as the following keywords in a second literature search of adult HGG patients . The same filters were applied as well as the same exclusion criteria for study selection. This approach reduced the number of publications meeting our study criterai from 580 publication to 36 publications (1–3,8,10,14–44) We removed 5 publications that were already considered for the pediatric analysis (1–3,8,10) leaving a total of 31 publications in adult HGGs.
From those 44 publications selected (13+31), we considered only patients who were relevant for our research objective and met the study criteria, including age between 15-25 years, high-grade tumours, and sufficient clinical characteristics and molecular data. These two different literature search
strategies allowed us to identify a large number of patients from the pediatric part as well as the adult part. However, 473 patients met the study criteria and were analysed.We collected all clinical
characteristics and molecular datas from those 473 patients that we regrouped under excel in Table 1. This table was used as main support in our data analysis to calculate distribution of different mutations alterations, tumors location, as well as patients mean age and mean survival depending on their tumor mutations. All statistic analysis were made under excel additionnaly to pie charts illustrating molecular alterations distribution in tumours. We also used GraphPad Prism software to illustrate survival of patients depending on tumors mutations by Kaplan-Meier survival curve.
High grade glioma PEDIATRIC 738 publications 6 - retrospective - 24 16 - review/articles - 36 3 - no access - 16 139 publicaions 26 - not related - 57 18 - radio/ treatment - 51 32 - LGG - 100
High grade glioma ADULT 580 publications 504 publications 296 publications 64 publications 14 publications 38 publications 32 - too young/ old - 210
4 - already used - 7 13 - excluded not enough
information - 41
547 cases
Flowchart from Pubmed research under « High-grade glioma mutation pediatric » and « High-grade glioma mutation adult » key words with several added filters.
Figure 1
General filters used: « Full text », « From 2015/10/01 to 2018/10/31 », « Age: 13-18, 19-24, 19+, 19-44 years old » Article types filters: « Case reports, Clinical trial, Comparative study, Controlled clinical trial, Dataset, Electronic supplementary materials, Evaluation study, Overall, Research support American recovery and reinvestement Act, research support NIH extramural, Research support NIH intramural, Research support non-US governmental, Research support US governmental non PHS, Research support US governmental PHS, Research support US governmental, Scientific integrity review, Systematic review, Twin study, Validation study »
164 52 8 42 35 473 295 6 13 31 12
Results
1.Study samples
We analyzed data from 473 AYA HGG patients identified during the literature search. The distribution of patients were the following: 191/430 (44,4%) were female, and 239/430 (55,6%) were male. Most of the tumours were located in cerebral hemispheres (186/280) 66,4%, while others were in the brainstem 63/280 (22.5%) and non-brainstem nor hemispheres 31/280 (11.1%). The mean age of our cohort was 19.78 years (range, 15-25 years).
Figure 2
MAIN CLINICAL INFORMATIONS IN OUR STUDY
CLINICAL CHARACHTERISTICS NUMBER PERCENTAGE
Grade Grade 3 147/473 31.1 % Grade 4 326/473 68.9 % Location Brainstem 63/280 22.5 % Non brainstem 31/280 11.1% Hemispheric 186/ 280 66. 4% Gender Femele 191/430 44. 4% Male 239/430 55. 6% Age (15-25) Mean age : 19.78 DISTRIBUTION IDH status
IDH wild type 300/405 74.1 % IDH mutation 105/405 25.9% H3F3A H3.3 wild type 287 /407 70.5 % H3.3 mutated 120/ 407 29.5% H3.3 K27M 75/407 18.4 % H3.3 G34 45/407 11.1 % CDKN2A CDKN2A intact 44/81 54.3 % CDKN2A deletion 37/81 45.7 % BRAF
BRAF wild type 304/366 83.1 % BRAF mutated 62/366 16.9 % PDGFRA
PDGFRA wild type 131 /166 78.9% PDGFRA amplification 26 /166 15.7% PDGFRA mutation 9/166 5.4% ATRX
ATRX wild type 65/ 120 54.2 % ATRX alteration 55 /120 45.8 % TP53
TP53 wild type 15/52 28.85% TP53 mutated 37/52 71.15% TERT
TERT wild type 128/153 83.7 % TERT mutated 25/153 16.3 % EGFR
EGFR amplification 15/163 9.2 % EGFR without amplification 148/163 90.8 % 1p/19Q 1p/19q codeleted 7/103 6.8 % 1p/19q non codeleted 96/103 93.2 % MGMT status MGMT promoter methylated 72/194 37.1 % MGMT promoter unmethylated 122/194 62.9 %
DIAGNOSIS NUMBER PERCENTAGE Anaplastic astrocytoma 98/473 20.7% Anaplastic oligoastrocytoma 3/473 0.6 % Anaplastic oligodendroglioma 16/473 3.4 % aPXA 23/473 4.9 % DIPG 14/473 3 % Glioblastoma 286/473 60.5 % Epithelioid GBM 27/473 5.7 % Others 6/473 1.2 %
In AYAs, we found out that 105/405 (25.9 %) of HGGs were IDH mutant, 75/407 (18.4%) were H3K27M mutant, 45/407 (11.1 %) were H3G34R mutant and 62/366 (16.9%) were BRAFv600 mutant.
IDH, H3K27M, H3G34R and BRAFv600E were mutually exclusive. The 27.7 % of tumors were either IDH wild type or not otherwise specified (Figure 3)
Figure 3
2. Molecular landscape of AYA HGGs
The main HGG subgroup in AYAs was Histone H3-mutant (H3K27M 18.4% and H3G34R 11.1%), which was the most common in « pediatric HGG subgroup».
A total of 118 H3.3 mutated tumors were distributed between 91 GBMs (77%), 15 anaplastic astrocytomas (12.7%), 6 DIPGs (5%), 4 anaplastic oligodendrogliomas, (3.4%), 1 anaplastic oligoastrocytoma (0.8%), and 1 high grade glioma not classified (0.8%).
The H3.3 K27M mutated tumours were the most frequent (n=75), and distributed between 52 GBMs (69.3%), 13 anaplastic astrocytomas (17.3%), 6 DIPGs (8%), 3 anaplastic oligodendrogliomas (4%) and 1 anaplastic oligoastrocytoma (1.3%). There tumours were in the brainstem (51.4%; 36/70), non-brainstem nor hemisphere (41.4% ; 29/70) and 5 were hemispheric. The mean age was 18.3 years (range: 15-25 years). We found that 13/24 (54.2%) of non-brainstem H3K27M mutated tumors were in the thalamus. 25,9% 18,4% 11,1% 16,9% 27,7%
HGG molecular distribution
IDH mutant H3K27 mutant H3G34 mutant BRAF mutant Not otherwise speciFiedWe identified 45 patients harboring H3.3 G34R mutations: 41 (91.1%) GBMs, 2 (4.5%) anaplastic astrocytomas, 1 (2.2%) anaplastic oligodendroglioma and 1 (2.2%) high grade glioma not classified. Most of our H3.3 G34R mutated gliomas were hemispheric (97% ; 37/38) and 1 was located in the brainstem. The mean age was 18.93 years (range : 15-25 years).
H3G34R mutant tumours co-occurring with other mutations
We identified 3/10 published cases of H3G34R mutant tumors also harboring PDGFRA amplification (30%). All were GBMs located in hemispheres and 1/3 (33%) also harbored concominant EGFR amplification. Mean age was 17.7 years (range : 16-19 years). We also identified 8 published cases of ATRX alterations 8/12 (66.7%) represented by 6 GBMs, 1 AOD and 1 HGG. Mean age was 18.9 years (range : 16-22 years). One published case of H3G34R mutant HGG harboured EGFR amplification, it was a GBM located in hemispheres. Finally, 6 cases of H3G34R mutant tumours also had TP53 mutation, all with concominant ATRX alteration, and these tumors distributed between 4 GBMs, 1 HGG and 1 AOD that were mainly hemispheric. The mean age of patients was 18.33 years (range : 16-22 years)
H3K27M mutant tumours co-occurring with other mutations
We have identified 3 cases of H3K27M mutant HGGs that also had PDGFRA amplification, 3/26 (11.5%) ; all tumors were GBMs and wetre located in brainstem with mean patient age of 16 years (range : 15-18 years).
We also have identified 6 cases with ATRX loss, 6/19 (31.6%) that were represented by 2 AAs (brainstem), 2 GBMs, 1 AOA and 1 AOD. 60% of tumors were located in brainstem, 2/5 (40%) were non-brainstem and 3/4 (75%) harbored TP53 mutation concomitantly (1 AOA, 1 AOD and 1GBM). Mean age was 17.67 years (range : 15-21 years).
We identified 9 HGG cases with TP53 mutation, 9/10 (90%). These tumors were represented by 2 AAs, 1 AOA, 3 AODs, 3GBMs ; all tumors were midline and 3/9 (33.3%) also had ATRX loss c (1GB, 1 AOA and 1 AOD). Mean patient age was 17.56 years (range : 15-22 years)
The second most frequent subgroup was IDH mutated tumours and they were the most commonly distrubuted in « adult diffuse glioma» subgroup.
We identified 105/405 IDH (25.9 %) mutant HGGs and they included 53 anaplastic astrocytomas (50.5 %), 43 GBMs (40.9 %), 7 anaplastic oligodendrogliomas (6.6 %), 1 anaplastic oligoastrocytoma (1%) and 1 mixed glioma (1%). Most of the tumors in this subgroup were located in the hemisphere, 25/26 (96.2%). The mean age of patients was 21.8 years (range 15-25).
Figure 4
IDH mutant tumours co-occurring with other mutations
All cases of IDH mutated HGGs were H3F3A wild type and BRAF wild type.
We have identified 4/18 (22.2%) cases of IDH mutated HGGs co-occurring with CDKN2A deletion, 2 were anaplastic astrocytomas and 2 were glioblastomas, with mean age of 21.75 years (range 15-25). Three cases of IDH mutated HGGs with PDGFRA amplification tumours were also reported, 3/14 (21.4%) ; all those cases were glioblastomas. One glioblastoma was located in hemisphere and 1 in the midline and one not specified. Mean age for IDH mutated PDGFRA amplificated glioblastoma
patients was 22.3 years (range 20-24).
6 /64 (9%) of IDH mutant HGGs harbouring 1p/19q codeletion appeared in our study : 1 was anaplastic oligoastrocytoma and 5 were anaplastic oligodendrogliomas. The anaplastic
oligoastrocytoma was hemispherically located. Three tumors were associated with TERT mutation (1AOA and 2 AODs). Mean age of patients with IDH mutant and 1p/19q co deleted tumours was 23.5 years (range 22-25).
We also identified 30/36 (83.3%) cases of IDH mutant tumours with ATRX alteration that included 20 anaplastic astrocytomas, 9 glioblastomas (mostly astrocytomas), 1 anaplastic oligodendroglioma,. 6/6 were hemispheric (AA) and 2/2 TP53 mutated (1AA,1 AOD), the mean age was 22.03 years old (range 18-25). There were 2/3 (66.7%) cases of IDH mutant and TP53 mutant tumors: 1 was anaplastic astrocytoma and 1 was anaplastic oligodendroglioma, all harbour ATRX mutation. Mean age was 21 years (range 19-23). We also identified 3 cases of TERT mutated tumors 3/34 (8.8%) : 1 was
anaplastic oligoastrocytoma, 2 were anaplastic oligodendrogliomas all associated with 1p/19q codel, mean age is 23 years old (range :22-25).
There was 1 case of EGFR amplification 1/28 (3.6%). It was hemispherically located AA in a 22 years old patient. EGFR was not present in IDH mutant GBM.
BRAF mutant tumours
We found that 62/366 (16.9%) of cases were BRAF mutant tumors : 24 eGBMs (38.7%), 21 GBMs (33.9%), 15 aPXAs (24.2%), 1 AA (1.6%) and 1ePXA (1.6%). Most of the BRAF mutant tumors were hemispheric 21/22 (95.5%). Mean age was 19.89 years (range 15-25 years).
BRAF mutant tumours co-occurring with other mutations All BRAF mutants were IDH and H3F3A wild type.
We identified that 22/33 (66.7%) of cases of BRAF mutant HGGs were co-occurring with CDKN2A deletion (8 aPXA and 14 eGBM) We only reported hemispheric location for this subgroup. Mean age was 19.96 years old (range: 15-25). Also, 12/30 (40%) of them harboured TERT. None of the BRAF mutated tumours harboured PDGFRA. 1/13 (7.8%) of BRAF mutant tumours harboured ATRX mutation (AA, hemispheric, 19 years old). 3/8 (37.5%) BRAF mutated tumours harboured TP53 mutation, all were eGBM, hemispheric located. The mean age was 22 years (range 19-25) and 1/3 case (33%) had TERT mutation concomitantly (hemispheric location, 22 years old). 15/45 (33.3%) of BRAF mutant tumors also had TERT mutation that was distributed between eGBMs and aPXAs. All tumors were found to be (4/4) hemispheric located. Mean age was 21.3 (range 15-25) and 12/14 (85.7%) of them had CDKN2A deletion concomitantly. Finally, 5.6% of BRAF mutant tumours harboured EGFR amplification, all were hemispheric eGBMs. Mean age was 17.5 years (range : 16-19 years).
3. Prognostic data
We analyzed mean survival for IDH, H3G34R, H3K27M and BRAF mutant tumours.
Mean survival for patients with IDH mutant tumours had the best prognosis with 52.4 months (range : 9.4 – 160.7 months) Patients with H3G34R mutated tumours had the second best prognosis with a mean survival of 27 months (range : 1-61 months) while patients with H3K27M mutated tumours had a mean survival of 12.2 months (range : 1- 48.2 months). On the other hand, patients with BRAF mutant tumours had an intermediate prognosis since we calculated a mean survival of 19.5 months (range : 1 – 60 months) which is worse than patients with IDH mutant tumours and patients with H3G34R tumours but better than patients with H3K27M mutant tumours. We drew Kaplan-Meier survival curve to illustrate our results (Figure 5).
Figure 5 0 50 100 150 200 0 50 100 IDH mutated months Probability of Survival 0 10 20 30 0 50 100 eGBM months Probability of Survival 0 20 40 60 80 0 50 100 H3.3 G34 mutation months Probability of Survival 0 100 200 300 0 50 100 Anaplastic astrocytoma months Probability of Survival 0 100 200 300 0 50 100 Glioblastoma months Probability of Survival 0 20 40 60 0 50 100 H3.3 K27 mutation months Probability of Survival
Kaplan-Meier curves of OS for H3.3 K27 mutated glioma (A), H3.3 G34 mutated glioma (B), IDH mutated glioma (C), Anaplastic astrocytoma (D), Epithelioid Glioma (E) and
Glioblastoma (F)
A B
C D
IDH mutated tumours had a mean survival of 52.4 months (range : 9.4 – 160.7 monhts). We calculated the mean survival of IDH mutant tumours co-occurring with other mutations and found out that
PDGFRA, 1p/19q and TP53 were adverse prognosis markers with a mean survival of 19, 35.98 and 34.3 months respectively. However, IDH mutant HGGs harbouring CDKN2A deletion or ATRX alteration showed a better mean survival with 53.56 months (range : 12.75-119 months) for IDH mutant tumours with CDKN2A deletions and 63.72 months (range : 5.03- 160.7 months) for IDH mutant tumours with ATRX alteration. This should suggest that CDKN2A deletions and ATRX are favourable prognosis markers in IDH mutant tumours.
Patients with H3G34R mutant tumours had a mean survival of 27 months (range : 1 – 61 months). We calculated the mean survival of H3G34R tumours co-occurring with other mutations and we only got datas for cases of H3G34R mutant tumors harbouring PDGFRA amplification with a mean survival of 17.7 months (range : 16-19 months). This would suggest as in IDH mutant tumours that PDGFRA amplification is also a bad prognosis marker for H3G34R mutant tumours.
Patients with H3K27M tumours had a mean survival of 12.2 months (range : 1 – 48.2 months). We calculated the mean survival of H3K27M mutant tumours co-occurring with other mutations. We had cases of H3K27M mutant tumours harbouring PDGFRA with a mean survival of 12.3 months (range : 12-16 months) and cases of H3K27M mutant tumours harbouring ATRX with a mean survival of 12.06 months (range : 1- 30.5 months). Opposite to IDH mutant tumours, ATRX and PDGFRA alteration had no impact on H3K27M mutant glioma.
Patients with BRAF mutant tumours had a mean survival of 19.5 months (range : 1- 60 months). We calculated mean survival for BRAF mutant tumours co-occurring with other mutations. BRAF mutant tumours co-occurring with TP53 mutation have a mean survival of 12.1 months (range 3.3-24) and BRAF mutant tumours co-occurring with TERT mutation had a mean survival of 9.58 months (range 3-24 months). Even worse, patients harboring HGGs with CDKN2A deletion had a mean survival of 5.65 months (range 4.3-7 months). This indicates that TERT, TP53 mutations and CDKN2A deletion are bad prognosis factors in BRAF mutant tumours. On the other hand, BRAF mutant tumours show a better prognostic factor when associated with EGFR amplification, with a mean survival of 21 months (range : 19-23 months).
Discussion
In our study, the most common alterations in HGG, in the specific subgroup of AYAs were the « pediatric histone mutant » subgroup with 29.5% of H3F3A mutated tumours. H3.3 mutation was previously reported to be specific for HGG and in particularly in glioblastoma in the pediatric subset (40%), while it is rare for adult GBMs to harbour H3.3 mutation (5%) (67). It suggests that H3.3 mutation is an early event of primary malignancy and this mutation frequency decrease with age, however it is still relativelly frequent in AYA’s patients.
H3K27M mutation was the most common genetic alteration with 17.9% of HGGs harboring H3K27M mutation in AYA’s patients. However, this mutation was less frequent in our AYA subset than the 30% H3K27M mutated HGG mentioned in pediatric publications (3,63,69) and more frequent than the H3K27M mutated tumors in adults which is a very rare event in this patient population (60). As
previously described H3K27M mutation mostly occurs in the midbrain which was confirmed also in our analysis (93%) (3,63,69). Also, publications mentioned that up to 78% of DIPGs are H3K27M mutant and less than 25% are non-brainstem gliomas (69) which were different to our results with H3K27M mutation reported in 51.4% brainstem tumors and 41.4% of non-brainstem H3K27M mutated tumours. We found an increased of non-brainstem located H3K27M tumors compared to pediatrics patients, mostly in thalamus which represented 54.2% of our non-brainstem H3K27M mutated tumors. Mean age of our AYA patients with H3K27M mutation was 18.3 years, which is more than pediatric patients with H3K27M mutated tumors mean age (10.5 years) (56) but less than adults with H3K27M mutated tumors with a mean age of 32 years old (68). This suggest that
H3K27M mutation could occur at any age even if it is more frequent in pediatric patients. Molecular alterations in the H3K27M mutated tumour subgroup were similar between childrens and AYAs with ATRX alterations appearing in 31.6% of our AYA cohort and it was previously mentioned that this mutation also occurs in 30% pediatric H3K27M mutated tumours (69). Also, TP53 was present in 90% of our H3K27M mutated tumours sample while the previous study mentioned this co-occurring
alteration present in only 82% of pediatric’s H3K27M mutated HGGs (67). MGMT promoter methylated and H3K27M mutated HGGs represented 12.5% of our AYA patients while MGMT methylation is known to be rare in pediatric H3K27M mutated tumours (70). This may indicate the difference of mean survival found between H3K27M mutated tumours in pediatrics, AYAs, and adults patients with an increasing mean survival with increasing age (less than a year, 12.2 months, 20
H3G34R mutant HGGs represented 11.1% of AYA which is also less than the 15% mentioned in prior studies in pediatrics poplation (69). As mentioned before H3G34R mutation was not found in DIPGs regardless age group and was nearly always in hemispheric location as we found in our AYA’s cohort (97%) and this corrresponds to previous publications (69). Pediatric patients with H3G34R mutated tumors had a mean age of 18 years old (56) while in our AYAs with H3G34R mutated tumors mean age is 18.93 years old . This is relevant that H3G34R mutation appears at an older age than H3K27M mutated pediatric HGG but are restricted to young age patients and never occur in adults tumours as H3K27M mutated tumours do. Also, alterations in H3G34R mutated tumours diverge with ATRX alteration known to be found in almost all pediatric H3G34R mutated tumours (67) while it was less frequent in AYA with only 66.7% AYA H3G34R mutated tumors. This could indicate ATRX alterations as an early event on gliomagenesis of H3.3 mutated tumours and more specifically to H3K27M mutated tumors.
MGMT promoter methylation is present in 73% of AYA which is quite frequent as in pediatric's H3G34R mutated tumours according to literature (9,10).
Second most frequent alterations in HGGs were « IDH mutant adult subgroup » with 25.9% IDH mutated tumours which are more than adults 10% IDH mutant gliomas and more than pediatrics tumours who almost never harbour IDH mutation, mentioned in literature (56,60).
Oligodendroglioma
IDH mutated and 1p/19q co-deleted tumors were reported to comprise 30-40% of adult HGGs (56) and represented (16/473) only 3.4% of our AYA cohort. This suggest that oligodendrogliomas are more frequent in older patiens. TERT mutation was found in 94% of ODs in adults literature (32) while it was only present in (2/4)50% of AYAs OD IDH mutant 1p/19q co deleted tumors and didn't appear in children IDH mutated 1p/19q co deleted OD (77) . Therefore, this suggest that TERT mutation rate in oligodendrogliomas increases with age. This could indicate that TERT is a secondary alteration in the progression of oligodendroglioma to their mature state.
Astrocytoma
TP53 mutation was present in 80% of IDH mutant AAs and IDH mutant GBMs (56) but we only had information for 1 case so could not make a conclusion about this alteration in GBMs in AYA. Also grade 2-3 IDH mutant astrocytoma harbor ATRX mutation in 78% of case and TERT mutation was present in only 20% of cases (32) while we found that ATRX alteration was present in 95.2% of cases and TERT mutation was not detcted in our AYA cases.
Similarly, IDH mutant GBMs frequently harbor ATRX mutation (up to 60%) and TERT mutation is present only in 12% of cases (32) and only in 2% of pediatric DIPGs (66) that is relevant to our findings in AYA with the manority of IDH mutant GBM’s harboring ATRX alteration (90%) and there were no TERT mutations. Therefore, this suggests that the rate of TERT mutations increases with age regardless IDH status (34) and that our AYAs subset is more similar to the pediatrics GBMs. ATRX alteration was found in 20% pediatric GBM (56) however IDH mutation in pediatric HGGs does not appear in children bellow 14 years (5) this suggest that ATRX alteration is rare in pediatric patients bellow 14 years old. ATRX mutation also increases with age and is specific to IDH mutation. BRAFv600E mutation is present in 3% of adults GBMs and 20% of pediatric GBMs (74). Our AYA GBM patients harbored BRAF in only 9.7% of cases, which indicate that BRAFv600E mutations is more common in young population.
Also, BRAFv600E mutation occurs mostly in children, and it was previously mentioned that the majority of eGBM harbor BRAFv600E mutation (up to 93%), TERT promoter mutation (at 71%) and CDKN2A deletion (at 79%) (38). AYA eGBMs were harboring BRAFv600E mutation in 88.9% of cases, TERT promoter mutation in 41.7% of cases and CDKN2A deletion in 76.2% of cases. PXA also harbor BRAFv600E mutation in 50-78% of cases, CDKN2A deletion in 60-83% of cases while TERT mutation only occurs in only 4% of PXAs and 23% of aPXAs (38). AYAs patients with aPXA harbor BRAFv600E in 68.2% of cases, TERT mutation in 31.6% of cases and CDKN2A deletion in 55% of cases.
Conclusion
Our work had been part of the GLIADOME study (78), analyzing specifically molecular alterations in HGGs of AYAs patients. This work allows us to find that AYA’s HGGs representing an
heterogeneous group of tumors harboring pediatrics as well as adults molecular mutations with a predomance for pediatrics subgroup’s alterations.
1. AYAs HGGs are heterogeneous clinicopathological and molecular subgroup of tumors. Main molecular alteration was H3F3A mutation (29.5%) with predominance for H3K27M mutation (17.9%) representing « pediatric histone mutant » subgroup. H3K27M mutation found in 41.4% non-brainstem HGGs with a predominance for thalamus location (54.2%). We also had 25.9% IDH mutated HGGs which represents the « adult IDH mutant » subgroup. IDH mutant 1p/19q codeleted tumors were rare (3.4%). Also we found out some BRAF mutant tumors (9.7%) which are mostly from « pediatric subgroup ».
2. Adults HGGs and pediatrics HGGS have distinct molecular alteration patterns. Pediatric HGGs have a predominance for H3.3 mutation. 30% of pediatric HGGs harbor H3K27M mutation and 15% harbor H3G34R mutation. H3K27M mutated tumors appear mostly in brainstem at 78% and only 25% in nonbrainstem location while H3G34R appear almost exclusively in hemisphere. Also H3K27M mutated tumors are affecting youngest pediatric population with a mean age of 10.5 years, while H3G34R mutated tumors have a mean age of 18 years old at diagnosis. Adults HGGs almost never harbor this H3.3 alteration (5%) which is specific to pediatrics gliomas. However, IDH1 mutated tumors are more present in adult group than pediatric patients who only harbor IDH mutated tumors in less than 10% cases. Also, BRAFv600E mutation is present in only 3% of adults GBMs and 20% of pediatric GBMs, defining BRAFv600E mutation as a pediatric mutation mostly.
3. AYA gliomas represent a heterogeneous group of tumours, mostly pediatric. It seems more important to treat those young adults based on their molecular characteristics and then refer them to neuro-oncologist in pediatry or specialized in adult neuro-oncology, more than treating them based on their age of diagnosis.
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