ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis

TL;DR: In this article, the role of X-linked (ATRX) status in the molecular classification of anaplastic gliomas and its impact on survival in the biomarker cohort of the NOA-04 clinical trial was explored.

Abstract: Mutation/loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) expression has been described in anaplastic gliomas. The present study explored the role of ATRX status in the molecular classification of anaplastic gliomas and its impact on survival in the biomarker cohort of the NOA-04 anaplastic glioma trial. Patients (n = 133) of the NOA-04 trial were analyzed for ATRX expression using immunohistochemistry. ATRX status was correlated with age, histology, isocitrate dehydrogenase (IDH), 1p/19q, alternative lengthening of telomeres (ALT) and O6-methylguanine-DNA methyltransferase (MGMT) status, and the trial efficacy endpoints. Loss of ATRX expression was detected in 45 % of anaplastic astrocytomas (AA), 27 % of anaplastic oligoastrocytomas (AOA) and 10 % of anaplastic oligodendrogliomas (AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive with 1p/19q co-deletion. The ALT phenotype was significantly correlated with ATRX loss. ATRX and 1p/19q status were used to re-classify AOA: AOA harboring ATRX loss shared a similar clinical course with AA, whereas AOA carrying 1p/19q co-deletion shared a similar course with AO. Accordingly, in a Cox regression model including ATRX and 1p/19q status, histology was no longer significantly associated with time to treatment failure. Survival analysis showed a marked separation of IDH mutant astrocytic tumors into two groups based on ATRX status: tumors with ATRX loss had a significantly better prognosis (median time to treatment failure 55.6 vs. 31.8 months, p = 0.0168, log rank test). ATRX status helps better define the clinically and morphologically mixed group of AOA, since ATRX loss is a hallmark of astrocytic tumors. Furthermore, ATRX loss defines a subgroup of astrocytic tumors with a favorable prognosis.

Summary

Introduction

• ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis.
• The prognostic or predictive impact of mutations of the isocitrate dehydrogenase 1 and 2 genes (IDH1/2), hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter and co-deletion of 1p and 19q have been extensively characterized [26].
• The prognostic value of ATRX status in different tumor entities has remained controversial:.

ATRX immunohistochemistry

• ATRX immunohistochemistry polyclonal rabbit antibody, dilution 1:400, product code HPA001906, Sigma-Aldrich, St. Louis, MO, USA) was performed using an automated immunostainer (Benchmark Ultra, , Tucson, AZ, USA) and standard protocols including pretreatment using Cell Conditioning 1 buffer for 52 min and standard signal amplification.
• Evaluation was performed by two observers (BW and DC) simultaneously on a multi-headed microscope and scoring was done in consensus.
• Endothelial cells, Wiestler et al. ‐6‐    cortical neurons and infiltrating inflammatory cells were generally positive and served as internal positive controls.
• Cases with negative tumors cells in which vessel cells and neurons were not stained were not evaluated and not considered for further statistical evaluation (n = 13 cases).
• ALT fluorescence in situ hybridization Telomere specific fluorescence in situ hybridization (FISH) was done using a standard formalin-fixed paraffin-embedded FISH protocol [20], using a FITC peptide nucleic acid telomere probe from Dako.

Statistics

• The co-occurrence of ATRX loss and reference histology, ALT, IDH1/2 and p53 mutations, MGMT promoter methylation and 1p/19q co-deletion was assessed using Fisher’s exact test.
• ATRX loss occurred almost exclusively in tumors harboring IDH mutations (42 / 98 IDH mutated tumors, 43%).
• In a univariate Cox regression model, reference histology was significantly associated with time to treatment failure (TTF), the primary endpoint of the NOA-04 trial (Table 3a).
• Point mutations in the promoter of the TERT gene increase telomerase expression and have been shown to occur frequently in gliomas.

Figures

Table 2. Molecular characteristics of anaplastic gliomas by ATRX status

Table 1. Baseline patient characteristics

Table 3. Uni- and multivariate Cox regression models for TTF a) Univariate Cox model of reference histology

Full text

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Year:2013
ATRXlossrenestheclassicationofanaplasticgliomasandidentiesa
subgroupofIDHmutantastrocytictumorswithbetterprognosis
Wiestler,B;Capper,D;Holland-Letz,T;Korshunov,A;vonDeimling,A;Pster,SM;Platten,M
;Weller,M;Wick,W
Abstract:Mutation/lossofalpha-thalassemia/mentalretardationsyndromeX-linked(ATRX)expres-
sionhasbeendescribedinanaplasticgliomas.ThepresentstudyexploredtheroleofATRXstatusin
themolecularclassicationofanaplasticgliomasanditsimpactonsurvivalinthebiomarkercohortof
theNOA-04anaplasticgliomatrial.Patients(n=133)oftheNOA-04trialwereanalyzedforATRX
expression using immunohistochemistry.ATRX status was correlatedwith age,histology,isocitrate
dehydrogenase(IDH),1p/19q,alternativelengtheningoftelomeres(ALT)andO6-methylguanine-DNA
methyltransferase(MGMT)status,andthetrialecacyendpoints.LossofATRXexpressionwasde-
tectedin45%ofanaplasticastrocytomas(AA),27%ofanaplasticoligoastrocytomas(AOA)and10
%ofanaplasticoligodendrogliomas(AO).ItwasmostlyrestrictedtoIDHmutanttumorsandalmost
mutuallyexclusivewith1p/19qco-deletion.TheALTphenotypewassignicantlycorrelatedwithATRX
loss.ATRXand1p/19qstatuswereusedtore-classifyAOA:AOAharboringATRXlosssharedasimilar
clinicalcoursewithAA,whereasAOAcarrying1p/19qco-deletionsharedasimilarcoursewithAO.
Accordingly, inaCoxregression modelincludingATRXand1p/19qstatus, histologywasno longer
signicantlyassociatedwithtimetotreatmentfailure. Survivalanalysisshowedamarkedseparationof
IDHmutantastrocytictumorsintotwogroupsbasedonATRXstatus:tumorswithATRXlosshada
signicantlybetterprognosis(mediantimetotreatmentfailure55.6vs.31.8months,p=0.0168,log
ranktest).ATRXstatushelpsbetterdenetheclinicallyandmorphologicallymixedgroupofAOA,since
ATRXlossisahallmarkofastrocytictumors.Furthermore,ATRXlossdenesasubgroupofastrocytic
tumorswithafavorableprognosis.
DOI:https://doi.org/10.1007/s00401-013-1156-z
PostedattheZurichOpenRepositoryandArchive,UniversityofZurich
ZORAURL:https://doi.org/10.5167/uzh-79916
JournalArticle
PublishedVersion
Originallypublishedat:
Wiestler,B;Capper, D;Holland-Letz, T;Korshunov,A;vonDeimling,A;Pster, SM;Platten, M;
Weller,M;Wick,W(2013).ATRXlossrenestheclassicationofanaplasticgliomasandidentiesa
subgroupofIDHmutantastrocytictumorswithbetterprognosis.ActaNeuropathologica:Epubaheadof
print.
DOI:https://doi.org/10.1007/s00401-013-1156-z

Wiestleretal.‐1‐

ATRX loss refines the classification of anaplastic gliomas and identifies a
subgroup of IDH mutant astrocytic tumors with better prognosis
Benedikt Wiestler
1,4
, David Capper
2,5
, Tim Holland-Letz
7
, Andrey Korshunov
2,5
,
Andreas von Deimling
2,5
, Stefan Michael Pfister
3,6
, Michael Platten
1
, Michael Weller
8
and Wolfgang Wick
1,4
1
Departments of Neurooncology,
2
Neuropathology and
3
Pediatric Hematology and
Oncology, University of Heidelberg, Im Neuenheimer Feld, and
4
Clinical Cooperation
Unit Neurooncology,
5
Clinical Cooperation Unit Neuropathology,
6
Divisions of
Pediatric Neurooncology and
7
Biostatistics, German Cancer Research Center, D-
69120 Heidelberg, Germany;
8
Department of Neurology, University Hospital Zurich,
Frauenklinikstrasse, CH-8091 Zürich, Switzerland
Corresponding Author:
Wolfgang Wick, Department of Neurooncology, Neurology Clinic & National Center
for Tumor Disease, University of Heidelberg and German Cancer Research Center,
Im Neuenheimer Feld 400, D-69120 Heidelberg, phone +49 (0)6221 56 7075, fax
+49 (0)6221 56 7554, email: wolfgang.wick@med.uni-heidelberg.de
Key words: ATRX, IDH, 1p/19q, anaplastic glioma, MGMT

Wiestleretal.‐2‐

ABSTRACT
Mutation / loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX)
expression has been described in anaplastic gliomas. The present study explored the
role of ATRX status in the molecular classification of anaplastic gliomas and its
impact on survival in the biomarker cohort of the NOA-04 anaplastic glioma trial.
Patients (n = 133) of the NOA-04 trial were analyzed for ATRX expression using
immunohistochemistry. ATRX status was correlated with age, histology, isocitrate
dehydrogenase (IDH), 1p/19q, alternative lengthening of telomeres (ALT) and O6-
methylguanine-DNA methyltransferase (MGMT) status, and the trial efficacy
endpoints.
Loss of ATRX expression was detected in 45% of anaplastic astrocytomas (AA), 27%
of anaplastic oligoastrocytomas (AOA) and 10% of anaplastic oligodendrogliomas
(AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive
with 1p/19q co-deletion. The ALT phenotype was significantly correlated with ATRX
loss. ATRX and 1p/19q status were used to re-classify AOA: AOA harboring ATRX
loss shared a similar clinical course with AA whereas AOA carrying 1p/19q co-
deletion shared a similar course with AO. Accordingly, in a Cox regression model
including ATRX and 1p/19q status, histology was no longer significantly associated
with time to treatment failure. Survival analysis showed a marked separation of IDH
mutant astrocytic tumors into two groups based on ATRX status: Tumors with ATRX
loss had a significantly better prognosis (median time to treatment failure 55.6 vs.
31.8 months, p = 0.0168, log rank test).
ATRX status helps to better define the clinically and morphologically mixed group of
AOA, since ATRX loss is a hallmark of astrocytic tumors. Furthermore, ATRX loss
defines a subgroup of astrocytic tumors with a favorable prognosis.

Wiestleretal.‐3‐

INTRODUCTION
The WHO classification, based solely on morphological criteria [16], may be
increasingly supplemented with defined molecular aberrations [5, 19, 22, 24]. These
might help to resolve the discrepancy between classification and clinical outcome. A
prototypical example for this discrepancy are anaplastic oligoastrocytomas, which are
histologically and molecularly mixed gliomas, displaying both astrocytic and
oligodendroglial features [17, 18]. As a result, the diagnosis of oligoastrocytomas is
subject to high interobserver variation [12].
The prognostic or predictive impact of mutations of the isocitrate dehydrogenase 1
and 2 genes (IDH1/2), hypermethylation of the O6-methylguanine-DNA
methyltransferase (MGMT) promoter and co-deletion of 1p and 19q have been
extensively characterized [26]. Recently, mutations and (consequently) loss of
expression of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) have
been reported in one third of pediatric glioblastomas [20] and 7% of adult
glioblastomas [7]. Virtually all mutations are inactivating and lead to a loss of protein
expression [7, 15]. Mechanistically, loss of ATRX and death-domain associated
protein (DAXX) are important factors for a telomere maintenance mechanism not
involving telomerases (alternative lengthening of telomeres (ALT)). A number of
studies have analyzed the frequency of ATRX mutation / loss of expression and its
correlation with molecular markers in glioma: In pediatric diffuse intrinsic pons
gliomas, ATRX mutation occurred in 2 of 22 cases [10]. High-throughput
resequencing revealed ATRX mutations to be present in 12 of 32 (37.5%) grade II
and III gliomas examined. In this cohort, ~ 70% of IDH mutant, 1p/19q intact tumors
carried ATRX mutations [9]. ATRX loss assessed by immunohistochemistry has been
reported as 27% in grade II and 41% in grade III astrocytomas in adults, compared to
a mutation rate of 33% and 46%, respectively [15]. Another recent study described a

Wiestleretal.‐4‐

significantly higher mutation rate of 73% in 44 anaplastic astrocytoma [8]. Notably,
these studies showed that loss of ATRX expression is more prevalent in astrocytic
than in mixed glial tumors and rare in pure oligodendrogliomas. Loss of ATRX
expression is highly associated with IDH mutation and almost mutually exclusive with
1p/19q co-deletion, the hallmark of oligodendroglial tumors.
The prognostic value of ATRX status in different tumor entities has remained
controversial: In pancreatic neuroendocrine tumors, altered ATRX expression has
been associated with a more aggressive phenotype [25]. In childhood acute myeloid
leukemia carrying FLT3 mutations, higher ATRX expression was associated with a
superior outcome [13]. In gliomas, ATRX mutation has been associated with a better
prognosis in a retrospective cohort of grade II, III and IV tumors [8].
The Neurooncology Working Group of the German Cancer Society (NOA)-04 trial
explored the optimal sequence of radiotherapy and alkylating chemotherapy in
patients with newly diagnosed, centrally confirmed anaplastic gliomas. NOA-04
revealed initial radiotherapy or chemotherapy to be comparable and IDH mutations
as a novel positive prognostic factor in anaplastic gliomas [27]. In the NOA-04 trial
cohort, we sought to determine the prognostic value of ATRX status as well as its
implications for molecular classification of anaplastic gliomas, especially mixed
oligoastrocytomas.

Frequently asked questions

Q1. What are the contributions in "Atrx loss refines the classification of anaplastic gliomas and identifies a subgroup of idh mutant astrocytic tumors with better prognosis" ?

The present study explored the role of ATRX status in the molecular classification of anaplastic gliomas and its impact on survival in the biomarker cohort of the NOA-04 anaplastic glioma trial. 1007/s00401-013-1156-z Wiestler et al. ‐1‐ ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis Benedikt Wiestler, David Capper, Tim Holland-Letz, Andrey Korshunov, Andreas von Deimling, Stefan Michael Pfister, Michael Platten, Michael Weller and Wolfgang Wick Departments of Neurooncology, Neuropathology and Pediatric Hematology and Oncology, University of Heidelberg, Im Neuenheimer Feld, and Clinical Cooperation Unit Neurooncology, Clinical Cooperation Unit Neuropathology, Divisions of Pediatric Neurooncology and Biostatistics, German Cancer Research Center, D69120 Heidelberg, Germany ; Department of Neurology, University Hospital Zurich, Frauenklinikstrasse, CH-8091 Zürich, Switzerland Corresponding Author: Wolfgang Wick, Department of Neurooncology, Neurology Clinic & National Center for Tumor Disease, University of Heidelberg and German Cancer Research Center, Im Neuenheimer Feld 400, D-69120 Heidelberg, phone +49 ( 0 ) 6221 56 7075, fax +49 ( 0 ) 6221 56 7554, email: wolfgang. Furthermore, ATRX loss defines a subgroup of astrocytic tumors with a favorable prognosis. 

Q2. How did the authors assess ATRX expression in a subset of the NOA-04 trial?

Using immunohistochemistry to assess ATRX expression in a subset of the NOA-04 trial patient population, the authors detected ATRX loss in approximately 40% of AA and 25% of mixed AOA. 

Q3. What was the t test used to compare the mean age of the two groups?

One-way ANOVA was employed to compare mean age between different groups followed by a pairwise t test comparison with Bonferroni correction. 

Q4. What is the way to evaluate the prognostic value of ATRX status?

Given the superior clinical course of oligodendroglial tumors, it seems to be important to evaluate the prognostic value of ATRX status only in astrocytic tumors as the authors did here. 

Q5. How many patients were available for the NOA-04 biomarker cohort?

The present NOA-04 biomarker cohort comprised 133 of the 274 patients of the NOA-04 intention-to-treat (ITT) population for which unstained paraffin slides were available. 

Q6. What is the prevalence of ATRX deficiency in IDH mutants?

While two studies reported a ~ 70% prevalence of ATRX deficiency in IDH mutant, 1p/19qWiestler et al. ‐11‐    intact tumors [8, 9], another study reported a < 50% prevalence [15]. 

Q7. What is the significance of the ATRX loss in the NOA-04 trial?

The high prevalence of ATRX loss in this tumor subset, as reported by others, argues for ATRX deficiency as a class-defining feature. 

Q8. How many patients were IDH wild type?

The authors grouped the 133 NOA-04 samples accordingly, resulting in 40 patients with ATRX loss, 40 patients with 1p/19q co-deletion, 17 patients with IDH mutation onlyWiestler et al. ‐12‐    and 31 patients who were IDH wild type. 

Q9. What is the way to classify mixed gliomas?

ATRX in conjunction with 1p/19q status may help to unequivocally classify mixed gliomas, whose diagnoses are subject to relevant inter-observer variation, as either astrocytic or oligodendroglial. 

Q10. What was the prognostic value of reference histology?

Upon incorporation of both ATRX and 1p/19q status into the model (Table 3b), the prognostic value of reference histology was no longer significant, while both ATRX loss and 1p/19q co-deletion were associated with TTF. 

Q11. What was the primary endpoint of the NOA-04 trial?

In a univariate Cox regression model, reference histology was significantly associated with time to treatment failure (TTF), the primary endpoint of the NOA-04 trial (Table 3a). 

Q12. What is the significance of ATRX status in pediatric gliomas?

To this extent, ATRX status is important as it both helps to re-classify mixed oligoastrocytic tumors and defines a subgroup of astrocytic tumors with a superior clinical course. 

Q13. What was the consensus of the panel review of the NOA-04 trial?

A retrospective panel review of 150 patients from the EORTC 26951 trial, which did not impose such a restrictive central histology, confirmed on consensus only 8% of the local AOA diagnoses [12]. 

Q14. What is the recent discovery of ATRX in gliomas?

ATRX mutation and loss of expression, which has just recently been discovered in gliomas [7], might further refine this classification. 

Q15. What is the association between ATRX loss and TTF?

Patients with tumors with ATRX loss had a longer time to treatment failure (median TTF 55.6 months [95% CI 45.1 months – to not reached], n = 40) than patients with IDH mutant tumors with ATRX expression (median TTF 31.8 months [95% CI 2.8 months – to not reached], n = 9, p = 0.0168, log rank test; Fig. 3e).