Brain Tumour Epidemiology in Austria

and the Austrian Brain Tumour Registry

Doctoral thesis at the Medical University of Vienna

for obtaining the academic degree

Doctor of Medical Science

Submitted by

Dr. Adelheid Wöhrer

Supervisor:

Ao. Univ. Prof. Dr. Johannes A. Hainfellner

Institute of Neurology

Medical University of Vienna

Waehringer Guertel 18–20, 1097 Vienna

Vienna, 01/2012

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CONTENTS

Abstract……………..………………………………………………………………...………….…. 3

Zusammenfassung……..…………………………………………………………………….…… 4

Definition of Epidemiological Terms …………………………………………………….……. 5

Background………………………..………………………………………………………….……. 6

1. Cancer control and cancer registries………………………………………………………............ 6

2. Primary Brain Tumours………………..……………………………………………………….……. 7

Summary of Results – Austrian Brain Tumour Registry………………………………....... 9

1. Set-up of a population-based brain tumour registry in Austria…..………………………….…... 9

2. Population-based epidemiology and tissue-based analyses.……………………………….…..12

3. Neuropathological characterisation of new tumour entities..………..…………..………….…...15

4. Increasing use of molecular markers in brain tumour epidemiology……………………………18

5. Outcome Surveillance………………………………………………………………………………. 20

Added Value & Future Perspectives……………………………….…………………………. 26

Peer-reviewed publications…..…………………………………………………….……………40

1. The Austrian Brain Tumour Registry: a cooperative way to establish a population-based

brain tumour registry. Wöhrer et al, Journal of Neurooncology 2009, 95:401–11…………… 40

2. Incidence of atypical teratoid/rhabdoid tumours in children: a population-based study by the

Austrian Brain Tumour Registry, 1996–2006. Wöhrer et al, Cancer 2010, 116:5725–32……67

3. Embryonal Tumour with Abundant Neuropil and True Rosettes (ETANTR) with loss of

morphological but retained genetic key features during progression. Wöhrer et al,

Acta Neuropathologica 2011, 122:787–90……………………………………………………….. 85

4. FISH-based detection of 1p 19q codeletion in oligodendroglial tumours: procedures and

Protocols for neuropathological practice – a publication under the auspices of the

Research Committee of the European Confederation of Neuropathological Societies.

Wöhrer et al, Clinical Neuropathology 2011, 30:47–55………………………………………… 95

Curriculum vitae………………………………………………………………………………… 113

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ABSTRACT

Cancer registries provide incidence and mortality data on patients with cancer at the

population-level. Cancer registration is most often restricted to the group of malignant

neoplasms, whereas information on benign and intermediate tumours is generally not

available. Brain tumours however differ from other sites by 1., the large spectrum of different

tumour types, and 2., the exclusive localisation in proximity to eloquent areas with

considerable neurological comorbidity and mortality irrespective of their biological behaviour.

In order to obtain a comprehensive overview of the brain tumour burden specialised brain

tumour registries, which provide information on all brain tumours types, have emerged in

several countries.

Within the frame of this thesis we summarise the Austrian experience on the establishment

of such a specialised brain tumour registry – the Austrian Brain Tumour Registry (ABTR). We

report on its initial steps – from consensus and commitment of the Austrian Society of

Neuropathology, formation of an interdisciplinary team of experts, setup of the infrastructure

including data confidentiality issues, to the sustained support of the Austrian neurooncology

community and major cooperation with the Austrian National Cancer Registry. ABTR differs

from other registries by its scientific setting and neuropathological background warranting

strong expertise in brain tumour typing and tissue-based research. Thereby, ABTR

constitutes also a virtual brain tumor biobank.

By having achieved these steps we further demonstrate that ABTR provides valid and

accurate population-based incidence and survival data for individual brain tumour types. First

scientific contributions by ABTR address various neuroepidemiological issues of national and

international relevance. We first estimate the exact incidence of rare tumour entities, refine

key diagnostic criteria of newly proposed tumour entities, advocate common standards for

testing of molecular markers, and assess medical progress via real-life outcome analyses.

The diverse scientific contributions highlight the enormous scientific potential of ABTR for

continued work.

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ZUSAMMENFASSUNG

Krebsregister erheben Inzidenz- und Mortalitätsdaten zu Krebserkrankungen, die in einer

definierten Population (populations-basiert) auftreten. Die Erfassung beschränkt sich meist

auf die Gruppe der malignen Neoplasien, während gutartige oder intermediäre Neubildungen

nicht regelmäßig erfasst werden. Hirntumore unterscheiden sich von anderen

Krebserkrankungen durch 1., ihr außerordentlich großes Spektrum an verschiedenen

Tumorentitäten und 2., ihre Lokalisation im Zentralen Nervensystem in Nähe zu eloquenten

Arealen und der damit verbundenen neurologischen Komorbidität und Mortalität, unabhängig

von ihrem intrinsischen biologischen Verhalten. Um ein komplettes Bild zum tatsächlichen

Krankheitsaufkommen zu erhalten, sind in mehreren Ländern spezialisierte Hirntumorregister

entstanden, die epidemiologische Daten zu allen Hirntumor-Typen zur Verfügung stellen.

Innerhalb dieser Arbeit fassen wir die Erfahrungen mit der Gründung eines derartigen

Registers in Österreich zusammen – das Österreichische Hirntumorregister (ABTR). Wir

beschreiben die ersten Schritte – von der Zustimmung und Konsensfindung innerhalb der

Österreichischen Gesellschaft für Neuropathologie, der Bildung eines interdisziplinären

Expertengremiums, der Infrastrukturentwicklung einschließlich Datenschutz-rechtlich

relevanter Fragestellungen, bis hin zur kontinuierlichen Unterstützung durch die nationale

Neuroonkologie-Gemeinschaft und der Zusammenarbeit mit dem Österreichischen

Nationalen Krebsregister. Einer der wesentlichen Punkte, in denen sich ABTR von anderen

Registern seiner Art unterscheidet, ist sein wissenschaftliches, neuropathologisch geprägtes

Umfeld, das diagnostische Expertise und Erfahrung mit gewebsbasierten Untersuchungen

einbringt. Auf diese Art wird ABTR auch zu einer populations-basierten, virtuellen Hirntumor-

Biobank.

Nachdem die Grundvoraussetzungen geschaffen worden sind, zeigen wir weiter, dass ABTR

qualitativ hochwertige populations-basierte Daten generiert, die erstmals umfassende

Analysen zum Auftreten und Überleben von Hirntumorpatienten in Österreich zulassen. Die

ersten wissenschaftlichen Beiträge von ABTR adressieren unterschiedliche

neuroepidemiologische Fragestellungen von nationaler und internationaler Bedeutung. Wir

erheben erstmals exakte Inzidenzdaten zu seltenen Tumortypen, präzisieren diagnostische

Kriterien von neu vorgeschlagenen Tumorentitäten, schlagen einheitliche Standards zur

molekulargenetischen Testung von Biomarkern vor, und zeigen durch populations-basierte

Überlebensanalysen den medizinischen Fortschritt im Zeitverlauf auf. Die unterschiedlichen

wissenschaftlichen Beiträge illustrieren das enorme wissenschaftliche Potential von ABTR

für weiterführende künftige wissenschaftliche Tätigkeit.

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DEFINITION OF EPIDEMIOLOGICAL TERMS

Morbidity

Morbidity refers to the incidence or prevalence of a disease within a defined population and

time period.

Incidence

Number of newly diagnosed cases over a specified time period. Incidence rates for cancer

are mostly expressed as n / 100,000 person-years.

Prevalence

Number of diseased cases in a defined population at a particular time point.

Mortality

Mortality rates quantify the number of cases who have died from the disease during a

specified time period.

Survival

Survival percentages express the probability of surviving for a specified time period.

Age-adjustment

Diseases occur at different rates in different age groups. Age-adjusting is a statistical method

that warrants comparability of rates across different populations with different age structures.

Age adjustment of rates is achieved by weighting crude rates with a common standard (see

Standard population).

Standard population

Different standard populations are in use for age adjustment of rates. The US 2000 and

WHO standard populations are used by ABTR to warrant international comparability.

Death-certificate-only (DCO) cases

DCO cases are cancer cases, which are only identified from death certificates.

ICD-O classification

The International Classification of Diseases for Oncology provides a standardised coding

scheme for individual neoplasms based on tumour morphology, localisation, and biological

behaviour.(Fritz et al., 2000)

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BACKGROUND

Cancer control & cancer registries

The global monitoring of variations in cancer incidence and survival is a major issue of the

International Agency for Research on Cancer (IARC)� at the World Health Organisation

(WHO).(Curado et al., 2007; http://www.iarc.fr) Cancer surveillance aims at the prevention of

cancer, improved patient care, risk factor analysis, and help for policy decision makers. At

the community level, population-based registries provide data on newly diagnosed cancer

cases. In Austria, the Austrian National Cancer Registry is in charge of cancer registration,

which is legally mandatory for the group of malignant neoplasms (Austrian National Cancer

Registration Law, 1969). Regional or national incidence data are compiled at the IARC and

made available to the general public and research communities (CANCERMondial

http://www-dep.iarc.fr/).

Basic principles of cancer registration

Cancer registration comprises the standardised collection of disease-relevant information on

affected individuals. Essential information includes personal identifiers, tumour

characteristics, basis and date of diagnosis, and date of death. The quality of the recorded

data largely depends on the completeness of case ascertainment, which relates to active and

passive case reporting from all available sources (hospital admission records, pathology

reports etc).(Fritz et al., 2000; IACR)

ICD-O codes & WHO classification as basis for global cancer registration

Information on tumour characteristics with regard to its morphology, topographical

localisation, and behaviour is essential. In order to warrant direct comparability across cancer

registries common registration standards have been implemented i.e. International

Classification of Diseases for Oncology (ICD-O).(Fritz et al., 2000) The gold standard for the

recording of tumour morphology remains the histopathological diagnosis (basis of diagnosis:

microscopically verified). It is primarily based on morphological tumour characteristics such

as cytology, grade of dedifferentiation, and proliferative activity. The WHO classification in

turn provides consensus diagnostic criteria for individual tumour entities, which serve as a

reference for pathologists globally.(Louis et al., 2007) According to the grade of malignancy

and the associated prognosis of the patients, tumours are assigned to WHO grades I-IV.

Only in cases without histological confirmation, the clinically or radiologically suspected

tumour diagnosis is recorded (non-microscopically verified).(Fritz et al., 2000)

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Primary brain tumours

Primary brain tumours comprise a large spectrum of clinically and genetically heterogeneous

disorders. According to the latest WHO classification more than 120 distinct brain tumour

entities are recognised (Louis et al., 2007 see also http://www.pubcan.org). Based on their

biological behaviour, brain tumours fall into three broad categories: 1. benign tumours [ICD-

O0], 2. tumours with intermediate or uncertain behaviour [ICD-O1], and 3. malignant tumours

[ICD-O3].(Fritz et al., 2000)

Both children and adults are affected. In childhood, brain tumours comprise the most

common group of solid neoplasms and constitute the most common cause of cancer-related

death in this age group.(Peris-Bonet et al., 2006; Stiller, 2007)�The vast majority of brain

tumours occur sporadically.(Louis et al., 2007) Only a minor proportion of 5% of all brain

tumours are attributable to genetic tumour syndromes, including the more common Li

Fraumeni syndrome (mutations in the TP53 gene), neurofibromatosis types 1 and 2 (caused

by NF1 and NF2 mutations respectively), or the rhabdoid tumour predisposition syndrome

(SMARCB1/INI1 mutations).(Farrell and Plotkin, 2007; Louis et al., 2007) So far, only few

environmental risk factors have been established e.g. long-term sequelae of ionising

radiation.(Wrensch et al., 1993; Sadetzki et al., 2005; Umansky et al., 2008) An additional

potential risk from low-frequency electromagnetic fields (e.g. mobile phone use) has been

suggested and is currently under debate.(Inskip et al., 2010; The Interphone Study Group,

2011; Aydin et al., 2011; Cardis et al., 2011; Feychting, 2011) In the vast majority of brain

tumours the underlying aetiological risk factors remain however obscure.

Affected individuals most often present with headache, focal neurological deficits, epileptic

seizures, or signs of increased intracranial pressure.(Pallud et al., 2010; de Groot et al.,

2011) The prognosis of brain tumour patients varies markedly according to the histological

and behavioural properties of the tumour.(Louis et al., 2007) Whereas benign brain tumours

may be cured by neurosurgical resection only, tumours with uncertain or malignant behaviour

typically require adjuvant therapy i.e. chemo- and/or radiation therapy. However, also non-

malignant tumours, which account for more than 50% of all brain tumours (Figure 1), may be

associated with a significant neurological comorbidity and mortality due to their proximity to

eloquent areas and space occupying effects within the brain. Benign tumours may show

malignant transformation over time and patients may suffer considerable treatment-related

side effects e.g. long-term cognitive decline following radiation therapy.(Dirks et al., 1994;

McCarthy et al., 2009; Soussain et al., 2009; Pflugbeil et al. 2011) Moreover, there has been

growing scientific interest over the last years not only in the group of malignant brain tumours

but also in certain benign tumour types such as acoustic neuroma.(Larjavaara et al., 2011)

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Figure 1. Distribution of primary brain tumours according to tumour behaviour.

More than 50% of primary brain tumours are either benign neoplasms or tumours of intermediate behaviour,

which have not been systematically registered prior to ABTR. Source: ABTR 2011.

In Austria, cancer registration is legally mandatory only for the group of malignant brain

tumours. Yet, no epidemiological data on benign and intermediate neoplasms are available.

In order to obtain a comprehensive overview of the brain tumour burden in Austria

registration of all brain tumour types is essential.

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SUMMARY OF RESULTS – THE AUSTRIAN BRAIN TUMOUR REGISTRY

Step 1. Set-up of a population-based brain tumour registry in Austria

The Austrian Brain Tumour Registry (ABTR) has been initiated in 2005 under the auspices of

the Austrian Society of Neuropathology. Its primary objectives include

1. the generation of comprehensive incidence data on all primary brain tumours in Austria,

2. the conduction of descriptive epidemiological studies on individual tumour entities,

3. the maintenance of high data-quality through a high degree of case ascertainment,

4. the direct linkage of case registration to medical practise and research.

The central coordination of ABTR is held with the Institute of Neurology at the Medical

University of Vienna. Cooperation partners include all Austrian neuro-oncology units i.e.

departments of neuropathology, neurosurgery, neurooncology, and neurology. A list of

participating institutions is provided in the appendix (page 37). Furthermore, ABTR is closely

cooperating with the Austrian National Cancer Registry with regard to mutual case

complementation. A detailed structure of ABTR is provided in figure 2.

Figure 2. ABTR structure.

The flexible ABTR structure provides direct interaction between the coordination centre, its partner institutions and

the Austrian National Cancer Registry.

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Definition of cases of interest is a crucial factor for every cancer registry. ABTR has

established the following case entry criteria based on the current ICD-O classification (Fritz

et al., 2000):

1. ICD-O morphology: all primary brain tumours

2. ICD-O topography: brain (C71.0-C71.9), meninges (C70.0-C70.9), spinal cord, cranial

nerves and other parts of the central nervous system (C72.0-C72.9), pituitary and pineal

glands (C75.1-C75.3), and olfactory tumours of the nasal cavity (C30.0)

3. ICD-O behaviour: benign (ICD-O0), intermediate (ICD-O1), and malignant (ICD-O3)

tumours

4. Permanent residence in Austria

ABTR multidisciplinary team

A multidisciplinary team of experts from various disciplines has emerged including surgical

neuropathologists, epidemiologists, biomedical statisticians, consulting clinicians, information

technology experts, data protection officials, and lawyers. ABTR is primarily run by

neuropathologists, who provide strong expertise in brain tumour typing. This pathology-

based approach further enables direct linkage of population-based brain tumour

epidemiology with tissue-based research (virtual brain tumour biobank).

ABTR data confidentiality and case registration procedures

The cooperation with ABTR partner institutions is reglemented via bilateral data protection

and service contracts, which were elaborated together with data confidentiality officials and

lawyers in accordance to the Austrian law and approved by the Austrian Data Protection

Commission (http://www.dsk.gv.at/DesktopDefault.aspx?alias=dsken). Ethical approval is

provided by local ethics committees. ABTR follows the guidelines on confidentiality for

population-based cancer registration by the IARC (http://www.encr.com.fr/

confidentiality.pdf). The list of abstracted parameters for cancer cases include: personal

identifiers, place of permanent residence, gender, date of birth, date of diagnosis, original

histopathological diagnosis, topographic localisation of the tumour within the CNS, and date

of death. ABTR data are abstracted from each partner institution, checked for plausibility and

duplicate registrations, pseudonymised, stored at the central database, and notified with the

Austrian National Cancer Registry.

ABTR database and data analyses

The ABTR database was programmed with the Filemaker® software. It is continuously

adapted, reprogrammed, and extended according to current needs and research projects.

For data analyses current versions of the following statistical software programmes are in

use: SPSS®, SAS®, and Microsoft Excel®. Spatial epidemiological analyses are conducted

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with the Rapid Inquiry Facility tool, a freely available software application for ArcGIS®,

provided by the Small Area Health Statistics Unit within the Department of Epidemiology and

Biostatistics at Imperial College, London (http://www.sahsu.org). Its implementation and

adaptation for ABTR purposes has been elaborated together with the medical information

scientist Christian Auer MSc (diploma thesis available at http://permalink.obvsg.at/

AC08363185).

Subsequent to the basic setup of ABTR, the systematic case registration process has

started. The first year dataset (2005) was carefully analysed and validated against the rates

of the largest primary brain tumour registry i.e. the Central Brain Tumor Registry of the

United States (CBTRUS http//www.cbtrus.org):

The Austrian Brain Tumour Registry: a cooperative way to establish a population-based

brain tumour registry

Wohrer et al, J Neurooncol. 2009; 95:401-11.

In 2005, a total of 1,688 incident brain tumour cases were registered in the Austrian

population (8.2 million inhabitants), resulting in an age-standardised overall brain tumour

incidence rate of 18.1 per 100,000 person-years. Less than 9% of the cases were identified by

death certificates (DCO-rate 8.4%). The incidence was slightly higher in females (18.6 per

100,000 person-years) compared with males (17.8 per 100,000 person-years). The proportion

of non-malignant brain tumours (i.e. benign and intermediate lesions) accounted for 51.3%.

Median age at diagnosis was 58.4 years, whereas 5.6% of brain tumours were diagnosed in

children and adolescents under the age of 18 years. The most common brain tumour types

included benign meningioma (WHO grade I, 25.2%), followed by glioblastoma (WHO grade

IV, 20.1%), and pituitary adenoma (8.9%). Direct comparison of initial ABTR findings with

the by far larger and representative CBTRUS dataset showed similar or slightly higher

incidence rates in Austria.

In sum, initial ABTR incidence rates were highly similar to the representative CBTRUS rates,

indicating a high degree of case ascertainment and validity of ABTR data.

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Step 2. Combining population-based brain tumour epidemiology with tissue-based

research

Paediatric brain tumours

The neuropathological spectrum of paediatric brain tumours differs markedly from that of

adults. In children and adolescents embryonal brain tumours (e.g. medulloblastoma, CNS

PNET), low-grade gliomas (e.g. pilocytic astrocytoma), ependymomas, epilepsy-associated

tumours (e.g. gangliogliomas), and germ-cell tumours are far more prevalent.(Rickert and

Paulus, 2001; Louis et al., 2007) As special attention needs to be paid to the developing

brain, the clinical management of paediatric patients differs considerably with regard to

diagnostic procedures and therapeutic options.(Pollack and Jakacki, 2011)

One peculiar brain tumour type predominantly arising in young children is atypical

teratoid/rhabdoid tumour (ATRT). ATRT is an exceedingly rare, highly malignant embryonal

brain tumour (WHO grade IV), which was defined an entity in 1996 (Rorke et al., 1996) and

subsequently introduced to the WHO classification in 2000.(Kleihues and Cavanee, 2000) It

was associated with a highly aggressive disease course with reported survival times from 0.5

to 11 months.(Bonnin et al., 1984; Rorke et al., 1995; Rorke et al., 1996; Burger et al., 1998;

Tekautz et al., 2005; Haberler et al., 2006) However, patients with ATRT benefit from novel

intensified treatment protocols.(Hilden et al., 1998; Hilden et al., 2004; Chen et al., 2006;

Gardner et al., 2008; Chi et al., 2009) These consist of maximal safe neurosurgical resection,

followed by adjuvant radiation therapy, conventional and intrathecal chemotherapy, as well

as high-dose chemotherapy and stem cell rescue (Slavc et al., 2009; von Hoff et al., 2011) –

treatment protocols, which differ substantially in intensity from standard regimens for other

embryonal brain tumours such as medulloblastoma. As further therapeutic decisions are

primarily based on the histopathological diagnosis, a correct initial diagnosis of ATRT is

crucial. However, the histopathological features of ATRT are complex with divergent

differentiation along neuroectodermal, mesenchymal, and epithelial lineages (Figure

3).(Louis et al., 2007) The morphological hallmark of ATRT are ´rhabdoid´ tumour cells,

which however may be scarce or even absent, leaving the tumour with an uncharacteristic

primitive neuroectodermal ´PNET-like´ appearance (Figure 4). Indeed, ATRTs have been

frequently mistaken for other primitive neuroectodermal tumours notably

medulloblastoma.(Burger et al., 1998; Haberler et al., 2006)

The characteristic genetic alteration of ATRT is the mutation and/or deletion of the

SMARCB1 (hSNF5/INI1) gene at chromosomal locus 22q11.23 (Biegel et al., 1999), which

leads to the loss of the SMARCB1/INI1 protein expression within the tumour cells. The recent

introduction of a monoclonal antibody against the SMARCB1/INI1 protein (Ab No. 612110,

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BD Transduction Laboratories) has proven a sensitive and specific tool for the diagnosis of

ATRT.(Haberler et al., 2006; Judkins et al., 2004)

Figure 3. Complex histopathological features of ATRT.

Characteristic histopathological features of ATRT include rhabdoid tumour cells (Haematoxylin & Eosin stain, left)

with divergent differentiation along primitive neuroectodermal (Immunoreactivity IR for neurofilament NFP and

glial fibrillary acidic protein GFAP), mesenchymal (IR for vimentin VIM and smooth muscle actin SMA), and

epithelial lineages (IR for epithelial membrane antigen EMA and cytokeratin CK). Meanwhile, the

immunohistochemical confirmation of the SMARCB1/INI1 protein loss plays a key-role in the diagnosis of ATRT

(Note the retained SMARCB1/INI1 protein expression in endothelial cells which serve as internal control).

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Figure 4. Characteristic versus uncharacteristic histopathological features of ATRT.

Rhabdoid tumour cells (left) with eccentric eosinophilic cytoplasms are the pathological hallmark of ATRT but may

be scarce or absent in some cases, leaving the tumour with an uncharacteristic primitive neuroectodermal

morphology (right). However, immunonegativity for the SMARCB1/INI1 protein (small inserts) points to the correct

diagnosis of ATRT in both cases.

Both the absolute rarity of ATRT according to prior single- and multicentre experiences

together with the problem of frequent misdiagnoses constituted limiting factors for the

generation of accurate population-based epidemiological data. This lack of knowledge was

addressed within the following ABTR study:

Incidence of atypical teratoid/rhabdoid tumours in children: a population-based study

by the Austrian Brain Tumour Registry, 1996-2006

Woehrer et al, Cancer 2010; 116:5725-32.

By combining a large population-based data set of malignant paediatric brain tumours with a

central histopathology review including the systematic immunohistochemical analysis of the

SMARCB1/INI1 protein expression, ABTR was able to first estimate the exact incidence of

these rare tumours. The age-standardised incidence rate of ATRTs refers to 1.38 / 1,000,000

person-years in children. Of note, peak incidence was found in very young children under the

age of two years. In this age group, ATRTs were as frequent as the more common embryonal

brain tumours CNS primitive neuroectodermal tumour and medulloblastoma. Of interest,

approximately 50% of ATRTs have not been initially recognised but were only identified

upon histopathology review. This underlines the importance of the systematic analysis of the

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SMARCB1/INI1 protein expression as diagnostic tool for ATRT. Most importantly, those

children, whose tumours were initially correctly diagnosed as ATRTs and who received an

appropriate intensified treatment, had a significantly better outcome compared with those,

whose tumours were initially misdiagnosed (p=0.0469).

In sum, the population-based ABTR study on the incidence of ATRTs raised awareness among

clinicians and pathologists of the high incidence of ATRTs in very young children and thereby

aimed at the optimisation of the diagnostic and therapeutic management of the affected

patients.

Step 3. Neuropathological characterisation of new brain tumour entities

The WHO classification of brain tumours comprises meanwhile more than 120 distinct

entities and the list is still expanding.(Louis et al., 2007) The definition of new tumour entities

requires distinct, independently confirmed and reproducibly recognisable morphological key

features and clinical characteristics (e.g. age, localisation, growth behaviour). The

introduction as distinct disease entity to the WHO classification and the subsequent

assignment of a unique ICD-O code is the prerequisite for the systematic registration of

these tumours and enables population-based epidemiological studies.(Fritz et al., 2000;

Louis et al., 2007)

Entities such as pilomyxoid astrocytoma or rosette-forming glioneuronal tumour of the fourth

ventricle have been added to the current 7th edition of the WHO classification.(Louis et al.,

2007) Yet, other tumour entities were only recently proposed and are still under debate.

Among these candidate entities is ´Embryonal Tumour with Abundant Neuropil and True

Rosettes´ (ETANTR). Like ATRT, ETANTR is a highly aggressive embryonal brain tumour,

which predominantly affects young children. Its morphological key features i.e. neuropil

islands and multilayered ´ependymoblastic´ rosettes (Figure 5) were first described as

peculiar growth pattern of CNS primitive neuroectodermal tumour by Eberhart et al in

2000.(Eberhart et al., 2000) Since then, its distinct morphological features have been

independently recognised by different groups. To date, approximately 50 cases have been

reported in the literature.(Eberhart et al., 2000; Spina et al., 2006; Fuller et al., 2006;

Dunham et al., 2007; Al-Hussain and Dababo, 2009; Gessi et al., 2009; Pfister et al., 2009;

Buccoliero et al. 2010; Korshunov et al., 2010; Ferri Niguez et al., 2010; Kleinschmidt-

Demasters et al., 2011; La Manjila et al., 2011; Wang et al., 2011; Al-Hussaini et al., 2011).

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Figure 5. Morphological key-features of ETANTR.

Morphological key features of ETANTR include primitive neuroectodermal tumour cells (left) with the formation of

hypocellular neuropil islands (centre) and multilayered rosettes (right).

Consequently, the debate has arisen whether ETANTR constitutes a distinct disease entity

rather than an unusual growth pattern.

The most striking morphological key feature of ETANTR is the presence of its characteristic

multilayered rosettes. However, these rosettes are not entirely specific for ETANTR as they

are also found in another embryonal brain tumour i.e. ependymoblastoma. The latter shares

a primitive neuroectodermal morphology but lacks the formation of paucicellular neuropil

islands. Thus, the morphological delineation of ETANTR from ependymoblastoma seems

problematic.(Judkins and Ellison, 2010) Only recently, new genetic findings provided insights

in the biology of both tumours. The amplification of the microRNA cluster C19MC at

19q13.42 was reported as genetic hallmark lesion of ETANTR.(Li et al., 2009; Pfister et al.,

2009; Korshunov et al., 2010) However, the same amplification is present in the vast majority

of ependymoblastomas, which suggests a common origin of both lesions.(Korshunov et al.,

2010) Thus, the fusion of ETANTR and ependymoblastoma to a single disease entity

(associated with one ICD-O morphology code) has been proposed.(Korshunov et al., 2010;

Woehrer et al., 2011)

The picture is further complicated by reports on single cases of ETANTR with

uncharacteristic or inconsistent morphological features, i.e. initial absence, only focal

presence, or secondary loss of neuropil islands and ependymoblastic rosettes.(Dunham et

al., 2007; Buccoliero et al., 2010) This variable morphology renders ETANTR prone to

misdiagnosis. As patients with ETANTR however require an intensified treatment approach,

an accurate histopathological diagnosis is of high clinical relevance. Whether and to what

extent the molecular-genetic testing of the hallmark amplification at 19q13.42 contributes to

diagnostic accuracy has not been addressed so far.

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The following case observation contributed considerably to a better understanding of the

morphology and genetics of ETANTR, thereby refining key diagnostic criteria of this tumour:

Embryonal Tumour with Abundant Neuropil and True Rosettes with loss of

morphological but retained genetic key features during progression: a case report.

Woehrer et al, Acta Neuropathol 2011; 122:787–90.

We have had the opportunity to follow a case of ETANTR in a 33-month-old female patient

from initial diagnosis to post-mortem examination. The clinical characteristics of the patient,

including young age at diagnosis and unfavourable outcome despite intensified treatment

were in accordance with previous reports. However, careful morphological evaluation of

primary and recurrent tumour tissues demonstrated marked malignant progression during the

disease course with evolution of a large cell / anaplastic phenotype, which might in parts be

attributable to the administered cytotoxic treatment. Of note, all morphological key features of

ETANTR i.e. neuropil islands and multilayered rosettes were secondarily lost during the

disease course. However, in parallel to the morphological evaluation matched genetic

analyses on initial and recurrent tumour tissues were performed. Genetic findings were

validated by the use of multiple techniques i.e. fluorescence in situ hybridisation and SNP

array (Affymetrix SNP Array 6.0). Both techniques independently demonstrated, that the

genetic hallmark amplification at 19q13.42 remained stable throughout the disease course.

Moreover, the comprehensive genetic approach enabled a better delineation of ETANTR from

the more common medulloblastoma by the lack of MYCC or MYCN gene involvement. In

sum, the detailed clinical, morphological, and genetic follow-up of this case demonstrated,

that morphological key features of ETANTR are less consistent when compared with the

genetic hallmark lesion.

Hence, systematic genetic testing of 19q13.42 contributes to diagnostic accuracy of ETANTR

and is recommended in every tumour with primitive neuroectodermal morphology in infants

in order to identify cases of ETANTR.

To date, accurate population-based epidemiological data on ETANTR are not available. So

far reported cases and small case series indicate a slight female preponderance and point to

a highly aggressive disease course.(Eberhart et al., 2000; Spina et al., 2006; Fuller et al.,

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2006; Dunham et al., 2007; Al-Hussain and Dababo, 2009; Gessi et al., 2009; Pfister et al.,

2009; Buccoliero et al. 2010; Korshunov et al., 2010; Ferri Niguez et al., 2010; Kleinschmidt-

Demasters et al., 2011; La Manjila et al., 2011; Wang et al., 2011; Al-Hussaini et al., 2011)

ABTR will address this issue in an upcoming study. As a first step, morphological

reevaluation of the population-based series of malignant paediatric brain tumours newly

diagnosed from 1996 – 2006 will be performed. As the morphological key features of

ETANTR are less consistent compared with the genetic hallmark lesion, systematic genetic

testing of 19q13.42 will be performed using fluorescence in situ hybridisation. As adequate

DNA probes are not yet commercially available, in-house probes are currently being

developed. This comprehensive morphological and genetic approach will first generate

accurate incidence and survival data of children with ETANTR, which is of relevance not only

for pathologists but also clinicians.

Step 4. Increasing use of molecular markers in brain tumour epidemiology

Over the last years an increasing number of molecular candidate markers, which provide

diagnostic, prognostic and/or predictive information on individual patients, has emerged in

cancer research.�Genetic testing of these markers has become increasingly relevant within

the setting of clinical trials and epidemiological studies.(Brody et al., 2011; Grossmann and

Samowitz, 2011; Loi et al., 2011; Petera et al., 2011; Tang et al., 2011) Still, whether and

how fast these candidate markers translate from basic research into routine diagnostic use

largely depends on their analytical and clinical test performances.(Hainfellner and Heinzl,

2010) With regard to primary brain tumours candidate markers include the 1p 19q codeletion

in oligodendrogliomas (Cairncross et al., 1998), the MGMT promoter methylation status in

glioblastoma (Hegi et al., 2005), the IDH1 mutation in diffuse low-grade gliomas (Parsons et

al., 2008; Yan et al., 2009), and the BRAF fusion gene in pilocytic astrocytoma.(Jones et al.,

2008)

Among the abovementioned markers, the 1p 19q codeletion probably constitutes the best

characterised marker so far. Its presence is strongly associated with oligodendrogliomas and

therefore also of diagnostic value.(Aldape et al., 2007; Gadji et al., 2009) It constitutes a

strong favourable prognostic and predictive marker, as patients, whose tumours harbour the

combined deletion, typically show a prolonged progression-free survival and enhanced

response to adjuvant therapies.(Cairncross et al., 1998; Kros et al., 2007)

The histopathological diagnosis of oligodendroglial tumours is based on the characteristic

clear-celled appearance of the tumour cells, which discernes oligodendrogliomas from

astrocytomas.(Louis et al., 2007) This ´honey-comb´ morphology might however be less

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accentuated or in case of mixed oligoastrocytoma intermingled with non-clear celled, fibrillary

astrocytic tumour cells, which results in considerable interobserver variability (Figure 6).

Hence, over the last years neuropathologists tended to favour the diagnosis of

prognostically favourable oligodendrogliomas over astrocytomas in order to ensure further

genetic testing. On a national scale the incidence of oligodendrogliomas seemed to increase

over the last years, while at the same time the proportion of astrocytomas

declined.(McCarthy et al., 2008)

Figure 6. Histopathological features of diffuse gliomas.

Oligodendroglioma (left) displays the characteristic clear-celled appearance, composed of round cells with

perinuclear halos. Oligoastrocytoma (centre) with mixed oligodendroglial and astrocytic tumour components.

Fibrillary astrocytoma (right) displays small cells with scant cytoplasm embedded in a dense fibrillary matrix.

After the positive prognostic value of the 1p 19q codeletion was recognised by

neurooncologists, neuropathologists have found themselves increasingly confronted with the

demand for genetic testing. Yet, several molecular techniques are available to analyse the 1p

19q status. Commonly used tests include fluorescence in situ hybridisation (FISH), PCR-

based loss-of-heterozygosity (LOH) analysis, bacterial artificial chromosome (BAC)-array

comparative genomic hybridisation (aCGH), and multiplex ligation-dependent probe

amplification (MLPA). However so far, no common standards for genetic testing exist with

regard to the different techniques.

Within the following work, we proposed common standard procedures for FISH-based 1p

19q testing, as FISH is a robust and cost-efficient technique which is available to the majority

of neuropathological laboratories:

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FISH-based detection of 1p 19q codeletion in oligodendroglial tumors: procedures and

protocols for neuropathological practice – a publication under the auspices of the

Research Committee of the European Confederation of Neuropathological Societies

(EURO-CNS).

Woehrer et al, Clin Neuropathol 2011; 30:47–55.

Within this work we provided a practical ´hands on´ approach to FISH-based 1p 19q testing

by summarising standard protocols and procedures – from commonly used locus-specific

probes and technical protocols to the neuropathological interpretation of hybridisation results.

Thereby we aimed at the implementation of common standards across various

neuropathological laboratories, including those non-academic laboratories, which do not have

a research focus on brain tumours.

Hence, common standards for FISH-based 1p 19q testing warrant comparability of test

results across the various neuropathological laboratories, which in turn enables a better

refinement of the diagnosis of oligodendroglioma and stratification into homogeneous patient

populations within clinical trials.

Step 5. Outcome surveillance

While controlled, randomised clinical trials are indispensable to confirm a benefit of a

therapeutic intervention, population-based outcome analyses enable a valid assessment of

the effect size, which actually reaches common practise. In contrast to clinical trials, which

represent the outcome of a highly preselected and favourable patient cohort, cancer

registries estimate survival at the population-level irrespective of inclusion and exclusion

criteria. With regard to brain tumours ABTR provides such real-life survival estimates of brain

tumour patients at the population level.

Real-life survival of glioblastoma patients after introduction of a new therapy standard

– Preliminary results

Background

Glioblastoma (GBM) constitutes the most common malignant brain tumour in adults. In

Austria, approximately 300 individuals are affected each year (age-adjusted incidence rate

3.4 / 100,000 person-years).(Wohrer et al., 2009) From the 1970s onwards, the standard

therapy of GBM consisted of maximal-safe neurosurgical resection followed by

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radiotherapy.(Walker et al., 1980) A population-based study on 987 GBM patients in the

Canton of Zurich from 1980–1994 documented the exceedingly poor outcome with median

overall survival (OS) times of only 4.9 months.(Ohgaki and Kleihues, 2005) Three years after

diagnosis only 1.2% of the patients were alive (long-term survivors). Age at diagnosis was

found a strong prognostic factor, older age being associated with a significantly shorter

survival (age < 50 years: median OS 8.8 months, age > 80 years: median OS 1.6 months).

While the role of radiotherapy was clearly established, the role of chemotherapy was much

more controversial. The interest in chemotherapy changed only in the late 1990s after the

novel alkylating agent temozolomide became available and was shown active against GBM.

New standard of care in 2005

In 2005, the EORTC-NCIC NCT00006353 randomised phase III trial in newly diagnosed

GBM patients demonstrated a significant improvement in survival (median OS 14.6 months),

when concomitantly treated with temozolomide and radiation.(Stupp et al., 2005) Eligibility

criteria for this trial consisted of age 18–70 years, WHO performance status < 2, and

adequate haematologic, renal, and hepatic function. Combined treatment was well tolerated

by the patients, increased the 2-year survival from 10% to 26%, and hence, set a new

therapy standard. Since then, the survival benefit from combined treatment has been

independently confirmed by single- and multicentre studies.(Bauchet et al., 2009; Erpolat et

al., 2009; Gauden et al., 2009) First population-based studies indicate an increase in survival

in the general population (median OS approximately 14 months) suggesting that the

beneficial effect of the combined therapy has translated to the general population.(Johnson

and O'Neill, 2011; Koshy et al., 2011; Lam and Chambers, 2011) However, current practice

and patient outcome have not yet been evaluated in the Austrian population and compared

with the Stupp trial population.(Stupp et al., 2005)

Patients & Methods

A total of 696 primary GBM cases in patients aged > 18 years newly diagnosed in 2005 and

2006 were identified in the Austrian population. Less than 5 percent of the cases were

reported through death certificates. The total patient cohort was followed until 31st December

2010. Follow-up data on the vital status and/or date of death were obtained from the Austrian

mortality statistics. Kaplan-Meier survival analyses were calculated using SPSS® version

16.0. Spatial survival analysis was performed, rates were displayed in tertiles and plotted for

individual Austrian health care districts using RIF® software.

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Preliminary results

OS of Austrian GBM patients was 32.8% at 1 year, 10.9% at 2 years, 4.6% at 3 years and

1.0% at 4 years (figure 6). Median OS of the total patient cohort was 6.0 months. Patients

aged 18–69 years had a significantly better outcome (p<0.0001) when compared with

patients above 70 years of age with median OS of 8 months (95%CI 6.5, 9.5) versus 5

months (95%CI 4.1, 5.9), and a 3-year OS rate of 6.2% versus 1.0%.

Figure 7. Population-based outcome of Austrian GBM patients

Kaplan-Meier survival curves of the total cohort of Austrian GBM patients (n=696, 2005–2006) and stratified

according to age (0–69 years, 70+ years).

When compared to the historic population-based cohort of the canton of Zurich (1980—1994)

a two–threefold increase in survival rates was noted in the contemporary ABTR patient

cohort (2005–2006) (Figure 8, Table 1). This increase in survival was most pronounced in

the fraction of long-term survivors (three years after diagnosis 4.2 % versus 1.2% of patients

were alive). Direct comparison of ABTR data with results of the Stupp trial population (2000-

2002) showed an inferior patient outcome in the Austrian population (Figure 8, Table 1). At 1-

and 2-years after diagnosis survival rates were twice as high within the Stupp trial population.

At 3-years after diagnosis (long-term survivors) the difference in survival was even most

pronounced (fourfold higher rates).

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Figure 8. Survival comparison of the contemporary ABTR patient cohort with the historic

population-based cohort from Zurich and the Stupp trial population.

Kaplan Meier survival curves illustrate an increase in survival in the ABTR cohort (n=696, 2005–2006) (centre)

when compared to the historic population-based cohort by Ohgaki et al (n=987, 1980-1994) (left) but less

favourable outcome compared to the combined treatment arm (blue line) of the Stupp trial population (n=573,

2000-2002) (right).

Table 1. Comparison of overall survival rates of GBM patients at 1 year, 2 years, and 3 years

after diagnosis in three different patient cohorts.

Overall

survival

Zurich

1980–1994

ABTR

2005–2006

Stupp

2000–2002

1 yr 17.7 % 33.0 % 61.1 %

2 ys 3.3 % 10.9 % 27.2 %

3 ys 1.2 % 4.2 % 16.0 %

Preliminary spatial epidemiological analyses of 1-year and 3-year overall survival rates of the

ABTR patient cohort indicate potential regional disparities across Austrian healthcare regions

(Figure 9).

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Figure 9. Regional variation in survival of glioblastoma patients across Austrian health care

regions.

1-year (A) and 3-year (B) overall survival rates of GBM patients indicate potential regional disparities across

Austrian healthcare regions. Red dots: Austrian neurooncology units (n=11). Survival rates are displayed in

tertiles, light-green: favourable outcome, dark-green: unfavourable outcome.

Interpretation of preliminary findings and next steps

In 2005, the Stupp trial set a new therapy standard for GBM patients by introducing

concomitant and adjuvant temozolomide in addition to radiotherapy to the postoperative

patient management.(Stupp et al., 2005) The observed increase in survival (median overall

survival 14.6 months) was confirmed by single- and multicentre studies thereafter.(Erpolat et

al., 2009; Gauden et al., 2009) However, due to restricted patient cohorts and relevant

selection bias these studies cannot be considered representative for the general unselected

population. Population-based survival analyses are only provided by means of cancer

registries. Indeed, early reports of cancer registries from France, the United States, and

Canada indicate an increase in survival at the population-level, which almost equals the

results of the Stupp trial.(Bauchet et al., 2010; Johnson and O'Neill, 2011; Koshy et al., 2011;

Lam and Chambers, 2011) Based on those results we figured a similar increase in survival

in Austrian GBM patients after 2005. Indeed, we found a two- to threefold increase in survival

when compared to a historic cohort (Ohgaki and Kleihues, 2005), which confirms a

considerable medical progress since the 1980ies. However, when compared to the Stupp

trial, survival of GBM patients in the Austrian population is still significantly worse. In contrast

to the findings from Canada (Lam and Chambers, 2011) and the United States (Johnson and

O'Neill, 2011; Koshy et al., 2011) our results show a delay in the translation of the new

therapy standard to the Austrian population.

In order to determine the underlying factors, we now aim to evaluate the various patterns of

care for Austrian GBM patients from 2005 onwards. The following prognostic factors, which

were shown to be associated with survival, will be analysed for the total GBM patient cohort:

age at diagnosis, extent of resection, tumour localisation within the CNS, perioperative

performance status, and information on the administered therapy modalities. Thereby, we will

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have the opportunity to determine the lag time from first introduction of the new therapy

standard to the nation-wide implementation in the various Austrian neuro-oncology centres.

These patterns of care will be analysed for different age cohorts (e.g. frequency of adjuvant

therapy versus palliation in elderly patients as compared to younger patients) and according

to gender (differences in patterns of care between males and females). Furthermore,

outcome of those Austrian GBM patients, who were recruited on clinical trials will be

compared to those treated off trial. As preliminary spatial analyses indicate potential regional

variations in GBM outcome across Austrian healthcare regions, which might be due to

differences in access to specialised tertiary care centres, this regional variation will be

readdressed with a larger patient cohort and longer follow-up times.

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ADDED VALUE OF THE THESIS & FUTURE PERSPECTIVES

Cancer registries provide incidence and mortality data on patients with cancer at the

population-level. Thereby, they estimate the burden and force of disease and allow for

continuous monitoring of time trends. Cancer registration is usually restricted to the group of

malignant neoplasms, whereas benign and intermediate lesions are not reported at a regular

basis. Brain tumours differ from other cancer types with regard to their exclusive localisation

within the brain, proximity to eloquent areas, and related morbidity and mortality, irrespective

of their biological behaviour. In order to obtain a comprehensive overview of the burden of

disease specialised brain tumour registries have emerged such as the Central Brain Tumour

Registry of the United States (http://www.cbtrus.org) or the French brain tumour data

bank.(Bauchet et al., 2007) Within this thesis we summarised the Austrian experience while

implementing a population-based brain tumour registry – ABTR.

National and international relevance of ABTR

The implementation of ABTR constituted a major effort of the Austrian neurooncology

community. The project has been joined and supported by all Austrian neurooncology units.

Thereby it has emerged as important communication platform between the various centres,

and has contributed considerably to a national neurooncology identity. A multidisciplinary

team of experts has been assembled under the guidance of neuropathologists, who provide

strong expertise in brain tumour typing and tissue-based research. This unique concept of a

population-based brain tumour databank and virtual biobank of tumour tissues discernes

ABTR from other cancer registries. Indeed, ABTR might serve as a role model for other

countries and its scientific contributions are not only recognized by national authorities but

also by the international neurooncology community.

ABTR scientific contributions

After the successful implementation of ABTR all relevant methodical aspects including the

infrastructure and multidisciplinary setting were published together with its initial

epidemiological findings.(Wohrer et al., 2009) For the first time comprehensive incidence

data on all types of brain tumours were generated in Austria. Rates were critically compared

with internationally available data and their validity was confirmed. We then continued to

address various epidemiological aspects of high scientific and clinical relevance.

As brain tumours are especially prevalent in children, where they constitute the most

common cause of cancer-related death (Peris-Bonet et al., 2006; Stiller, 2007), we focused

on this age cohort. Atypical teratoid/rhabdoid tumour (ATRT) is a rather rare but highly

malignant brain tumour, which predominantly occurs in young children.(Burger et al., 1998)

Due to its rarity and complex histomorphology, which carries the problem of frequent

misdiagnoses, there has been a lack of valid epidemiological data on this tumour. This

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problem was addressed by combining a large, population-based dataset of paediatric brain

tumours (spanning an eleven years period) with a central histopathology review. Thereby, we

were first able to estimate the incidence and survival of patients with ATRT.(Woehrer et al.,

2010) Our results were intriguing and raised awareness among clinicians and pathologists of

the high prevalence of ATRT in infants aged less than two years, and relevance of a correct

initial diagnosis for further patient management and outcome.

Likewise, Embryonal Tumour with Abundant Neuropil and True Rosettes (ETANTR) is a rare

paediatric brain tumour, which is associated with a highly aggressive disease

course.(Eberhart et al., 2000) Its distinct morphological features have only recently been

recognised and our experiences are still limited. The distinct clinical and genetic

characteristics however suggest its introduction as disease entity to the upcoming WHO

classification.(Korshunov et al., 2010) The latter lists all tumour types and serves as a

reference for pathologists worldwide. Thereby, it enables systematic research on specific

tumour types including epidemiological and clinical studies. By reporting an exceptional case

of ETANTR in a young female patient we contributed to a better understanding of the

interrelation between morphology and genetics of this tumour and thus aimed at the

refinement of advocated diagnostic criteria and subsequent ICD-O coding.(Woehrer et al.,

2011)

Molecular-genetic biomarkers are increasingly recognised in brain tumour research as they

provide diagnostic, prognostic, and/or predictive information for individual patients. With

regard to brain tumours, candidate molecular markers include among others the MGMT

promoter methylation (Hegi et al., 2005), IDH1 mutation (Parsons et al., 2008), and

codeletion of chromosomal arms 1p and 19q (Cairncross et al., 1998). The 1p 19q codeletion

is highly associated with oligodendroglial tumours and constitutes a strong positive

prognostic factor. As the characteristic clear-celled morphology of oligodendroglial tumours is

however subject to interobserver variability, neuropathologists tended to favour the

prognostically favourable diagnosis of an oligodendroglial over an astrocytic tumour, and the

incidence of oligodendrogliomas seemed virtually to increase.(McCarthy et al., 2008)

Moreover, neuropathologists have been increasingly confronted with the demand of genetic

testing by their medical oncologists. However, no commonly accepted standards for genetic

testing exist so far. Various techniques are available and the interpretation of results varies

considerably among neuropathological laboratories. Within the subsequent work, we

suggested common standards for fluorescence in situ hybridisation-based testing of the 1p

19q codeletion.(Woehrer et al., 2011) These guidelines aim to serve as a practical manual,

which also addresses the needs of non-academic neuropathological laboratories without

special research focus on brain tumours.

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Ongoing projects and future perspectives

The aforementioned findings raised in turn new scientific questions, which led to the

definition of a number of ongoing ABTR projects:

ETANTR: Single case reports and small case series point to an absolute rarity of the

disease. However, incidence data are not yet available. We will conduct a retrospective

histopathology review of the large, population-based paediatric tumour cohort. As the

morphological features are not as consistent as the genetic hallmark lesion, systematic

genetic testing of 19q13.42 will be performed. Thereby, we will first be able to determine the

exact incidence and survival of ETANTR in the paediatric population.

Glioblastoma: Cancer registries are the only source for population-based outcome analyses,

which are considered the gold standard as they represent ´real-life´ survival within a given

population. Increases in survival reflect medical progress through innovations in diagnosis

and therapy, which translated from basic research to the population-level. We focused on the

most common malignant brain tumour in adults i.e. glioblastoma, which is still associated

with an exceedingly poor outcome.(Wohrer et al., 2009a) Preliminary ABTR survival analysis

indicates a two- to threefold increase in survival from the 1980ies to 2005.(Ohgaki and

Kleihues, 2005) However, when compared with the Stupp trial population (Stupp et al.,

2005), which set the current therapy standard, survival of Austrian patients is still

considerably poorer. Moreover, preliminary spatial epidemiological analyses raise the issue

of differences in survival across Austrian health care regions, which might have implications

for national policy makers.

Summary

The so far described findings exemplarily highlight the enormous and diverse scientific

potential of ABTR. It is an invaluable source for accurate incidence and mortality data on

individual brain tumour types, which serve as basis for basic and clinical research. Since the

implementation of ABTR in 2005 more than 8,000 brain tumour cases have been registered

so far. The sustained maintenance of ABTR is of major importance for the national

neurooncology community, Austrian society and national policy makers. Its scientific

contributions however are not only of national relevance but may be of benefit to the entire

brain tumour research community.

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Appendix

List of contributing institutions

Medical University of Vienna

Institute of Neurology, Department of Internal Medicine I, Department of Neurosurgery,

Department of Paediatrics, Department of Radiotherapy

Währinger Gürtel 18–20, 1090 Vienna

Medical University of Graz

Departments of Pathology, Neurosurgery, and Paediatrics

Auenbruggerplatz 2/4, 8036 Graz

Medical University of Innsbruck

Departments of Pathology, Neurosurgery, and Paediatrics

Christoph-Probst-Platz 1, Innrain 52, 6020 Innsbruck

State Neuropsychiatric Hospital Wagner-Jauregg

Departments of Pathology and Neuropathology, Neurosurgery, and Medical Oncology

Wagner-Jauregg-Weg 15, 4020 Linz

Private Medical University Salzburg

Department of Neurosurgery and Paediatrics

Müllner Hauptstraße 48, 5020 Salzburg

State Hospital Klagenfurt

Departments of Pathology and Neurosurgery

St. Veiter Straße 47, 9020 Klagenfurt am Wörthersee

State Hospital Feldkirch

Departments of Pathology, Neurosurgery and Paediatrics

Carinagasse 47, 6800 Feldkirch

Vienna Danube Hospital

Departments of Pathology and Neurosurgery

Langobardenstraße 122, 1220 Vienna

Krankenanstalt Rudolfstiftung

Departments of Pathology and Neurosurgery

Juchgasse 25, 1030 Vienna

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Kaiser Franz Josef Hospital

Department of Neurology

Kundratstraße 3, 1100 Vienna

State Hospital Wiener Neustadt

Departments of Pathology and Neurosurgery

Corvinusring 3-5, 2700 Wiener Neustadt

General Hospital St. Poelten

Departments of Pathology, Neurosurgery and Neurology

Propst-Führer-Straße 4, 3100 St. Pölten

Austrian National Cancer Registry, Statistics Austria

Guglgasse 13, 1110 Vienna

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Funding

The project was supported by the Anniversary fund of the Austrian National bank (project

12268) and MOBIKIDS (7th EU framework project)

Acknowledgements

My sincerest thanks go to the supervisors of this doctoral thesis, who perfectly

complemented each other as scientific mentors and personal characters: to Johannes A.

Hainfellner for his infinite commitment to the project and personal support, to Harald Heinzl

for his accurateness and constant drive, and to Thomas Waldhör, who provided substantial

scientific input with his own charm and humour. I further acknowledge all ABTR

collaborators, without whom this project would have not been possible, for investing their

time and continued efforts. My final thanks go to my host institution – the Institute of

Neurology – a circle of friends.

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PEER-REVIEWED PUBLICATIONS

Journal of Neurooncology 2009; 95: 401-11, IF 2.929

The Austrian Brain Tumour Registry: a cooperative way to establish a population-

based brain tumour registry

Adelheid Wöhrer, MD1,2, Thomas Waldhör, PhD3, Harald Heinzl, PhD4, Monika Hackl5,

Johann Feichtinger, MD6, Ulrike Gruber-Mösenbacher, MD7, Andreas Kiefer, MD8, Hans

Maier, MD9, Reinhard Motz, MD10, Angelika Reiner-Concin, MD11, Bernd Richling, MD12,

Carmen Idriceanu13, Michael Scarpatetti, MD14, Roland Sedivy, MD15, Hans-Christian Bankl,

MD15, Wolfgang Stiglbauer, MD16, Matthias Preusser, MD17, Karl Rössler, MD18, Johannes

Andreas Hainfellner, MD1,2

1Institute of Neurology, Medical University of Vienna, A-1097 Vienna, Währinger Gürtel 18-

20, Austria

2For the Austrian Society of Neuropathology, www.oegnp.at

3Centre for Public Health, Department of Epidemiology, Medical University of Vienna, A-1097

Vienna, Währinger Gürtel 18-20, Austria

4Core Unit for Medical Statistics and Informatics, Section of Clinical Biometrics, Medical

University of Vienna, A-1097 Vienna, Währinger Gürtel 18-20, Austria

5Austrian National Cancer Registry, Statistics Austria, A-1110 Vienna, Guglgasse 13, Austria

6Department of Pathology, Krankenanstalt Rudolfstiftung, A-1030 Vienna, Juchgasse 25,

Austria 7Department of Pathology, Feldkirch State Hospital, A-6807 Feldkirch, Carinagasse 47,

Austria 8Institute of Pathology, State Hospital Klagenfurt, A-9020 Klagenfurt, St. Veiter Strasse 47,

Austria 9Department of Pathology, Medical University of Innsbruck, A-6020 Innsbruck, Christoph-

Probst-Platz Innrain 52, Austria

10Department of Pathology and Neuropathology, State Neuropsychiatric Hospital Wagner-

Jauregg, Linz, A-4020 Linz, Wagner-Jauregg-Weg 15, Austria

11Institute of Pathology, Danube Hospital, A-1220 Vienna, Langobardenstrasse 122, Austria

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12Department of Neurosurgery, Christian Doppler Clinic, Paracelsus Private Medical

University, A-5020 Salzburg, Strubergasse 21, Austria 13Department of Neurology, Christian Doppler Clinic, Paracelsus Private Medical University,

A-5020 Salzburg, Strubergasse 21, Austria 14Institute of Pathology, Medical University of Graz, A-8036 Graz, Auenbruggerplatz 25,

Austria

15Department of Clinical Pathology, General Hospital St. Pölten, A-3100 St. Pölten, Probst-

Führer-Strasse 4, Austria 16Institute of Pathology, General Hospital Wiener Neustadt, A-2700 Wiener Neustadt,

Corvinusring 3-5, Austria 17Department of Internal Medicine I, Medical University of Vienna, A-1097 Vienna, Währinger

Gürtel 18-20, Austria

18For the Task Force for Neurosurgical Oncology, Austrian Society of Neurosurgery

Corresponding author

Johannes A. Hainfellner

Institute of Neurology, Medical University of Vienna

Waehringer Guertel 18-20, 1097 Vienna, Austria

Phone: +43/1/40400-5507

Fax: +43/1/40400-5511

E-mail: johannes.hainfellner@meduniwien.ac.at

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ABSTRACT

In Austria, registration of malignant brain tumours is legally mandatory, whereas

benign and borderline tumours are not reported. The Austrian Brain Tumour Registry (ABTR)

was initiated under the auspices of the Austrian Society of Neuropathology for the

registration of malignant and non-malignant brain tumours. All Austrian neuropathology units

involved in brain tumour diagnostics contribute data on primary brain tumours. Non-

microscopically verified cases are added by the Austrian National Cancer Registry to ensure

a population-based dataset.

In 2005 we registered a total of 1,688 newly diagnosed primary brain tumours in a

population of 8.2 million inhabitants with an overall age-adjusted incidence rate of

18.1/100,000 person-years. Non-malignant cases constituted 866 cases (51.3%). The

incidence rate was higher in females (18.6/100,000) as compared to males (17.8/100,000).

95/1,688 (5.6%) cases were diagnosed in children (<18 years). The most common histology

was meningioma (n=504, 29.9%) followed by glioblastoma (n=340, 20.1%) and pituitary

adenoma (n=151, 8.9%). Comparison with the Central Brain Tumour Registry of the United

States (CBTRUS) database showed high congruency of findings.

The ABTR model led by neuropathologists in collaboration with epidemiologists and

the Austrian National Cancer Registry presents a cooperative way to establish a population-

based brain tumour registry with high quality data. This setting links cancer registration to the

mission of medical practice and research as defined by the World Medical Association in the

Declaration of Helsinki. The continued operation of ABTR will aid in monitoring changes in

incidence and in identifying regional disease clusters or geographic variations in brain tumour

morbidity/mortality.

Keywords:

Brain neoplasm, Epidemiology, Incidence, Survival, Cancer registry

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Definition of epidemiological terms

Incidence rates measure the occurrence of newly diagnosed cases of disease within a

defined time period. Rates are expressed per 100,000 person-years and age-adjusted to the

US 2000 standard population. Age-specific rates are calculated for a subset of a population

and describe the rate of disease in defined age groups. Smoothed incidence rates (SIR) are

calculated by incorporating population size using random effect models (see Methods).

Survival rates are the probability (percents) of surviving for a specified time period.

Death-certificate-only (DCO) cases are cancer cases only identified from the death

certificate.

INTRODUCTION

Global cancer registration is a major concern of the International Agency for

Research on Cancer (IARC, http://www.iarc.fr/) at the World Health Organization (WHO,

http://www.who.int/en/). Its objective is to monitor the global burden, force and management

of disease [1]. In many developed countries, cancer registration including malignant brain

tumours is legally mandatory [2-5]. At the national or regional level, cancer registries are in

charge of this task [6].

With regard to brain tumours, these registries are often the only source of

epidemiological information. Unfortunately, the majority of these registries provide incidence

rates only for malignant brain tumours. Benign and borderline brain tumours are usually not

reported. Furthermore, collective rates are presented for brain tumours as a group, whereas

detailed information on individual tumour types is not easily available (e.g. [1]). Only few

specialized registries provide systematic and detailed data on individual brain tumour entities

[7-10].

In Austria, as in other European countries, cancer registration is a governmental

issue [Austrian National Cancer Registry, Statistics Austria (www.statistik.at/web-en/)] and is

restricted to malignant brain tumours (ICD-O /3). However, there is growing public and

scientific interest in malignant and non-malignant brain tumour entities, e.g. due to the

reported increase of incidence rates [11-13] or the possible risk of mobile phone use [14-17].

The Austrian Brain Tumour Registry (ABTR) was initiated in 2005 under the auspices of the

Austrian Society of Neuropathology, with the aim of establishing a comprehensive brain

tumour registry in Austria. ABTR is led by neuropathologists and epidemiologists in

interaction with the Austrian National Cancer Registry. In this paper we show that the ABTR

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model is a cooperative way to establish a population-based brain tumour registry that can

provide high quality and reliable data for epidemiological studies.

METHODS

The Austrian Brain Tumour Registry (ABTR) was initiated under the auspices of the

Austrian Society of Neuropathology in 2005. The coordination centre of ABTR is at the

Institute of Neurology (Neuropathology), Medical University of Vienna. Cooperative partners

contributing data to ABTR comprise all Austrian pathology or neuropathology

departments/units involved in brain tumour diagnostics (10 departments in 2005).

The spectrum of collected tumours comprises all malignant and non-malignant

primary brain tumour types including lymphomas and haematopoietic neoplasms (Louis et

al., 2007). Hamartomas, benign cysts and vascular malformations are not reported. Cases

are reported according to the International classification of diseases for oncology (ICD-O) 3rd

version. Behaviour is coded /0 for benign tumours, /1 for low or uncertain malignant potential

or borderline malignancy, (/2 for in situ lesions), /3 for malignant tumours, and /10 death-

certificate-only cases [19]. Tumours at any of the following sites are registered: brain (C71.0-

C71.9), meninges (C70.0-C70.9), spinal cord, cranial nerves and other parts of the central

nervous system (C72.0-C72.9), pituitary and pineal glands (C75.1-C75.3), and olfactory

tumours of the nasal cavity [C30.0 (as defined by ICD-O morphology codes 9522-9523)].

Published ABTR data may slightly differ from those of the Austrian National Cancer

Registry for the following reason: ABTR is reporting through ICD-O-3 codes, whereas the

Austrian National Cancer Registry uses ICD-10. Discrepancies of summary data are mainly

due to different coding of primary CNS lymphomas: according to ICD-O-3, CNS lymphoma is

reported as a brain tumour, whereas ICD-10 classifies it as a haematopoietic disease.

Data collection

The following parameters are provided for each case by the local neuropathology

departments: name, social insurance number, date of birth, gender, date of surgery, original

histopathological diagnosis of the neurosurgical biopsy report. Personal identifiers are

secondarily made anonymous (see below). In addition, the following data are obtained for

each case from the Austrian National Cancer Registry: tumour site, date of death, federal

state of residence, and healthcare region. Patients without permanent residence in Austria

are excluded.

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In cases without histopathological diagnosis (clinically diagnosed cases and DCO

cases), epidemiological data are added by the Austrian National Cancer Registry rendering

the completed ABTR dataset population-based.

Ethics and confidentiality issues

Ethical approval for all ABTR activities has been obtained from the ethics committee

of the Medical University of Vienna. Cooperation of ABTR and its partners is regulated by

bilateral service and confidentiality contracts in accordance to the Austrian law. All

documents are generated by lawyers and data confidentiality officials, and are approved by

the Austrian Data Protection Commission (http://www.dsk.gv.at/DesktopDefault.

aspx?alias=dsken). Furthermore, ABTR follows the guidelines on confidentiality for

population-based cancer registration edited by the International Association of Cancer

Registries (IACR, http://www.iacr.com.fr/) [20]. This approach protects the privacy of

individuals while at the same time allowing access to complete and accurate original data.

ABTR database

All data are stored on a FileMaker®-based (version 6.0) database placed on the local

IT server of the Institute of Neurology. The server is protected by firewalls of the Institute of

Neurology and the Medical University of Vienna. Access to data is restricted to authorized

members of the ABTR task force who are bound by confidentiality contracts.

Collection process

In each ABTR partner centre a contact person is defined who is in charge of the

preparation and transmission of the respective local dataset. Once a year, the completed

dataset of the preceding calendar year is electronically transferred to the ABTR database.

For data transfer, public key cryptography software (PGP Corporation Encryption

Applications® version 8.0, http://www.pgpi.org/) is used. In the ABTR coordination centre,

encrypted data are decoded and imported into the ABTR database. Incoming data are

checked for plausibility and duplicate-registrations. Thereafter data are made anonymous by

replacement of personal identifiers by identification numbers (ID).

Data analysis

Analysis and interpretation of all data are performed on basis of interdisciplinary

interaction of neuropathologists, biostatisticians and epidemiologists. For ABTR data

processing, the following software packages are used: Microsoft® Excel® version 12.1.0,

SPSS® version 16, SAS® 9.2, R2.7.0®, ArcGis® (licenses are provided by the Medical

University of Vienna). In order to provide direct comparability of ABTR incidence rates to

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those of the Central Brain Tumour Registry of the United States (CBTRUS,

http://www.cbtrus.org/), all rates were age-standardized to the Year 2000 US standard

population [21].

Population-based incidence rates were calculated for brain tumours as a group as

well as for individual tumour types. Further, we performed spatial epidemiological analyses

on the incidence and survival of Austrian glioblastoma patients. Indirectly standardized

incidence rates (SIR) were smoothed by a spatially unstructured random effect using Markov

Chain Monte Carlo methods in Winbugs® Version 1.4.3 leading to substantial shrinkage in

the estimated rates. Choropleth maps of SIR’s were drawn by SAS®.

RESULTS

Basic characteristics of collected brain tumours

A total of 1,807 brain tumour cases was registered in the Austrian population of 8.2

million inhabitants within the time period January 1st – December 31st 2005. 1,688 cases

were incident cases, whereas the remaining 119 cases referred to tumour recurrences.

ABTR partner centres (=Austrian neuropathology departments involved in brain tumour

diagnostics) contributed 1,366/1,688 (80.9%) cases. Each of these cases was histologically

confirmed. In addition, the Austrian National Cancer Registry contributed another 322/1,688

(19.1%) cases. Among these, 141/1,688 (8.4%) accounted for death-certificate-only (DCO)

cases. The remaining 181/1,688 (10.7%) tumours were diagnosed either clinically, by clinical

investigation, by specific tumour markers, or the basis of diagnosis was unknown. We

considered all these cases as non-microscopically verified [18]. Considering the total cohort

of 1,688 patients, median age at diagnosis was 58.4 years (standard deviation 19.2 years,

range 0 - 92.9 years). 95/1,688 (5.6%) cases were diagnosed in children and adolescents

aged <18 years, whereas 1,593/1,688 (94.4%) were diagnosed in individuals aged >18

years. 777/1,688 (46.0%) patients were male, whereas 911/1,688 (54.0%) patients were

female.

Brain tumour incidence (see also Table 1)

The overall incidence rate of brain tumours was 18.1/100,000 person-years (95%C.L.

17.4-18.9). The rate was somewhat higher in females (18.6/100,000 person-years (95%C.L.

17.6-19.7)) as compared to males (17.8/100,000 person-years (95%C.L. 16.8-18.9)). Benign

(ICD-O /0) and borderline (ICD-O /1) brain tumours constituted 51.3% with incidence rates of

7.4/100,000 person-years (95%C.L. 7.0-7.9) and 2.0/100,000 person-years (95%C.L. 1.7-

� ���

2.2) respectively, whereas malignant brain tumours (ICD-O /3) accounted for the remaining

48.7% with an incidence rate of 8.8/100,000 person-years (95%C.L. 8.3-9.3).

Direct comparison to recently published CBTRUS statistics including the CBTRUS

Supplement Report on 2004 data [7, 8], shows that Austrian incidence rates for malignant

and non-malignant tumours are comparable or slightly higher (table 1).

Incidence rates of brain tumours by histology (Table 2) [7, 8]

Neuroepithelial tumours constituted the largest group of primary brain tumours

(38.3%), followed by tumours of the meninges (30.3%). Together, both groups accounted for

approximately 2/3 of all central nervous system neoplasms. Gliomas of astrocytic,

oligodendroglial and ependymal origin accounted for 35.2% of all primary brain tumours.

Among these, glioblastomas constituted the largest group (56.3%) followed by diffuse

astrocytomas (10.1%) and oligodendroglial tumours (9.7%).

Table 3 illustrates incidence rates for individual tumour types. Meningiomas and

glioblastomas accounted for approximately 1/2 of all tumours, whereas most other tumour

entities were rare.

Figure 1 illustrates and compares the distribution of relative frequencies for ABTR

and CBTRUS histology groupings. Both distributions are highly similar, indicating the validity

of ABTR data despite the relative small sample size of a one-year-period (n=1,688) as

compared to the much larger CBTRUS dataset of a five-year-period (n=73,583).

Gender distribution

As a total, brain tumours were more frequent in females as compared to males,

mainly due to the predominance of meningiomas in females (m/f ratio = 1/2.3). In contrast,

gliomas were 1.3 times more common in males. Figure 2 illustrates the gender distribution

for different tumour behaviour.

Age distribution

Age-specific incidence rates illustrate the age distribution of all brain tumours (figure

3). Age-specific incidence rates were low in childhood, adolescence and young adulthood. In

later adulthood, incidence rates steadily increased and showed a peak in the age group 70-

<80 years. At higher age, the rates for brain tumours declined (in contrast to other cancer

types, e.g. prostate, lung or breast cancer, where incidence rates show a steady increase

(Curado et al., 2007; IARC)[1, 21]). In childhood (0-<15 years) the age-specific incidence

rates were highest in the very young (0-<5 years of age).

� ��

A total of 95 tumours occurred in children and adolescents (0-<18 years). The most

common histological diagnoses in this age cohort comprised pilocytic astrocytomas (n=24,

25.3%), followed by diffuse astrocytomas (n=12, 12.6%) and glioblastomas (n=7, 7.4%). The

group of embryonal tumours accounted for 9.5% (n=9), including 5 medulloblastoma cases

(5.3% of paediatric cases).

A total of 1,593 tumours occurred in adults (>18 years of age). The most frequent

histologies included meningiomas (n=421, 26.4%), followed by glioblastomas (n=328, 20.6%)

and pituitary adenomas (n=149, 9.4%).

The most common histologies for individual age cohorts are shown in table 4.

Spatial epidemiology

Spatial epidemiological analysis for individual tumour types is currently limited by the

relatively small sample size and short follow-up. We analysed the regional distribution of

glioblastoma incidence and survival, the most common malignant brain tumour.

Figure 4A illustrates the spatial pattern of smoothed incidence rates for individual Austrian

healthcare regions. No remarkable variations in smoothed incidence rates were detected,

which might be due to the currently low number of cases.

Figure 4B shows the spatial pattern of 1-year survival rates of glioblastoma patients for

individual Austrian healthcare regions. Healthcare regions in the vicinity of neurooncology

units show a more favourable 1-year survival, and none of the regions with poor 1-year

survival harbours a neurooncology unit. This congruency might reflect differential survival

due to benefits of targeted services located close by. However, definite conclusions are

currently limited due to the small sample size (n=340) and considerable variability.

DISCUSSION

ABTR – a cooperative way to establish a population-based brain tumour registry

Brain tumour classification is based on histogenetic principles and is a domain of

neuropathology. In 1979, the WHO convened for the first time an international panel of

neuropathologists for the development of the WHO classification of tumours of the central

nervous system [23]. Since then, neuropathology consensus conferences have been held in

several-year-periods for updating and further development of the classification system [24,

25]. Meanwhile, the WHO brain tumour classification comprises a spectrum of more than 110

tumour entities or variants [18]. Each new tumour entity or variant adopted by the WHO

� ��

consensus panel receives an International Classification of Diseases for Oncology (ICD-O)

morphology code [19]. These ICD-O morphology codes form the basis for cancer

registration. Considering the large spectrum of brain tumour entities and variants,

neuropathological expertise warrants high quality standards of data. Therefore, ABTR has

been initiated by neuropathologists in cooperation with epidemiologists, biostatisticians and

the Austrian National Cancer Registry. This setting links cancer registration to the mission of

medical research as defined by the World Medical Association in the Declaration of Helsinki

[26]. We are not aware of any other national brain tumour registry developed in such a

setting. Thus, we consider ABTR as cooperative and promising way to population-based

brain tumour registration.

Trend to registration of all brain tumour types

In most countries including Austria, brain tumour registration is restricted to malignant

tumour types [1]. Only a few countries (e.g. Scandinavian countries, Canada, US) report

incidence rates on benign and borderline brain tumours as well [27-30]. These tumours

constitute approximately 50% of all brain tumours(CBTRUS, 2008) [7]. However, benign and

borderline lesions may be associated with significant neurological deficits (e.g. due to

unfavourable tumour site), and may show malignant biological progression over time [31].

Therefore, increasing attempts to register all brain tumours have been made. In the US it has

already become legally mandatory to report all brain tumour types (Benign Brain Tumour

Cancer Registries Amendment Act; Public Law 107-260). In line with these developments in

other countries, ABTR registers all brain tumour types, including benign and borderline

lesions.

Registration of non-microscopically verified brain tumours

In brain tumour diagnostics, histology remains the gold standard. However, according

to the literature, only 60 - 80% of all brain tumours are histologically verified [7, 27]. In the

remaining patients who do not undergo neurosurgery for various reasons, the diagnosis is

usually based on neuroradiological findings. These patients are either followed-up or receive

primary conservative treatment (e.g. advanced age and poor performance status -->

palliative care; diffuse brainstem glioma --> irradiation; acoustic neurinoma --> stereotactic

radiosurgery; germ cell tumour --> chemotherapy). In the case of ABTR, data on malignant,

non-microscopically verified brain tumours are retrieved from the Austrian National Cancer

Registry. Benign brain tumours, that are not operated upon but are seen by neurosurgeons,

are currently neither reported to ABTR nor to the Austrian National Cancer Registry, which

causes a certain underestimation of respective rates. Therefore, in the future, Austrian

� ���

neurosurgical departments will be involved in ABTR activities, thus providing access to the

entire cohort of brain tumour cases.

Public and scientific benefits provided by the population-based ABTR

The population-based ABTR provides the following public and scientific benefits:

1. Surveillance of brain tumour incidence. Recent scientific evidence suggests an

increase in the incidence of some brain tumour entities (e.g. acoustic neurinoma,

meningiomas) [12]. Continuous surveillance of population-based brain tumour incidence as

intended by ABTR is the only objective tool to monitor changes.

2. Surveillance of brain tumour survival. In the past decades, diagnostic and

therapeutic innovations have significantly improved survival of brain tumour patients (e.g.

radiotherapy, operating microscope/micro-neurosurgery, MRI – treatment response

surrogate, etc.) [32-39]. Before introduction as new medical standard regimen, candidate

diagnostic or therapeutic procedures are systematically tested in clinical trials on selected

patient samples. In case of a clinically meaningful effect observed in such trials, new

procedures may translate into standard clinical use. Subsequently, the efficacy of the new

medical procedure still needs to be shown in the non-selected, general population (phase IV

– post-approval surveillance studies). In case of brain tumours, appreciable effects of

diagnostic/therapeutic innovations on patient survival can be shown only by means of

population-based cancer registries.

3. Spatial epidemiology. The ABTR dataset allows for spatial epidemiological

analyses of brain tumours at the level of individual Austrian healthcare regions. Such

analyses may reveal geographic variations of brain tumour incidence and patient survival.

The results may be of particular relevance for health-political considerations [40, 41]. Spatial

epidemiological analyses have been performed for the incidence and the survival of the most

common malignant brain tumour – glioblastoma (figures 4A and 4B). Initial findings indicate

that some regions show a more unfavourable outcome (0-25% 1-year survival) as compared

to others. Interestingly, healthcare regions in the vicinity of neurooncology units show a more

favourable outcome, and none of the regions with poor outcome harbours a neurooncology

unit. This might reflect differences in survival due to benefits of targeted services located

close by. However, there are no remarkable variations in SIR, which might be due to the

currently small sample size (n=340). A larger sample size and a longer follow-up period are

needed before any valid conclusions can be drawn.

4. International collaborations. Brain tumours are relatively rare and therefore

considered as orphan diseases. To obtain a sufficient number of cases for clinical studies,

� ���

international collaboration is often mandatory. The design of international clinical trials

necessitates detailed knowledge of brain tumour frequencies in a given population. ABTR will

provide such data for the Austrian population and thus facilitate participation in international

trials.

5. Aetiological brain tumour research. So far, established aetiological risk factors for

brain tumours include rare germline mutations associated with familial brain tumour

syndromes (e.g. neurofibromatosis, Li-Fraumeni syndrome), exposure to ionizing radiation

and immunosuppressants giving rise to brain lymphomas [42-46]. However, only a small

fraction of brain tumours can be explained by these risk factors. In the vast majority of cases,

the aetiology is still unknown. Epidemiological risk factor research (analytical epidemiology)

may lead to a better understanding of brain tumour aetiology serving as a basis for future

therapeutic approaches [47]. ABTR will provide a resource for aetiological brain tumour

research in Austria.

Synergistic neuropathological and epidemiological expertises in the ABTR setting

Brain tumour registries facilitate the design of epidemiological risk factor research

(case-control, cohort studies, etc.). Such studies necessitate synergistic cooperation of

neuropathologists and epidemiologists [47]. Neuropathological expertise is needed for

histopathological panel review (to overcome interobserver variability [48]) and for reasonable

grouping of tumour entities (due to the rarity of brain tumours different entities are frequently

grouped together). Epidemiological expertise is needed for the definition of a sufficiently

large sample size (e.g. in case-control studies), for modalities of data acquisition (e.g.

interviews) and for statistical analyses. In the ABTR setting synergistic neuropathological and

epidemiological expertises are provided by involvement and tight interaction of both

disciplines.

Comparison of ABTR and CBTRUS brain tumour incidence rates

ABTR has been recently established. Therefore, the time-span of data acquisition

and analysis is restricted to a one-year-period with a relatively small sample size. As brain

tumours comprise a large spectrum of rare entities, incidence rates may show slight

variations from year to year. Therefore, more reliable incidence rates can be calculated over

a longer time period (e.g. five-year interval). To assess whether the ABTR data of the first

year are representative, we compared our findings for benchmark purposes with the latest

CBTRUS statistics, as CBTRUS is the largest brain tumour registry in the world with a high

quality dataset. For comparison, CBTRUS data of the Statistical Report (2000-2004) as well

as the Supplement Report (2004) were used (see Table 1). The CBTRUS Supplement

Report provides one year of data and was analyzed after the collection of non-malignant

� ���

brain tumours had become mandatory in the United States (2004 dataset). Of note, ABTR

uses data of children and adolescents aged < 18 years, whereas CBTRUS uses data < 20

years. Comparing the datasets, we found considerable congruency of relative frequencies of

tumours (figure 1) and of the incidence rates (table 1). Interestingly, the ABTR incidence

rates are comparable or slightly higher than the CBTRUS rates (table 1). It needs to be

clarified whether this remains a single year observation or whether there are consistent

differences, e.g. due to different ethnic structures in the US and Central European

populations. However, the high congruency between ABTR and CBTRUS data indicates a

high quality of our initial one-year ABTR dataset. Thus, our findings can be considered as

representative and confirm the ABTR concept as a suitable approach to population-based

cancer registration.

Outlook

Most importantly, ABTR needs to become an established resource for monitoring

changes in brain tumour morbidity/mortality. In this way, ABTR can be utilized in the public

health system in Austria as a high quality management tool for brain tumour surveillance.

From a global perspective, having a resource such as ABTR will provide additional data

needed for epidemiology studies of small incidence diseases such as brain tumours.

Acknowledgements

ABTR is supported by the Anniversary Fund of the Österreichische Nationalbank

(project no: 12268). The study is part of the doctoral thesis Brain Tumour Epidemiology in

Austria (www.meduniwien.ac.at/clins). The authors are grateful to Andreas Jurkowitsch for IT

support, Markus Neumann for support with regard to data confidentiality issues, and

Hermann Hayn for legal advice.

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�

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Tables & Figures

Table 1. Comparison of ABTR and CBTRUS age-adjusted incidence rates (US 2000

standard population).

Table 1

INCIDENCE RATE ABTR (2005) CBTRUS (2000-2004)a CBTRUS (2004)b

Total 18.1 16.5 17.2

Male 17.8 15.8 16.3

Female 18.6 17.2 18.0

Malignant 8.8 7.3 7.2

Non-malignant 9.4 9.2 10.0

aPrimary brain tumours in the United States, CBTRUS Statistical Report 2000-2004 years data collected, issue 2007-2008

bPrimary brain tumours in the United States, CBTRUS Statistical Report Supplement 2004

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ndym

al tu

mou

rs

93

83/1

, 939

4/1,

939

3/3,

939

1/3,

93

92/3

49

2.

9

0.39

0.

24-0

.60

0.

76

0.55

-1.0

3

0.57

0.

44-0

.73

Cho

roid

ple

xus

tum

ours

9390

/0, 9

390/

1, 9

390/

3

4

0.2

0.

05

0.01

-0.1

5

0.06

0.

01-0

.18

0.

05

0.02

-0.1

2

Neu

rona

l and

mix

ed n

euro

nal-g

lial

tum

ours

9493

/0, 9

412/

1, 9

413/

0, 9

492/

0,

9505

/1, 9

505/

3, 9

506/

1, 9

506/

1,

9509

/1, 8

680/

1

23

1.

4

0.27

0.

15-0

.45

0.

31

0.18

-0.5

0

0.29

0.

20-0

.41

Pin

eal p

aren

chym

al tu

mou

rs

93

61/1

, 936

2/3,

939

5/3

5

0.

3

0.09

0.

02-0

.23

0.

05

0.01

-0.1

7

0.07

0.

03-0

.15

Em

bryo

nal t

umou

rs

94

70/3

, 947

1/3,

947

4/3,

947

3/3,

95

00/3

, 949

0/3,

950

1/3,

939

2/3,

95

08/3

18

1.

1

0.26

0.

13-0

.45

0.

24

0.12

-0.4

2

0.25

0.

16-0

.37

Tu

mo

urs

of

cran

ial n

erve

s

9560

/0, 9

540/

0, 9

550/

0, 9

571/

0,

9571

/3, 9

540/

3

125

7.

4

1.30

1.

03-1

.61

1.

46

1.17

-1.7

9

1.36

1.

17-1

.58

Tu

mo

urs

of

the

men

ing

es

51

1

30.3

3.57

3.

11-4

.07

6.

96

6.35

-7.6

2

5.31

4.

93-5

.72

Tum

ours

of m

enin

goth

elia

l cel

ls

95

30/0

, 953

1/0,

953

2/0,

953

7/0,

95

33/0

, 953

4/0,

953

8/1,

953

9/1,

95

38/3

, 953

0/3

50

4

29.9

3.45

3.

00-3

.95

6.

91

6.30

-7.5

5

5.23

4.

85-5

.63

���

Mes

ench

ymal

, non

-men

ingo

thel

ial

tum

ours

8815

/0, 8

900/

3, 9

220/

0, 9

150/

1,

9364

/3, 9

161/

1

7

0.4

0.

11

0.04

-0.2

4

0.06

0.

01-0

.18

0.

08

0.04

-0.1

6

Tu

mo

urs

of

hae

mat

op

oet

ic

syst

em

95

90/3

56

3.

3

0.65

0.

46-0

.89

0.

51

0.36

-0.7

1

0.57

0.

45-0

.71

Ger

m C

ell T

um

ou

rs

90

64/3

,907

0/3,

910

0/3,

908

0/1,

90

85/3

6

0.4

0.

11

0.04

-0.2

5

0.06

0.

01-0

.19

0.

09

0.04

-0.1

7

Tu

mo

urs

of

the

sella

r re

gio

n

93

50/1

, 943

2/1,

943

2/1,

827

2/0

16

7

9.9

2.

02

1.69

-2.4

1

1.66

1.

35-2

.01

1.

81

1.58

-2.0

6

Oth

erc

80

00/0

, 800

0/1,

800

0/3,

937

0/3

17

7

10.5

1.93

1.

59-2

.33

1.

65

1.38

-1.9

7

1.78

1.

56-2

.02

a Tum

our

grou

ping

s ac

cord

ing

to th

e W

HO

bra

in tu

mou

r cl

assi

ficat

ion

syst

em

b AB

TR

age

-sta

ndar

dize

d in

cide

nce

rate

s (U

S 2

000

stan

dard

pop

ulat

ion)

c "Oth

er"

incl

udes

: DC

O c

ases

, neo

plas

ia n

ot o

ther

wis

e sp

ecifi

ed (

NO

S),

cho

rdom

a

Ta

ble

3.

AB

TR

20

05

: In

cid

en

ce

ra

tes

fo

r in

div

idu

al

bra

in t

um

ou

r ty

pe

s l

iste

d a

cc

ord

ing

to

th

eir

to

tal

fre

qu

en

cie

s.

Ra

tes

fo

r ra

re b

rain

tum

ou

r ty

pe

s (

<1

% o

f th

e c

oh

ort

an

d <

3 i

nd

ivid

ua

ls a

ffe

cte

d)

are

no

t s

ho

wn

fo

r c

on

fid

en

tia

lity

re

as

on

s.

Tab

le 3

Ad

just

ed r

ates

B

rain

tu

mo

ur

typ

e

ICD

-O M

orp

ho

log

y co

des

M

F

To

tal

%

rate

a 95

% C

.L.

Men

ingi

oma

(I)b,

c

9530

/0, 9

531/

0, 9

532/

0, 9

537/

0,

9533

/0, 9

534/

0, 9

530/

0, 9

530/

0

114

31

1

425

25

.2

4.

39

4.04

-4.7

6

Glio

blas

tom

a (I

V)c

94

40/3

191

14

4

340

20

.1

3.

40

3.10

-3.7

3

Pitu

itary

ade

nom

a

8272

/0

93

68

15

1

8.9

1.

63

1.41

-1.8

7

Neu

rinom

a

9560

/0

54

60

11

4

6.8

1.

24

1.05

-1.4

5

Diff

use

astr

ocyt

oma

(II)

c

9400

/3, 9

420/

3, 9

411/

3, 9

410/

3

35

26

61

3.

6

0.75

0.

60-0

.93

Aty

pica

l men

ingi

oma

(II)

b,c

95

38/1

, 953

8/1,

953

9/1

30

24

54

3.2

0.

58

0.45

-0.7

2

Mal

igna

nt ly

mph

oma

95

90/3

25

22

47

2.

8

0.48

0.

37-0

.62

Ana

plas

tic a

stro

cyto

ma

(III)

c

9401

/3

25

14

39

2.3

0.

44

0.33

-0.5

8

Pilo

cytic

ast

rocy

tom

a (I

)c

9421

/1, 9

425/

3 [p

ilom

yxoi

d

astr

ocyt

oma

(II)

c incl

uded]

21

18

39

2.3

0.

57

0.43

-0.7

5

Epe

ndym

oma

(II)

c

9391

/3, 9

393/

3

9

17

26

1.5

0.

29

0.20

-0.4

0

Mal

igna

nt m

enin

giom

a (I

II)b,

c

95

38/3

, 953

0/3

9

16

25

1.

5

0.26

0.

18-0

.36

Olig

oast

rocy

tom

a (I

I)c

93

82/3

[an

apla

stic

olig

oast

rocy

tom

a

(III)

c exc

lude

d]

14

9

23

1.

4

0.27

0.

19-0

.39

���

Olig

oden

drog

liom

a (I

I)c

94

50/3

12

5

17

1.0

0.

20

0.13

-0.3

0

a AB

TR

age

-sta

ndar

dize

d in

cide

nce

rate

s (U

S 2

000

stan

dard

pop

ulat

ion)

b Sum

s of

men

ingi

oma

subt

ypes

in th

e W

HO

cla

ssifi

catio

n fo

r gr

ades

I, II

, or

III

c WH

O g

rade

s in

par

enth

eses

if a

ppro

pria

te

���

Ta

ble

4.

AB

TR

20

05

: T

he

fiv

e m

os

t c

om

mo

n b

rain

tu

mo

ur

typ

es

fo

r a

ge

co

ho

rts

.

Tab

le 4

0-<1

8 ye

ars

(n=9

5)

18

-<40

yea

rs (

n=2

55)

40-<

65 y

ears

(n

=754

)

65+

year

s (n

=584

)

Mos

t com

mon

his

tolo

gy

P

ilocy

tic a

stro

cyto

ma

(I)a

25.3

%

P

ituita

ry a

deno

ma

14

.1%

Men

ingi

oma

(I)a

30.6

%

M

enin

giom

a (I

)a 27

.2%

Sec

ond

mos

t com

mon

his

tolo

gy

D

iffus

e as

troc

ytom

a (I

I)a

12.6

%

S

chw

anno

ma

12.2

%

G

liobl

asto

ma

(IV

)a 20

.7%

Glio

blas

tom

a (I

V)a

24.7

%

Thi

rd m

ost c

omm

on h

isto

logy

Glio

blas

tom

a (I

V)a

7.4%

Men

ingi

oma

(I)a

12.2

%

P

ituita

ry a

deno

ma

9.8%

Pitu

itary

ade

nom

a 6.

7%

For

th m

ost c

omm

on h

isto

logy

Med

ullo

blas

tom

a (I

V)a

5.3%

Glio

blas

tom

a (I

V)a

11.0

%

S

chw

anno

ma

7.6%

Mal

igna

nt ly

mph

oma

5.3%

Fift

h m

ost c

omm

on h

isto

logy

Ana

plas

tic a

stro

cyto

ma

(III)

a 4.

2%

D

iffus

e as

troc

ytom

a (I

I)a

9.4%

Men

ingi

oma

(II)

a 3.

4%

S

chw

anno

ma

4.3%

a WH

O g

rade

s in

par

enth

eses

if a

ppro

pria

te

Figure 1. Comparison of ABTR and CBTRUS relative frequencies of brain tumour

types shows highly similar distributions.

Figure 2. ABTR 2005: Gender distribution of brain tumours according to ICD-O

behaviour (benign, borderline, malignant).

� ���

Figure 3. ABTR 2005: Age-specific incidence rates of brain tumours.

Figure 4A. ABTR 2005: Spatial pattern of smoothed glioblastoma incidence rates for

individual Austrian healthcare regions (SIR = smoothed incidence rates expressed in

quartiles < 0.980 = lower quartile, 0.980-1.008 = the two median quartiles, > 1.008 upper

quartile; black dots: location of the currently 11 Austrian neurooncology units).

� ���

Figure 4B. ABTR 2005: Spatial pattern of 1-year survival rates of glioblastoma patients

in individual Austrian healthcare regions (0-25% = poor 1-year survival; >25-100% =

more favourable 1-year survival; black dots: location of the currently 11 Austrian

neurooncology units).

� ���

Cancer 2010; 116: 5725-32, IF 5.131

Incidence of atypical teratoid/rhabdoid tumours in children: a population-based study

by the Austrian Brain Tumour Registry, 1996-2006

Running title: Incidence of AT/RT in children

Woehrer Adelheid, MD1, Slavc Irene, MD2, Waldhoer Thomas, PhD3, Heinzl Harald, PhD4,

Zielonke Nadine, MSc5, Czech Thomas, MD6, Benesch Martin, MD7, Hainfellner Johannes A,

MD1, Haberler Christine, MD1, on behalf of the Austrian Brain Tumour Registry

Other group members

Azizi Amedeo A, MD2, Scarpatetti Michael, MD8, Ebetsberger Georg, MD9, Weis Serge,

MD10, Jones Neil, MD11, Klein-Franke Andreas, MD12, Sterlacci William, MD13, Jauk Barbara,

MD14, Kiefer Andreas, MD15, Mueller Guido, MD16, Gruber-Moesenbacher Ulrike, MD17,

Reiner-Concin Angelika, MD18, Feichtinger Hans, MD19

1Institute of Neurology, Medical University of Vienna, 2Department of Paediatrics, Medical

University of Vienna, 3Centre of Public Health, Department of Epidemiology, Medical

University of Vienna, 4Centre for Medical Statistics, Informatics and Intelligent Systems,

Medical University of Vienna, 5Austrian National Cancer Registry, Statistics Austria, 6Department of Neurosurgery, Medical University of Vienna, 7Division of Paediatric

Hematology/Oncology, Department of Paediatrics, Medical University of Graz, 8Institute of

Pathology, Medical University of Graz, 9Department of Paediatrics, Children´s and Maternity

Hospital Linz, 10Department of Pathology and Neuropathology, State Neuropsychiatric

Hospital Wagner-Jauregg, 11Department of Paediatrics, Paracelsus Private Medical

University Salzburg, 12Department of Paediatrics, Medical University of Innsbruck, 13Institute

of Pathology, Medical University of Innsbruck, 14Department of Paediatrics, State Hospital

Klagenfurt, 15Institute of Pathology, State Hospital Klagenfurt, 16Department of Paediatrics,

State Hospital Feldkirch, 17Institute of Pathology, State Hospital Feldkirch 18Institute of Pathology, Danube Hospital Vienna, 19Department of Pathology, Krankenanstalt

Rudolfstiftung Vienna; Austria

Correspondence to

Christine Haberler, MD

Institute of Neurology, Medical University of Vienna

Waehringer Guertel 18-20, A-1097 Vienna, Austria

� ���

Phone: +43 1 40400 5595

Fax: +43 1 40400 5511

E-mail: christine.haberler@meduniwien.ac.at

Total number of text pages (25), tables (2), and illustrations (2).

Sources of support: This study was supported by the Anniversary Fund of the

Österreichische Nationalbank (grant no: 12268).

The authors declare no financial disclosures.

Condensed abstract

A population-based series of malignant, high-grade brain tumours in children reveals an age-

standardized incidence rate of 1.38/1,000,000 person-years for AT/RTs, with a particularly

high incidence in very young children under the age of three years. Increased awareness of

this high incidence in young children might help to optimize diagnostic and therapeutic

management of patients with AT/RT.

� � �

Abstract

Background Atypical teratoid/rhabdoid tumours (AT/RT) are highly malignant embryonal

CNS tumours, which were defined an entity in 1996. As compared to other malignant CNS

tumours their biological behaviour is particularly aggressive, but patients may benefit from an

intensified treatment. AT/RTs display a complex histomorphology, which renders them prone

to misdiagnosis. They occur predominantly in young children, with an estimated prevalence

of 1–2% among all paediatric CNS tumours. However, population-based data on the

incidence of these tumours are not yet available.

Methods A nation-wide survey on malignant high-grade CNS tumours (WHO grade III/IV),

diagnosed in children (0–14 years) from 1996–2006 was conducted by the Austrian Brain

Tumour Registry. A central histopathology review was performed including the assessment

of the SMARCB1 (INI1) protein status.

Results A total of 311 newly diagnosed, malignant CNS tumours were included. AT/RTs

constituted the sixth most common entity (6.1%), referring to an age-standardized incidence

rate of 1.38/1,000,000 person-years in children. Peak incidence was found in the 0–2 years

age group, where they were as common as CNS PNETs and medulloblastomas. 47.4% of

AT/RTs were initially diagnosed, whereas 52.6% were retrospectively detected by the central

review. The 5-year survival of AT/RT patients was 39.5%, with 66.7% in the correctly

diagnosed versus 15.0% in the not recognized group (p=0.0469).

Conclusion Clinicians and pathologists should be aware of the high incidence of AT/RTs in

young children in order to optimize diagnostic and therapeutic management of patients with

AT/RT.

Keywords

Atypical teratoid/rhabdoid tumour, CNS neoplasm, childhood, epidemiology, population-

based incidence, survival.

� ���

Introduction

Central nervous system (CNS) tumours represent more than 20% of all childhood

malignancies (0–14 years) in developed countries1,2 and constitute the most common cause

of cancer-related death in this age group.2

Atypical teratoid/rhabdoid tumours (AT/RTs) are rare, highly malignant, embryonal CNS

tumours, which occur sporadically, or in the context of a rhabdoid tumour predisposition

syndrome.3 Malignant rhabdoid tumours (MRT) were originally described in the kidney4 and

subsequently observed in soft tissues and the CNS.5,6 AT/RT was defined as an entity� in

1996.7 In 2000, AT/RTs were introduced to the WHO brain tumour classification8 and the

International Classification of Diseases for Oncology (ICD-O third edition).9

Histopathologically, AT/RTs are characterised by rhabdoid tumour cells and variable areas of

primitive neuroectodermal, epithelial and mesenchymal differentiation.3 This complex

morphology may render the differential diagnosis to other malignant CNS tumours including

CNS PNET/medulloblastomas, choroid plexus carcinomas, germ cell tumours, or malignant

gliomas difficult.10,11 Yet, AT/RTs display a distinct genetic profile characterised by the

biallelic inactivation of the SMARCB1 (hSNF5/INI1) gene at chromosomal locus

22q11.23,12,13 which causes the loss of SMARCB1 (INI1) protein expression. Since the recent

introduction of a sensitive and specific monoclonal antibody against the SMARCB1 protein

(Ab No. 612110, BD Transduction Laboratories), diagnostic means have been significantly

improved.11,14

The biological behaviour of AT/RTs is highly aggressive and the prognosis is exceedingly

dismal compared to other malignant brain tumours. Reported survival times ranged from 0.5

to 11 months with a particularly poor outcome for infants.(Bonnin et al., 1984; Burger et al., 1998; Haberler et al.,

2006; Rorke et al., 1995; Rorke et al., 1996; Tekautz et al., 2005)6,7,10, 11,15,16 However, recent studies provide

evidence that patients benefit from intensified multi-modal therapies17–20 and 2-year overall

survival rates of up to 70% have been reported.21

AT/RTs have been predominantly observed in children, particularly in very young children

under the age of three years.6,7,10,13,15,17,18 Only single cases were reported in adults.22–32

Several large, hospital-based series established an AT/RT prevalence of 1–2% among

paediatric brain tumours,8,33–35 but population-based data on the incidence of AT/RTs in

children are not available so far. Yet, such data provide important insights into the burden of

disease and might contribute to patient care. Hence, we present the first population-based

study on histologically confirmed AT/RTs in the paediatric population.

�

� ���

Methods

Study Design and Source of Data

A retrospective nation-wide survey on malignant, high-grade CNS tumours (WHO grade III

and IV tumours3) in children under the age of fifteen years was conducted by the Austrian

Brain Tumour Registry (ABTR)36. Since its establishment in 2005, the ABTR has evolved as

a national neuro-oncology network, with active contributions from various disciplines

(neurosurgeons, pathologists, neurooncologists) in all Austrian neuro-oncology units. The

ABTR is tightly cooperating with the population-based Austrian National Cancer Registry. For

this study all newly diagnosed cases from 1996 (after the definition of AT/RT as an entity)

through 2006 were included. To encompass a maximum of patients, the ABTR network was

used for retrospective case ascertainment. Active case reporting included all neuro-oncology

units (n=7), which are routinely engaged in the diagnosis and management of paediatric

brain tumour patients in Austria. A permanent Austrian residence was considered mandatory.

Tumours at any of the following sites were included9: brain (C71.0–C71.9), meninges

(C70.0–C70.9), spinal cord, cranial nerves and other parts of the CNS (C72.0–C72.9),

pituitary and pineal glands (C75.1–C75.3), and olfactory tumours of the nasal cavity (C30.0).

All metastatic tumours were excluded. Eligible cases were identified through local

paediatricians and pathologists. In a second step, data of these patients were matched with

those of the population-based Austrian National Cancer Registry. The following parameters

were abstracted for each case: personal identifiers, gender, date of birth, date of surgery,

histopathological diagnosis. For patients with AT/RT information on the extent of resection,

metastatic stage, treatment, and follow-up (last update May 2010) were retrieved from the

local paediatricians. Personal identifiers were pseudonymised and all data were stored in the

ABTR database.

Central Histopathology Review

In all available cases, paraffin-blocks or alternatively ten unstained sections were retrieved

from the local pathology departments. For the central histopathology review conventional

histological stainings and the systematic analysis of SMARCB1 protein expression were

performed, as previously described.11 All histopathological diagnoses were classified

according to the WHO 2007 diagnostic consensus criteria.3 Two neuropathologists (AW,

JAH) at the Institute of Neurology, Medical University of Vienna independently reviewed the

HE and SMARCB1 staining of the total series. In all SMARCB1 negative tumours a panel of

additional immunohistochemical markers, typically expressed in AT/RT (cytokeratin,

epithelial membrane antigen, neurofilament protein, glial fibrillary acidic protein, muscle actin,

vimentin) were performed. AT/RT was diagnosed when divergent differentiation along

epithelial, mesenchymal, neuronal, or glial lines was found in addition to complete loss of

SMARCB1 protein expression in tumour cell nuclei, but retained expression in preexisting

� ���

cells, e.g. endothelial cells. Additionally a third neuropathologist (CH), experienced in the

diagnosis of paediatric brain tumours reviewed all AT/RTs.

Statistical Methods

All statistical data analyses were performed with Statistical Analysis Software SAS® version

9.2, SPSS® version 17.0 and Microsoft Excel® version 12.1.0. All incidence rates were age-

adjusted to the WHO world standard population using direct methods and expressed per

1,000,000 person-years. National population estimates per single year were provided by

Statistics Austria. Special analysis was performed for 5 different age groups: 0–2 years, 3–5

years, 6–8 years, 9–11 years, and 12–14 years.

Differences in frequency of misdiagnoses before and after the introduction of AT/RTs to the

WHO brain tumour classification in 2000, as well as metastatic disease, extent of resection

(gross total, subtotal, biopsy), and localization (supratentorial, infratentorial, spinal) within

subgroups of initially recognized versus not recognized cases, were tested by the Fisher´s

exact test. Differences in age were tested by the Wilcoxon-Mann-Whitney test. The

distribution of annually observed numbers of AT/RTs was compared to a Poisson distribution

(Kolmogorov-Smirnov test). Survival probabilities were estimated according to Kaplan-Meier.

Univariate survival analyses were performed for age, localization, extent of resection, initial

establishment of the diagnosis (recognized versus non-recognized AT/RTs), and metastatic

disease. Differences in survival curves were tested with the log-rank test. To clarify the

impact of an intensified treatment regimen (MUV ATRT, EU rhab) on survival a Cox model

was estimated with intensified treatment regimen as time- dependent binary covariable and

initial establishment of the diagnosis as fixed covariable.

Ethical considerations

Ethical approval was obtained from the Ethics Committee of the Medical University of

Vienna. Reporting through the established ABTR warranted data confidentiality.

� ���

Results

During the period 1996–2006, a total of 319 primary malignant, high-grade CNS tumours

(WHO grade III and IV) were reported in the Austrian paediatric population (annual estimate:

1.4 million children). 225/319 (70.5%) were microscopically verified at diagnosis, whereas

29.5% were diagnosed either clinically (malignant brain stem gliomas, n=19), by specific

tumour markers (germ cell tumours, n=3), or the basis of diagnosis was unknown (n=72). In

202/225 (89.8%) microscopically verified cases, tissue was provided for a central

histopathology review. Upon central review another eight cases were excluded, due to

reclassification as low-grade gliomas. Thus, in total 311 tumours were included in the

analysis. Upon central histopathology review, 19 cases displayed morphological and

immunohistochemical features of AT/RT including complete loss of SMARCB1 protein

expression in tumour cell nuclei. In addition, two tumours with morphological features of

epithelioid sarcoma showed lack of SMARCB1 protein expression. As loss of SMARCB1

expression has been described in epithelioid sarcomas37, both tumours were not classified

as AT/RTs.

Median age at diagnosis of the total cohort was 7.5 years. 173/311 tumours were diagnosed

in males (55.6%), 138 (44.4%) in females (M/F ratio 1.25). Embryonal brain tumours

constituted the largest group (n=142, 45.7%), followed by astrocytic (n=86, 27.7%) and

ependymal tumours (n=24, 7.7%). Incidence data of the ten most common tumour entities is

provided in table 1. AT/RT constituted the sixth most common tumour entity; the age-

standardized incidence rate was 1.38/1,000,000 person-years (age-specific incidence rate:

1.28/1,000,000 person-years). As some variation in the incidence of AT/RTs was noted

(range 0–4 cases per year), the distribution of annually observed tumours was analyzed and

found to be consistent with the expected distribution for a rare event of 19/11=1.73 cases per

year (p=0.8531).

Classification of children into five age groups showed a peak incidence of AT/RTs among

children below the age of three years (figure 1). In this age group AT/RTs were as common

as CNS PNETs or medulloblastomas. In contrast, AT/RTs were rare in all other age groups.

9/19 (47.4%) AT/RTs were initially diagnosed, whereas 10/19 (52.6%) cases were

retrospectively detected by the central histopathology review. Initial diagnoses of those

tumours were medulloblastoma (n=6), ependymoblastoma (n=1), anaplastic meningioma

(n=1), small cell sarcoma (n=1), and Ewing sarcoma (n=1). Misdiagnoses of AT/RTs tended

to be more common in the period 1996–2000 (before the introduction of AT/RT to the WHO

brain tumour classification) than after 2000 (p=0.0698). In contrast, none of the initially

diagnosed AT/RTs was reclassified upon central review. Clinical characteristics and

treatment regimens of AT/RT patients are listed in table 2. The M/F ratio was 0.9. Median

age at diagnosis was 1.4 years, with a range from 0.2–14.4 years. 68.4% of the patients

� ���

were under the age of three years at the time of diagnosis. The groups of initially recognized

and not-recognized cases did not differ significantly in terms of age (p=0.7801), metastatic

disease (p=0.6381), extent of resection (p=0.4560), and localization (p=0.2762). Figure 2

illustrates the Kaplan-Meier survival curves of AT/RT patients, stratified by initially recognized

versus not recognized AT/RTs. Overall survival of not recognized patients was significantly

worse compared to initially recognized cases (p=0.0469, logrank test). 5-year survival of all

AT/RT patients was 39.5% (66.7% in the initially diagnosed group versus 15.0% in the not

recognized group). Extent of resection was significantly associated with survival (p=0.0127),

whereas no significant associations could be observed for age (p=0.3565), localization

(p=0.4928), or metastatic disease (p=0.1182). Analysis of treatment strategies revealed, that

the important factor for prolonged survival was an intensified treatment (MUV ATRT, EU rhab

protocol; p=0.0241, Cox model).

� ���

Discussion

First single-centre studies on AT/RTs documented their occurrence predominantly in very

young children under the age of three years.6,7,10,11,13,15,17,18 This was confirmed in larger

single-centre series, based on continuous paediatric CNS tumour collectives including all

types of brain tumours. These microscopically verified series established a low prevalence of

AT/RTs between 0.9–2.1%.33–35 A similar prevalence of 1.1% was found in a multi-centre,

hospital-based study on paediatric brain tumours in France.41 However, definite conclusions

on the population-based incidence can be provided only by cancer registries, in particular

specialized brain tumour registries such as the Central Brain Tumour Registry of the United

States (CBTRUS)42 or projects such as ACCIS, a European project on the incidence and

survival of paediatric tumours.1,43,44 Yet, data published by these organizations are mostly

presented as summary rates for groups of tumours (e.g. embryonal tumours or gliomas),

exact incidence rates of rare tumour entities (including AT/RTs) are not provided. Reliable

incidence data of AT/RTs will be available in cancer registries just in several years time with

the increasing use of the ICD-O third edition of 2000. Therefore, our approach was to actively

report cases from all Austrian neurooncology units from 1996 onwards, when AT/RTs were

defined as a distinct tumour entity. We screened for all malignant, high-grade paediatric CNS

tumours, entering into the differential diagnosis of AT/RT. As others and we have previously

shown, that AT/RTs are frequently misdiagnosed,10,11 a central histopathology review

including the assessment of SMARCB1 protein status was performed, which was feasible in

the majority of all tumours (70.5%). Using this strategy we found an AT/RT incidence rate of

1.38/1,000,000 person-years (95% C.I. 0.83–2.15) in children, in accordance with the low

AT/RT prevalence of single-centre studies.33–35 AT/RT is a rare CNS tumour. However, in

very young children (0–2 years) we found AT/RT to be as frequent as medulloblastoma and

CNS PNET (17.3%, 16.0%, and 13.3% respectively). In order to ensure a population-based

dataset, we included also non-microscopically verified cases. Thus, the actual incidence rate

of AT/RT might be even slightly higher as single non-microscopically verified cases might

have been missed. Comparison of the incidence rates of the most frequent paediatric CNS

tumours across Europe, England, and the United States shows highly similar rates in Austria,

thereby confirming a high degree of case ascertainment and validity of our findings.1,42,45

Half of the AT/RTs in our series were initially not recognized. Such misdiagnoses tended to

be more common in the first years after the definition of AT/RT as an entity and before the

introduction of the anti-SMARCB1 antibody as diagnostic tool. This finding emphasizes the

relevance of a systematic analysis of the SMARCB1 protein status in malignant paediatric

brain tumours. Furthermore, it underlines the importance of a central histopathology review in

epidemiological studies on tumours with a complex histopathology.

� ��

In the literature, single malignant rhabdoid tumours with retained SMARCB1 protein

expression have been reported.46 We did not detect such tumours in our series. Loss of

SMARCB1 protein expression is characteristic for malignant rhabdoid tumours including

AT/RTs, but not absolutely specific. Alterations of the SMARCB1 gene have been recently

implicated in several other tumours, such as epithelioid sarcomas, single cases of

gangliogliomas, familial schwannomas and cribriform neuroepithelial tumours.37,47–52 These

findings indicate that the morphology needs to be cautiously interpreted in conjunction with

the immunohistochemical profile for the differential diagnosis of AT/RT. Indeed, two tumours

in our series displayed features of epithelioid sarcoma and showed loss of SMARCB1 protein

expression. Whether these tumours fall in the spectrum of rhabdoid tumours or represent a

separate tumour entity with alteration of the SMARCB1 gene needs to be clarified in larger

patient cohorts.

Notably, the most frequent misdiagnosis of AT/RT was medulloblastoma. This might

implicate that the overall survival of patients with embryonal brain tumours, especially in the

young medulloblastoma age group improves, as not-recognized AT/RTs are no longer

included.

As previously reported,6,7,10,11,15,16 the overall survival of AT/RT patients is also in our series

poor. However, we could show, that patients whose tumours were initially diagnosed as

AT/RTs and consequently treated according to an intensified protocol showed a better

outcome compared to those, who were initially not recognized. This is in line with our

previous findings of a single-centre study11 and emphasizes the importance of a correct

diagnosis and appropriate treatment of AT/RT patients.

In conclusion, we present the first population-based study of histologically confirmed AT/RTs

in children, showing a high incidence in the very young (0–2 years), where they constitute

together with CNS PNETs and medulloblastomas the most frequent malignant, high-grade

CNS tumours. An increasing awareness among clinicians and pathologists of this high

disease occurrence will help to optimize the diagnostic and therapeutic management of

AT/RT patients.

� ���

Conflicts of Interest

The authors declared no conflicts of interest.

Acknowledgements

We are grateful to Irene Leisser for excellent technical assistance, and Andreas Jurkowitsch

for IT support. We acknowledge the assistance of Leo Karger, Reinhard Motz, Franz Wuertz,

Agnes Gamper, Hans Maier, Christian Urban, Gabriele Pammer, and Selma Hoenigschnabel

in case ascertainment. The ABTR is supported by the Anniversary Fund of the

Österreichische Nationalbank (project no: 12268). This study is part of the doctoral thesis

Brain Tumour Epidemiology in Austria (www.meduniwien.ac.at/clins).

� ��

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Figure 1. Relative frequencies of the six most common malignant, high-grade CNS tumour

entities in five different age groups.

Figure 2. Kaplan-Meier survival curves of AT/RTs stratified by initial diagnosis.

� ��

Acta Neuropathologica 2011; 122: 787-90, IF 7.695

Embryonal Tumour with Abundant Neuropil and True Rosettes (ETANTR) with loss of

morphological but retained genetic key features during progression

Adelheid Woehrer1, Irene Slavc2, Andreas Peyrl2, Thomas Czech3, Christian Dorfer3, Daniela

Prayer4, Susanne Stary5, Berthold Streubel5, Marina Ryzhova6, Andrey Korshunov7, Stefan

M Pfister8,9, Christine Haberler1

1Institute of Neurology, 2Department of Paediatrics, 3Department of Neurosurgery, 4Department of Radiology, 5Department of Pathology, Medical University of Vienna, Austria; 6NN Burdenko Neurosurgical Institute, Moscow, Russia; 7Clinical Cooperation Unit

Neuropathology, 8Division Molecular Genetics, German Cancer Research Centre,

Heidelberg, 9Department of Paediatric Oncology, Heidelberg University Hospital, Germany.

Correspondence to

Christine Haberler, M.D.

Institute of Neurology, Medical University of Vienna

Waehringer Guertel 18–20, A-1097 Vienna

Email: christine.haberler@meduniwien.ac.at

Phone: +43–1–40400–1629

Fax: +43–1–40400–5511

Running head

Loss of morphological but retained genetic key features in ETANTR

� ��

Embryonal tumour with abundant neuropil and true rosettes (ETANTR) is a recently

recognized, rare embryonal CNS tumour, which predominantly occurs in young children and

is associated with a highly aggressive disease course [1-3, 5-8, 13, 14, 18, 19, 23(Al-Hussain

and Dababo, 2009; Buccoliero et al.; Dunham et al., 2007; Eberhart et al., 2000; Ferri Niguez

et al.; Fuller et al., 2006; Gessi et al., 2009; Korshunov et al.; La Spina et al., 2006; Manjila et

al.; Pfister et al., 2009; Wang et al.). The histopathological diagnosis of ETANTR is based on

the presence of primitive neuroectodermal tumour cells forming distinct multilayered

´ependymoblastic´ rosettes and characteristic neuropil islands. Recently, genome-wide

analyses have revealed a novel amplification at 19q13.42 [16,19(Li et al., 2009; Pfister et al.,

2009), which is meanwhile considered the genetic hallmark of ETANTR [13, 16](Korshunov

et al.; Li et al., 2009). The characteristic clinical, morphological, and genetic features support

the concept of a distinct CNS PNET variant and suggest its introduction to the WHO

classification of Tumours of the Central Nervous System [17]. As the amplification at

19q13.42 has also been found in the vast majority of ependymoblastomas analyzed to date

[13, 16](Korshunov et al.; Li et al., 2009), the common genetic background suggests the

fusion of these two tumour types to a single entity.

Herein, we report for the first time the evolution of morphological features and genetic

aberrations during the disease course in a patient with ETANTR. A 33-month-old girl

presented with a six-month history of episodic headaches, increased head circumference

and mild gait disturbance. Magnetic resonance (MR) imaging showed a 9.6x8.6x11.7cm left

parieto-occipital, space-occupying, partly cystic lesion displaying T1- weighted hypo- to

isointense signals (Fig 1A) with cerebrospinal fluid-intense cysts on FLAIR sequence (Fig

1B), and marked Choline/Creatine increase as a sign of cell proliferation on single voxel

spectroscopy (Fig 1C). Near total macroscopic resection was performed. Histopathology

revealed a primitive neuroectodermal tumour with highly cellular areas. Furthermore,

hypocellular neuropil-islands and multilayered rosettes were encountered, prompting the

diagnosis of ETANTR (Fig 2A-C). Immunohistochemistry demonstrated widespread

expression of neuronal markers including synaptophysin and neurofilaments, EMA was

detected solely within the rosettes in a dot-like and luminal surface pattern. SMARCB1/INI1

nuclear protein expression was retained. Fluorescence in situ hybridization (FISH) and a

SNP array (Affymetrix SNP Array 6.0) demonstrated amplification at 19q13.42 (Fig 3A, B).

The SNP array revealed additional DNA copy number alterations including trisomies of

chromosomes 2, 14, 17, 19, and 20, as well as partial gain of 1q (Fig 3B). Postsurgical spinal

imaging and cerebrospinal fluid cytology were negative. The child was treated according to

an intensified therapy protocol (three cycles of a modified HIT 2000 SKK protocol [20]

including vincristine, cyclophosphamide, methotrexate, etoposide, and ifosfamide,

accompanied by intrathecal administration of etoposide and liposomal cytarabine) followed

� ��

by high-dose chemotherapy according to a modified Finlay protocol [4] with stem cell rescue

and cranial irradiation to a total of 54 Gy. After ten months the tumour was progressive with

cerebrospinal fluid dissemination. Subtotal resection was performed. Histopathology showed

a marked increase in tumour cell size, cellular pleomorphism with frequent nuclear moulding

and wrapping, and prominent nucleoli indicating malignant progression towards a large

cell/anaplastic phenotype (Fig 2D). The small biopsy specimen did neither contain areas of

neuropil nor ependymoblastic rosettes. FISH and SNP array revealed still amplification at

19q13.42. Interestingly, FISH demonstrated a significant increase in the proportion of cells

harboring the amplification, as well as condensation and fusion of the amplified signals (Fig

3A). Compared to the primary tumour, the SNP array revealed balanced profiles of

chromosomes 14, 17, 19, and 20, but loss of 17p, 18p and proximal 18q as well as a

complex rearrangement of chromosome 19q were detectable (Fig 3B). The child continued

on an anti-angiogenic treatment (Peyrl et al, manuscript in preparation) that consisted of

thalidomide, fenofibrate, celecoxib, etoposide, cyclophosphamide, and bevacizumab. Four

months later the clinical condition deteriorated. The patient died 15 months after the initial

diagnosis. Post-mortem examination of the brain revealed a left parietal resection cavity

surrounded by soft greyish tumour masses, which extended to the contralateral hemisphere,

covered the ventricular surfaces, and filled the basal cisterns. Upon histopathology, the large

cell/anaplastic tumour cells showed a striking nucleolar enlargement, which consisted

ultrastructurally of dense filamentous zones (Fig 2E-H). However, despite extensive sampling

procedures neither neuropil islands nor multilayered rosettes were detectable.

The clinical characteristics of this patient, including young age and unfavorable outcome

despite intensified treatment, as well as anaplastic progression of the tumour tissue are in

accordance with previous reports [13]. Whereas loss of neuropil islands has been already

observed in recurrences [13], the absence of multilayered rosettes has only been noticed in a

single recurrent tumour biopsy so far [8]. This case is unique, as we can exclude a sampling

error through extensive autoptic investigation, and confirm the loss of all morphological key

features of ETANTR during disease progression. Interestingly the amplification at 19q13.42

was retained at relapse, and moreover a significant increase in the proportion of cells

harboring the amplification was noted. The latter could correspond to the prominent nucleoli

in the recurrent tumour tissue. Enlarged nucleoli are a common feature of many cancers [11,

22](Koh et al.; Tornoczky et al., 2007), and have been linked to malignant transformation [12]

and cytotoxic treatment [21]. In cell cultures of peripheral paediatric neuroblastoma, activated

nucleoli indicate amplification of the MYCN gene [10]. In paediatric medulloblastoma

prominent nucleoli are typically found in the large cell subtype and are frequently associated

with MYCC or MYCN amplification [15] Neither MYCC nor MYCN amplification were noted in

this case and MYCN amplification does not seem to play a role in ETANTR [9].

� �

In the recurrent tumour tissue additional genomic aberrations were found, such as loss of

17p including tumour suppressor TP53, which could play a role in anaplastic progression.

The observed changes of genetic aberrations and morphological features support the

concept of selection of a therapeutically resistant, more aggressive clone during cytotoxic

treatment. Further paired genetic analyses could elucidate the mechanisms underlying the

biological evolution of the tumour.

In the literature single cases have been reported, which were initially diagnosed as central

neurocytoma or medulloepithelioma, but exhibited characteristic histopathological features of

ETANTR at relapse [2, 3]. This obviously variable morphology could lead to misdiagnosis of

ETANTR. Accurate diagnosis of ETANTR is of high clinical relevance because of its poor

response to current PNET treatment protocols and thus frequent fatal outcome. Therefore,

genetic analysis of 19q13.42 contributes to diagnostic accuracy and should be performed in

all CNS PNETs in children.

� �

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Deimling A, Wiestler OD, Giangaspero F, Rosenblum M, Pietsch T, Lichter P, Pfister

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19. Pfister S, Remke M, Castoldi M, Bai AH, Muckenthaler MU, Kulozik A, von Deimling A,

Pscherer A, Lichter P, Korshunov A (2009) Novel genomic amplification targeting the

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microRNA cluster at 19q13.42 in a paediatric embryonal tumour with abundant

neuropil and true rosettes. Acta Neuropathol 117 (4):457-464. doi:10.1007/s00401-

008-0467-y

20. Rutkowski S, Bode U, Deinlein F, Ottensmeier H, Warmuth-Metz M, Soerensen N, Graf

N, Emser A, Pietsch T, Wolff JE, Kortmann RD, Kuehl J (2005) Treatment of early

childhood medulloblastoma by postoperative chemotherapy alone. N Engl J Med 352

(10):978-986. doi:352/10/978 [pii] 10.1056/NEJMoa042176

21. Smetana K, Karban J, Trneny M (2010) To the nucleolar bodies (nucleoli) in cells of the

lymphocytic lineage in patients suffering from B - chronic lymphocytic leukemia.

Neoplasma 57 (6):495-500

22. Tornoczky T, Semjen D, Shimada H, Ambros IM (2007) Pathology of peripheral

neuroblastic tumours: significance of prominent nucleoli in undifferentiated/poorly

differentiated neuroblastoma. Pathol Oncol Res 13 (4):269-275.

doi:PAOR.2007.13.4.0269

23. Wang Y, Chu SG, Xiong J, Cheng HX, Chen H, Yao XH (2011) Embryonal tumour with

abundant neuropil and true rosettes (ETANTR) with a focal amplification at

chromosome 19q13.42 locus: Further evidence of two new instances in China.

Neuropathology. doi:10.1111/j.1440-1789.2011.01215.x

� ��

Captions for figures

Figure 1. Neuroimaging

A T1-weighted MRI with gadolinium contrast displays a hypo- to isointense, 9.6 x 8.6 x 11.7

cm left parieto-occipital, hypo- to isointense lesion with B cerebrospinal fluid–intense cysts

on the FLAIR sequence and C pathologic Cho/NAA ratio and lactate peak on single voxel

spectroscopy.

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Figure 2. Morphological evolution of the tumour

A First biopsy displays a primitive neuroectodermal tumour with B hypocellular neuropil

islands and C multilayered rosettes. D Recurrent tumour biopsy and E post-mortem tissue

show malignant evolution of a large cell / anaplastic phenotype with F striking enlargement of

intensely eosinophilic nucleoli, which G, H ultrastructurally consist of dense filamentous

zones.

� ��

Figure 3. Genetic findings

A FISH displays stable amplification at 19q13.42 (green signals) in the primary and recurrent

tumour. Note the increased and condensed green signals in the tumour recurrence. B SNP

array results are shown for chromosomes 19, 17, and 14. Copy number analysis confirms

the amplification at 19q13.42 in the primary and recurrent tumour (arrow heads). The primary

tumour reveals trisomies of chromosomes 19, 17, and 14 (blue lines), these extra copies are

lost in the tumour recurrence (yellow lines). A complex rearrangement of 19q and loss of 17p

are observed in the recurrent tumour.

� ��

Clinical Neuropathology 2011; 30: 47-55, IF 1.067

FISH-based detection of 1p 19q codeletion in oligodendroglial tumours: procedures

and protocols for neuropathological practice - a publication under the auspices of the

Research Committee of the European Confederation of Neuropathological Societies

(Euro-CNS)

Adelheid Woehrer1,2, Petra Sander3, Christine Haberler1, Sabine Kern1, Hans Maier3,

Matthias Preusser2,4, Christian Hartmann5,6, Johan M Kros7, Johannes A Hainfellner1,2

1Institute of Neurology, Medical University of Vienna, Waehringer Guertel 18-20, 1097

Vienna, Austria 2Comprehensive Cancer Centre Central Nervous System Unit (CCC-CNS), Medical

University of Vienna, Waehringer Guertel 18-20, 1097 Vienna, Austria 3Department of Pathology, Medical University of Innsbruck, Muellerstrasse 44, 6020

Innsbruck, Austria 4Department of Medicine I, Medical University of Vienna, Waehringer Guertel 18-20, 1097

Vienna, Austria 5Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University

Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germany 6Clinical Cooperation Unit Neuropathology, German Cancer Research Centre, 69120

Heidelberg, Germany 7Department of Pathology, Erasmus MC Rotterdam, Postbus 2040, 3000 CA Rotterdam, The

Netherlands

Correspondence to

Johannes A Hainfellner

Institute of Neurology, CCC-CNS, Medical University of Vienna

Waehringer Guertel 18–20, A-1097 Vienna, Austria

Telephone number: +43-1-40400-5507

Fax: +43-1-40400-5511

Email: johannes.hainfellner@meduniwien.ac.at

Running title

FISH-based analysis of 1p 19q status in oligodendroglial tumours

� ��

Abstract

The codeletion of chromosomal arms 1p 19q is a characteristic and early genetic event in

oligodendroglial tumours, that is associated with a better prognosis and enhanced response

to therapy. Over the last years, the increasing clinical demand to determine the 1p 19q status

has led to the implementation of its testing in many neuropathology laboratories. Several

different methods for 1p 19q testing are available: PCR-based loss of heterozygosity

analysis, multiplex ligation-dependent probe amplification, array comparative genomic

hybridization, and fluorescence in situ hybridization (FISH). Herein, we focus and critically

discuss the latter method because a detailed description of procedures and protocols for

FISH-based 1p 19q testing in practice is lacking. We present a practical approach to the

FISH-based assessment of the 1p 19q status in oligodendroglial tumours, from commonly

used locus-specific probes and technical protocols to the neuropathological interpretation of

results. Thereby, we aim to facilitate the implementation of FISH-based 1p 19q testing for

clinical purposes in standard neuropathology laboratories without special focus on brain

tumour research.

Keywords

Fluorescence in situ hybridization – oligodendroglial neoplasm – chromosomal arms 1p 19q

� ��

Introduction

In gliomas, a number of biomarkers that provide diagnostic or prognostic/ predictive

information are currently under debate, the most common being isocitrate dehydrogenase-1

(IDH1) mutation in low and high grade gliomas [1, 2], BRAF gene fusion in pilocytic

astrocytomas [3], O6-methylguanine methyltransferase (MGMT) promoter methylation status

in glioblastomas [4], and the codeletion of chromosomal arms 1p 19q in oligodendroglial

tumours [5, 6, 7]. However, whether and how fast a biomarker translates from preclinical

research to the routine diagnostic setting depends on both its clinical and analytical

performance [8]. Among the various candidate biomarkers in gliomas, the 1p 19q codeletion

in oligodendroglioma probably constitutes the best-characterized and most extensively

investigated marker up to date. The 1p 19q codeletion is a characteristic and early molecular

genetic event in oligodendroglial tumours, and 1p 19q codeleted tumours are associated with

a better prognosis and enhanced response to therapy [5, 9, 10]. Over the last years, the

increasing interest of medical oncologists in the 1p 19q status has led to the implementation

of its testing in many neuropathology laboratories. However, despite the huge body of

literature on this marker, no consensus guidelines or standard protocols for practical use

exist.

Various methods for 1p 19q testing are available: PCR-based loss of heterozygosity (LOH)

analysis, multiplex ligation-dependent probe amplification (MLPA), bacterial artificial

chromosome (BAC)-array comparative genomic hybridization (aCGH), and fluorescence in

situ hybridization (FISH) [11, 12, 13, 14]. The latter method has proven robust and cost-

efficient, and straightforward to implement, but a detailed description of procedures and

protocols for FISH-based 1p 19q testing in practice is lacking.

Herein, we present a detailed practical approach to FISH-based analysis of the 1p 19q

status, including commonly used locus-specific probes, technical protocols and the

interpretation of results, in order to facilitate implementation of FISH-based 1p 19q testing for

clinical purposes in standard neuropathology laboratories without special focus in brain

tumour research.

� �

Procedures and protocols

Tumour tissue

1p 19q FISH can be performed on either RCL2- (Preusser et al.), or formalin-fixed paraffin

embedded (FFPE), or fresh/frozen tumour tissue. It does not require additional blood

samples of the patient.

Regions of interest

Tumour areas need to be preselected under the light microscope. Adequate areas should

contain >60 % tumour cell infiltration [16], and no necrosis or hemorrhage. Further

processing of the block relies on the chosen approach: interphase nuclei may be isolated

from punch preparations of the block [17], or whole-block slides are cut at a thickness of 4-5

microns [18]. FISH analysis is performed using a dual-color approach for chromosomes 1

and 19 separately. Target probes hybridize to subtelomeric 1p36 and 19q13.3 in combination

with control probes on 1q and 19p, respectively (see figure 1). For evaluation, the signal ratio

is assessed for 100-200 adjacent, non-overlapping interphase nuclei, and the results are

expressed as percentage.

Probes

A number of companies provide locus-specific and fluorochrome-labeled DNA probes.

Widely used, commercially available probes include those of Vysis ® (Abbott Laboratories,

Illinois, USA) or Zytomed Systems ® (Berlin, Germany). Among other suppliers are

Qbiogene Inc ® (Carlsbad, USA), Cytocell Ltd ® (Cambridge, UK), and Kreatech Diagnostics

® (Amsterdam, The Netherlands). For some laboratories, in-house production of the probes

may be an adequate alternative.

Reagents and protocols

Many companies provide ready-to-use FISH kits that contain all necessary reagents for

pretreatment and washing procedures (e.g. the Histology FISH Accessory Kit K5599 by

Dako, Glostrup, Denmark). However, for those laboratories, which expect only a limited

number of investigations, preparation of fresh working solutions may be more suitable. A list

of required reagents and working solutions is provided in textbox 1. Along with the FISH

probes, manufacturers supply protocols and technical support. Exemplarily, the protocol of

the Medical Universities of Vienna and Innsbruck (MUV/MUI protocol) for hybridization on

paraffin-embedded sections is stated in table 1. This protocol incorporates useful adaptations

according to the institutional experiences. However, in addition to these standard protocols

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and procedures appropriate handling of the individual sample with slight variations based on

practical experience is important. For instance, critical steps such as the right protease

digestion time may differ from case to case, and require individual handling. For further

reading on this issue a recent publication by Horbinski et al [19] is recommended.

Furthermore, standardization of slide denaturation and hybridization can be achieved by the

use of automated systems (e.g. ThermoBrite system combined with the VP2000 Processor

by Abbott Molecular Europe, Wiesbaden, Germany).

Equipment

Examination of slides requires a fluorescence microscope equipped with adequate filters

(e.g. Vysis probes labeled with Spectrum Green and Spectrum Orange (Vysis Abbott

Laboratories, Illinois, USA) require Zeiss filter sets 49 (4´,6-diamidino-2-phenylindole –

DAPI), 15 (Cy3.5), 10 (FITC), 23 (red+green) (Carl Zeiss Microimaging, Germany).

Interpretation of FISH results

Signal ratios are assessed individually for chromosomes 1 and 19 and interpreted grossly

according to the guidelines of the International Society of Paediatric Oncology (SIOP) Europe

Neuroblastoma Pathology and Biology and Bone Marrow Group (Ambros and Ambros,

2001). Whereas normal, diploid nuclei show a signal ratio of 2/2, a nucleus is considered to

harbor a deletion, if the target signal is 0 or 1 in relation to normal or excess control signals

(e.g. 2/0, 2/1, 3/0, 3/1, etc). Such deletions most likely correspond to a loss of heterozygosity

(LOH) found by PCR. However, with increasing grade of malignancy genomic polyploidies

may be encountered. These chromosomal polysomies may be balanced (e.g. 3/3, 4/4, 5/5,

etc) or imbalanced (e.g. 3/2, 4/2, 3/5, etc), indicating relative gains or losses. Whether an

imbalance situation with relative loss of the target 1p or 19q corresponds to a hemizygous

deletion in presence of reduplication cannot be resolved by FISH. Further clarification

requires the use of ancillary tests such as PCR-based LOH [16]. For interpretation of FISH

results, signal ratios for 100–200 adjacent, non-overlapping nuclei are evaluated. The

number of nuclei exhibiting a balance, imbalance, or deletion are summed and expressed as

percentages. If the number of ´deleted´ nuclei exceed a certain cut-off value (see next

paragraph), the tumour is considered to show a ´deletion´ for the chromosome part targeted.

In case of an imbalance situation with relative loss of the target (1p or 19q), the prevailing

pattern (e.g. 4/2) and its prevalence (e.g. in 60 % of the evaluated nuclei) are reported as

such.

Cut-off definition

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For proper interpretation of test results, clear definition of a cut-off for deletion or imbalance

status is an essential prerequisite. Many neuropathology laboratories choose their cut-off

arbitrarily. Cut-offs in use for the overall target-to-control signal ratios range from 15–70 %

[20, 21]. Instead of summary ratios, the SIOP examination guidelines recommend the

evaluation of control-to-target signal ratios for individual nuclei and suggest a cut-off of 50 %

of nuclei that show a deletion [16]. An alternative approach is to determine a lab-specific cut-

off on non-neoplastic, white matter oligodendrocytes (e.g. on temporal lobe specimens).

Herein, the fraction of nuclei which deviate from the diploid 2/2 signal ratio

(=deletion/imbalance/polysomy) is determined on a series of 5-10 cases. The cut-off value is

then calculated by the mean plus 3 standard deviations [18, 22]. For example, the cut-off

calculated by this method at the Medical University of Vienna is 30% for 1p/1q and 19p/19q,

respectively.

Documentation

As fluorescence signals bleach over time, adequate photo documentation is required in the

routine clinical setting.

Discussion

Tumour biological background for combined 1p 19q assessment

Oligodendroglial tumours comprise pure oligodendrogliomas and mixed gliomas

(oligoastrocytoma, glioblastoma with an oligodendroglial component) [23]. Together they

account for approximately 35 % of all gliomas [24]. More than 70 % of oligodendrogliomas

and roughly 40 % of oligoastrocytomas display deletions of chromosomal arms 1p and 19q

(Fallon et al., 2004; Fuller and Perry, 2005). The deletion typically involves both

chromosomal arms, whereas solitary losses on either 1p or 19q are encountered only in a

small fraction of tumours, in particular mixed oligoastrocytomas [25]. Although the

characteristic codeletion is known since many years [27], the underlying mechanism that

mediates the combined loss has been proposed a few years ago [28, 29]. A balanced whole-

arm translocation between chromosomes 1 and 19 was described, which leads to the

formation of two derivative chromosomes. Subsequent loss of the derivative chromosome

composed of chromosomal arms 1p and 19q [der(1;19)(q10;p10)] results in the observed

codeletion.

As the chromosomal status for 1p/1q most often corresponds to 19p/19q and vice versa,

congruency of hybridization results on both chromosomal arms serves as internal validity

� ���

control. Therefore, for quality reasons testing of both chromosomal loci is recommended in

the routine clinical setting instead of 1p testing only [30, 31].

In contrast to the codeletion of whole chromosomal arms 1p and 19q, which is strongly

associated with an oligodendroglial morphology, small interstitial deletions on 1p have been

described in diffuse gliomas [14, 32, 33]. These partial 1p deletions were found to be more

prevalent in astrocytic as compared to oligodendroglial tumours and seem to be associated

with a worse prognosis (Idbaih et al., 2005; Iuchi et al., 2002) [32, 33]. For reliable detection

of partial deletions, FISH as proposed in this paper is not useful, and more sophisticated

methods such as aCGH need to be applied [14]. Such ancillary diagnostics are, however,

beyond standard needs of contemporary clinical neuro-oncology.

Role of 1p 19q status as diagnostic marker

The relevance of the 1p 19q deletion as a potential diagnostic marker for oligodendrogliomas

is controversially discussed [19, 34, 35, 36]. The issue of tumour typing on basis of the 1p

19q status has been raised [37, 38]. It has become clear, however, that the 1p 19q

codeletion is closely but not absolutely associated with the oligodendroglial phenotype. There

are a few morphologically typical oligodendrogliomas that do not show the characteristic

codeletion. According to the current World Health Organization (WHO) 2007 consensus

criteria, the diagnosis of an oligodendroglioma remains morphology-based irrespective of the

1p 19q deletion status [23]. Hence, 1p 19q losses constitute a prognostic molecular

cytogenetic marker, but do not per se define a distinct tumour entity or variant.

Methodological considerations of FISH-based 1p 19q testing

FISH is an approved method for molecular cytogenetic testing on routinely available, FFPE

tissues [39]. FISH analysis is independent from the age of the paraffin block, does not

require reference tissues such as autologous blood samples, and the hybridization result is

morphologically controlled. Thus, FISH constitutes a robust and straight-forward to

implement technique, suitable for the majority of neuropathology laboratories, including

standard laboratories without diagnostic/research focus on brain tumours.

FISH analysis can be performed either on isolated nuclei or on tissue sections. In case of

detecting a deletion such as 1p or 19q loss, the hybridization of isolated tumour cell nuclei

has been reported to be superior to conventional tissue sections due to the avoidance of

truncated nuclei [40]. However, according to our experience, hybridization of tissue sections

yields sufficiently reliable results as long as adequate thickness of the sections (4-5 microns)

is warranted and a sufficient number of nuclei (100–200) are evaluated. The delicate

pretreatment and digestion procedures for FISH on isolated nuclei is laborious and time-

consuming, requires a certain level of experience, and should be done only as second line

� ���

investigation in the rare situation, in which FISH on sections does not allow proper evaluation

of the test result because of nuclear overlap.

According to our experience, the neuropathological interpretation of the FISH results is

straight-forward in most cases. A classic deletion with a signal ratio of 2/1 prompts an instant

diagnosis. The evaluation of inconclusive, imbalance situations may require examination of a

larger number of nuclei. In the diagnostic setting in practice, the exact cut-off value is not a

major issue, as FISH usually yields clear-cut results far beyond the cut-off (defined as >30%

in our centres) in the case of a 1p 19q deletion status (e.g. >80% of the nuclei showing a

deletion). However, in case of an unclear FISH result additional hybridization of another

section may be necessary.

According to our experience, FISH yields interpretable results in the vast majority of cases

(>90%). However, we came across single cases that stayed hybridization-refractory despite

repetitive attempts with various adaptations of the protocol apparently due to poor DNA

quality in the tissue specimen. Moreover, continued analytics on such specimens with an

alternative method like a PCR-based LOH analysis, in our experience, will not likely yield an

interpretable result either.

Limitations of FISH-based 1p 19q testing

FISH has some methodological limitations neuropathologists need to be aware of. First,

commonly used and commercially available probes for 1p/1q and 19p/19q span a relatively

large region (400-600kb), and do not allow for detection of small interstitial deletions [14].

Secondly, imbalance situations with relative losses of the targets 1p 19q (e.g. 4/2, 4/3, 5/3

ratios) could correspond to hemizygous deletions in the presence of reduplication. Such

cases require further clarification by an alternate e.g. PCR-based method [16].

Analytical performance of FISH-based 1p 19q testing

FISH has been shown a reliable method for the analysis of the 1p 19q status in

oligodendroglial tumours [17]. In addition, several independent studies found good

concordance between FISH and PCR-based LOH or MLPA results [11, 13, 22, 41]. However,

inter-observer and inter-laboratory variability have not yet been addressed in a systematic

way, and common standards for 1p 19q FISH procedures across neuropathology

laboratories are still lacking. Similarly, no accepted standards exist for PCR-based LOH or

MLPA either [13]. There, the situation might be even more complicated as the interpretation

of results strongly depends on the choice and quantity of probes / microsatellite markers

along chromosomal arms 1p and 19q. Moreover, the number of microsatellite markers that

are required to show a deletion seems unclear [13]. Thus, quality assurance remains an

� ���

issue, and round-robin tests with regular participation of laboratories performing 1p 19q

testing in the clinical setting need to be set up in future.

Clinical performance of FISH-based 1p 19q testing

The codeletion of chromosomal arms 1p 19q is considered a strong prognostic factor in

oligodendroglial tumours, especially for the subset of anaplastic oligodendrogliomas, being

associated with a longer progression-free and overall survival of the patients. Up to date,

molecular genetic testing of the 1p 19q status is of particular relevance within the setting of

clinical trials, where it serves as an important stratification factor (e.g. European Organisation

for Research and Treatment of Cancer (EORTC) trial 22033-26033), and has meanwhile led

to the design of distinct clinical trials for 1p 19q intact and 1p 19q deleted tumours (EORTC

26053-22054 CATNON versus EORTC 26081-NCCTG N0577 CODEL). In contrast to the

role of the 1p 19q status as patient stratification factor or eligibility criterion in clinical trials, its

significance in the routine clinical setting is that of a prognostic factor which needs to be

weighed in conjunction with other prognostic factors (e.g. performance status, IDH1 status,

Ki67 index). So, knowledge of the 1p 19q status in practice is helpful for individual patient

counselling, but does not per se define a common therapy/patient management standard [8,

37].

Considerations with regard to the usefulness of 1p 19q testing in particular situations

Testing of the 1p 19q status is useful in the case of oligodendroglioma and mixed

oligoastrocytoma. In pure astrocytoma 1p 19q testing cannot be generally recommended, as

a deletion status is quite rare. According to the authors’ and others’ experience repeated

testing in case of tumour recurrence seems not useful, as the codeletion typically constitutes

an early genetic event [19]. In paediatric and adolescent oligodendroglial neoplasms, 1p 19q

testing is not so relevant due to the rarity of deletion in this age cohort.

Conclusions

FISH allows for fast and accurate detection of molecular cytogenetic alterations. It constitutes

a straightforward assay for implementation in routine diagnostic assessment of the 1p 19q

status in oligodendroglial tumours. This work is intended to serve as a manual for the

implementation of FISH-based 1p 19q testing for clinical purposes in the standard

neuropathology laboratory.

Conflict of Interest Statement

The authors declare no conflict of interest.

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Acknowledgments

We thank Renate Vesely (Krankenanstalt Rudolfstiftung, Vienna) and Peter and Inge Ambros

(St. Anna´s Children´s Cancer Research Institute, Vienna) for helpful comments and

technical advice, and Gerda Ricken and Sabine Kaindl for excellent technical assistance.

� ���

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Textbox, Tables & Figures

Textbox 1. FISH reagents.

REAGENTS

1 M NaSCN Natriumthiocyanat (pH 6.0–8.5)

0.2N HCl

Pepsin buffer (0.9% NaCl, pH 2.0)

0.05% Pepsin-protease solution

3.7% Formaldehyde in 1xPBS pH 7.2 (phosphate buffered saline)

Citrate buffer

2xSSC pH 7.0 (saline-sodium citrate buffer)

2xSSC/0.3% NP-40 wash solution

Aqua bidest

Fluorescence mounting medium (4´,6-diamidino-2-phenylindole – DAPI)

Ethanol 70% / 96%

�

� ���

Table 1. MUV/MUI protocol for FISH on formalin-fixed paraffin-embedded sections.

PRETREATMENT

Deparaffinize slides (4-5 microns thick) by incubating in xylene at 65°C for 3x

Step 1

10 min, dehydrate 2x 5 min in ethanol (100%) and air dry for 10 min

Step 2 Pretreat slides in 0.2N HCl for 20 min at room temperature

Step 3 Wash slides in 2xSSC for 2x 5 minutes at room temperature (shake slightly)

Incubate slides in 1M NaSCN or alternatively citrate buffer, both for 30 min

Step 4

at 80°C (water bath)

Step 5 Wash slides in 2xSSC for 2x 5 min at room temperature (shake slightly)

Incubate slides in 0.05% protease-pepsin solution for 20 minutes at 37°C

Step 6

(water bath)

Rinse slides briefly in 2xSSC and wash for 2x 5 min in 2xSSC at room

Step 7

temperature (shake slightly)

Step 8 Fix slides in 3.7% formaldehyde in 1xPBS for 10 min at room temperature

Wash slides in 2xSSC for 2x 5 min at room temperature (shake

Step 9

slightly) and air dry for 10 min

Dehydrate slides by a series of ethanol washes (70%, 90%, and 100%)

Step 10

each for 2 min and air dry for 10 min

CO-DENATURATION AND HYBRIDIZATION

Step 1 Add probe mixture, cover by a coverslip and seal with rubber cement

NOTE: Handle slides in reduced light after probes have been applied

Step 2 Co-denature slides on a hot plate at 78°C for 8 min

Step 3 Hybridize slides over night in a preheated humidity chamber at 37°C for at least 16 h

WASHING PROCEDURE

Step 1 Remove rubber cement and detach coverslip in 2xSSC carefully

Step 2 Wash slides in 2xSSC containing 0.3% NP-40 for 1min at 37°C

Step 3 Wash slides in 2xSSC containing 0.3% NP-40 for 1min at 72°C

Step 4 Wash slides in 2xSSC containing 0.3% NP-40 for 1min at room temperature

Wash slides briefly in distilled water, air dry for 10 min and cover with a fluorescence

Step 5

mounting medium containing DAPI

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Figure 1. Locus-specific probes on chromosomes 1 and 19 (Vysis, Abbott Laboratories,

Illinois, USA). Chromosomal target regions are indicated in red, control regions in green.

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Figure 2. Representative FISH images (chromosome 1, analogous signals are obtained with

probes for chromosome 19).

Target signal=red, Control signal=green, DAPI counterstained A. Normal diploid signal ratio

(2 controls / 2 targets), magnification 63x B. Deletion status (signal ratio 2 controls / 1 target),

magnification 63x. C. Imbalance with relative loss (4 control / 2 target signals), magnification

63x D. Fluorescence in situ hybridization of a 4 micron thick tissue section (magnification

40x) shows a deletion status (signal ratio 2 controls / 1 target) of the majority of nuclei. For

evaluation only non-overlapping nuclei (arrows) are analyzed, whereas clustered/overlapping

nuclei (*) are not considered.

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Curriculum Vitae

Adelheid WOEHRER

Date of Birth: September 6th, 1980

Place of Birth: Mödling, Niederösterreich, Austria

Nationality: Austria

Address private: Stolzenthalergasse 13/18, A-1080 Vienna

Academic Degree: Medical Doctor (MD)

Current Position: Resident in Neuropathology,

Institute of Neurology, Medical University of Vienna

Address: AKH 4J, Währinger Gürtel 18-20, A-1090 Vienna

Phone: +43 1 40400 5505

Fax: +43 1 40400 5511

E-mail: Adelheid.Woehrer@meduniwien.ac.at

Education

2006: Medical Doctor (MD), MUV

2007–: Resident training in Neuropathology, Institute of Neurology, MUV

2009–: Doctoral program Clinical Neurosciences at the MUV

PhD thesis ´Brain Tumor Epidemiology in Austria´

Publications

Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of

gastric cancer growth in vivo. Cancer Biol Ther 2008;7:1377-1385. Cejka D, Preusser M,

Woehrer A, Sieghart W, Strommer S, Werzowa J, Fuereder T, Wacheck V.

Ki67 index in intracranial ependymoma: a promising histopathological candidate biomarker.

Histopathology 2008;53:39-47. Preusser M, Heinzl H, Gelpi E, Hoftberger R, Fischer I, Pipp

I, Milenkovic I, Woehrer A, Popovici F, Wolfsberger S, Hainfellner JA.

Residual nonfunctioning pituitary adenomas: prognostic value of MIB-1 labeling index for

tumor progression. J Neurosurg 2009;111:563-571. Widhalm G, Wolfsberger S, Preusser M,

Fischer I, Woehrer A, Wunderer J, Hainfellner JA, Knosp E.

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O(6)-methylguanine DNA methyltransferase immunoexpression in nonfunctioning pituitary

adenomas: are progressive tumors potential candidates for temozolomide treatment? Cancer

2009;115:1070-1080. Widhalm G, Wolfsberger S, Preusser M, Woehrer A, Kotter MR,

Czech T, Marosi C, Knosp E.

The Austrian Brain Tumour Registry: a cooperative way to establish a population-

based brain tumour registry. J Neurooncol 2009;95:401-411. Woehrer A, Waldhor T,

Heinzl H, Hackl M, Feichtinger J, Gruber-Mosenbacher U, Kiefer A, Maier H, Motz R,

Reiner-Concin A, Richling B, Idriceanu C, Scarpatetti M, Sedivy R, Bankl HC,

Stiglbauer W, Preusser M, Rossler K, Hainfellner JA.

Elevated blood markers 1 year before manifestation of malignant glioma. Neuro Oncol

2010;12:1004-1008. Gartner W, Ilhan A, Neziri D, Base W, Weissel M, Wohrer A, Heinzl H,

Waldhor T, Wagner L, Preusser M.

Unclassifiable tauopathy associated with an A152T variation in MAPT exon 7. Clin

Neuropathol 2010;30:3-10. Kovacs GG, Woehrer A, Strobel T, Botond G, Attems J, Budka

H.

Primary central nervous system lymphoma: a clinicopathological study of 75 cases.

Pathology 2010;42:547-552. Preusser M, Woehrer A, Koperek O, Rottenfusser A,

Dieckmann K, Gatterbauer B, Roessler K, Slavc I, Jaeger U, Streubel B, Hainfellner JA,

Chott A.

5-Aminolevulinic acid is a promising marker for detection of anaplastic foci in diffusely

infiltrating gliomas with nonsignificant contrast enhancement. Cancer 2010;116:1545-1552.

Widhalm G, Wolfsberger S, Minchev G, Woehrer A, Krssak M, Czech T, Prayer D,

Asenbaum S, Hainfellner JA, Knosp E.

Incidence of atypical teratoid/rhabdoid tumors in children: A population-based study

by the Austrian Brain Tumor Registry, 1996-2006. Cancer 2010;116:5725-5732.

Woehrer A, Slavc I, Waldhoer T, Heinzl H, Zielonke N, Czech T, Benesch M, Hainfellner

JA, Haberler C.

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FISH-based detection of 1p 19q codeletion in oligodendroglial tumors: procedures and

protocols for neuropathological practice - a publication under the auspices of the

Research Committee of the European Confederation of Neuropathological Societies

(Euro-CNS). Clin Neuropathol 2011;30:47-55. Woehrer A, Sander P, Haberler C, Kern S,

Maier H, Preusser M, Hartmann C, Kros JM, Hainfellner JA.

Value of 1H-magnetic resonance spectroscopy chemical shift imaging for detection of

anaplastic foci in diffusely infiltrating gliomas with nonsignificant contrast-enhancement. J

Neurol Neurosurg Psychiatry 2011;82:512-20. Widhalm G, Krssak M, Minchev G, Woehrer

A, Traub-Weidinger T, Czech T, Asenbaum S, Marosi C, Knosp E, Hainfellner JA, Prayer D,

Wolfsberger S.

Prognostic value of Ki67 index in anaplastic oligodendroglial tumors – a translational study of

the European Organization for Research and Treatment of Cancer (EORTC) Brain Tumor

Group. Histopathology 2011 in press. Preusser M, Hoeftberger R, Woehrer A, Gelpi E,

Kouwenhoven M, Kros J, Sanson M, Idbaih A, Brandes A, Heinzl H, Gorlia T, Hainfellner J,

van den Bent M.

Current Concepts and Management of Glioblastoma. Ann Neurol. 2011;70:9-21. Preusser M,

de Ribaupierre S, Wöhrer A, Erridge SC, Hegi M, Weller M, Stupp R.

Value and limitations of immunohistochemistry and gene sequencing for detection of the

IDH1-R132H mutation in diffuse glioma biopsy specimens. J Neuropathol Exp Neurol.

2011;70:715-23. Preusser M, Wöhrer A, Stary S, Höftberger R, Streubel B, Hainfellner JA.

Longitudinal brain imaging of five malignant glioma patients treated with bevacizumab using

susceptibility-weighted magnetic resonance imaging at 7T. Magn Reson Imaging

2012;30:139-47. Grabner G, Nöbauer I, Elandt K, Kronnerwetter C, Woehrer A, Marosi C,

Prayer D, Trattnig S, Preusser M.

Immunohistochemical testing of BRAF V600E status in 1,120 tumor tissue samples of

patients with brain metastases. Acta Neuropathol 2011 (epub ahead of print). Capper D,

Berghoff AS, Magerle M, Ilhan A, Wöhrer A, Hackl M, Pichler J, Pusch S, Meyer J, Habel A,

Petzelbauer P, Birner P, von Deimling A, Preusser M.

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Multiple intracranial cavernomas with focal amyloid deposition – diagnostic pitfalls. Clin

Neuropathol 2011;30:324-7. Velnar T, Bunc G, Flisar D, Kulas D, Woehrer A, Budka H,

Popovic M.

Embryonal Tumor with Abundant Neuropil and True Rosettes (ETANTR) with loss of

morphological but retained genetic key features during progression. Acta Neuropathol

2011;122:787-90. Woehrer A, Slavc I, Peyrl A, Czech T, Dorfer C, Prayer D, Stary S,

Streubel B, Ryzhova M, Korshunov A, Pfister SM, Haberler C.

Strong 5-Aminolevulinic Acid Induced Fluorescence is a Novel Intraoperative Marker for

Representative Tissue Samples in Sterotactic Brain Tumor Biopsies. Neurosurgical Review

2011 in press. Widhalm G, Minchev G, Wöhrer A, Preusser M, Furtner J, Mert A, Di Ieva A,

Prayer D, Marosi C, Hainfellner JA, Knosp E, Wolfsberger S.