Detection of MAPK pathway activation in juvenile pilocytic astrocytomas as part of the neuropathological diagnosis. Master Thesis

Detection of MAPK pathway activation in juvenile pilocytic astrocytomas as part of the

neuropathological diagnosis

Master Thesis

Submitted in partial fulfilment of the requirements for the degree of Master of Science in Biomedical Science (MSc)

to the University of Applied Sciences FH Campus Wien Master Degree Program for Biomedical Science

Author:

Verena Kainz, BSc.

Student identification number:

1530003009 Supervisor:

Ao. Univ. Prof. Dr. Thomas Ströbel Reviewer:

Ao. Univ. Prof. Dr. DI Johannes Nimpf Date:

31. 08. 2018

Declaration of authorship:

I declare that this Master Thesis has been written by myself. I have not used any other than the listed sources, nor have I received any unauthorized help.

I hereby certify that I have not submitted this Master Thesis in any form (to a reviewer for assessment) either in Austria or abroad.

Furthermore, I assure that the (printed and electronic) copies I have submitted are identical.

Date: ... Signature: ...

Preface

Ich möchte mich an dieser Stelle ganz herzlich bei all jenen bedanken, die mich während meiner Masterarbeit begleitet und motiviert haben und mich während des Studiums unterstützt haben.

Zu aller erst möchte ich mich gerne bei meinem Betreuer Herrn Ao. Univ. Prof. Dr.

Thomas Ströbel für die Bereitstellung des Themas und seine verlässliche fachliche und persönliche Unterstützung und seinen Rückhalt während des gesamten Arbeitsverlaufes bedanken.

Ein weiterer besonderer Dank gilt Frau Michaela Kreiter, BSc, welche sehr viel Zeit aufgewendet hat, um mich sorgfältig in alle Techniken einzuschulen, mir wertvolle Tipps gegeben hat und auf deren Hilfestellung ich mich immer verlassen konnte. Sie hat mir mit viel Geduld und ihrer fachlichen Erfahrung stets weitergeholfen. Es waren sehr schöne und lustige Stunden, die ich mit euch im Labor verbringen durfte und die mir immer in Erinnerung bleiben werden.

Außerdem gilt ein besonderer Dank meiner Familie, insbesondere meinen Eltern, die mir dieses Studium ermöglicht, mir Selbstvertrauen gegeben haben und mir in dieser Zeit eine unglaubliche Stütze waren.

Nicht zuletzt möchte ich mich bei einem ganz besonderen Menschen bedanken, der mir während des ganzen Studiums zur Seite stand und dessen Zuwendung und Verständnis mir sehr geholfen haben.

Abstract

Central nervous system tumors represent about 25 % of neoplastic disease in childhood and the pilocytic astrocytoma is the most frequent brain tumor in the pediatric population.

Pilocytic astrocytomas are low-grade gliomas (LGG) and classified as WHO grade I. They are relative benign, slow growing tumors and can be cured due to surgery. Although this type of tumor is associated with excellent overall survival, children can suffer mortality due to recurrence and malignant progression even in the setting of optimal resection.

Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway drives the formation of the majority of LGGs and genetic defects including KIAA1549-BRAF fusions and BRAF mutations are responsible for it. The most common mechanism of MAPK pathway activation in LGGs is a tandem duplication in 7q34, resulting in a KIAA1549- BRAF fusion transcript. The identification of this fusion gene can be of diagnostic and prognostic value. The activation of the MAPK signaling pathway may open new therapeutic strategies for patients with pilocytic astrocytoma and so the detection of the gene fusions is important.

48 patients with well-documented pilocytic astrocytomas were examined. DNA and RNA were isolated from formalin-fixed, paraffin-embedded (FFPE) and corresponding frozen tumor samples. Polymerase chain reaction (PCR), sequencing and multiplex ligation- dependent probe amplification (MLPA) were used for the detection of the fusion transcripts.

Fusion transcripts were detected in 38/48 tumors from frozen tissue. KIAA1549-BRAF gene fusions confirmed by sequencing included 16-9 (24 cases), 15-9 (7 cases), 16-11 (6 cases) and 18-9 (1 case). The detection of the fusion genes from FFPE tissue was performed from 26 tumor samples. In none of these FFPE tissue samples we could determine with certainty any particular fusion or no fusion at all. Furthermore, DNA from 35 patients was isolated to detect a 2 Megabase tandem duplication at 7q34 leading to KIAA1549-BRAF fusion using MLPA analysis. Comparing the results obtained from RT- PCR analysis and MLPA analysis we observed that in 9 out of 22 cases with a DQ between 0,8 and 1,2 no KIAA1549-BRAF fusion could be detected using RT-PCR, whereas in the remaining 13 cases the KIAA1549-BRAF fusion was found. In all cases with a DQ between 1,21 and 1,29 the KIAA1549-BRAF fusion could be detected using RT-PCR. In one out of 6 cases with a DQ between 1,30 and 1,65 no KIAA1549-BRAF fusion could be detected using RT-PCR, whereas in the remaining 5 cases the KIAA1549-BRAF fusion was found. Taken together, we were not able to obtain clear and convincing results for the presence of a 2 Megabase tandem duplication at 7q34 in FFPE tissues as well as fresh frozen tissues. Since FFPE tissue with a storage time for at least one year at room temperature was used and since the time between the sample collection and the fixation are no longer transparent it needs further investigations to examine the influence of storage time and fixation on the quality of RNA and DNA isolated from FFPE tissue samples.

Kurzfassung

Tumore des zentralen Nervensystems umfassen etwa 25 % aller neoplastischen Krankheiten bei Kindern, wobei das pilozytische Astrozytom der häufigste Gehirntumor ist. Pilozytische Astrozytome sind niedriggradige Gliome und entsprechen WHO Grad I.

Es ist ein gutartiger und langsam wachsender Tumor, welcher bei vollständig operativer Entfernung günstige Prognosen aufweist. Obwohl dieser Tumortyp mit sehr gutem Gesamtüberleben assoziiert ist, besteht bei Kindern aufgrund von Rezidiven und bösartigem Verlauf trotz optimaler Resektion ein gewisses Sterblichkeitsrisiko.

Konstitutive Aktivierung des MAP-Kinase (MAPK)-Pathway fördert die Bildung der meisten niedriggradigen Gliome und genetische Defekte wie KIAA1549-BRAF Fusionen und BRAF Mutationen sind dafür verantwortlich. Der häufigste Mechanismus der Aktivierung des MAPK-Pathways ist eine Tandemduplikation des Locus 7q34 und daraus resultierend die Entstehung des KIAA1549-BRAF Fusionstranskripts. Die Identifizierung dieses Transkripts ist von diagnostischer und prognostischer Bedeutung und eröffnet eventuell neue therapeutische Strategien für Patienten mit pilozytischen Astrozytomen.

Es wurden 48 Patienten mit dokumentierten pilozytischen Astrozytomen untersucht. DNA und RNA wurden aus Formalin fixiertem und in Paraffin eingebettetem Gewebe und aus korrespondierendem Gefriermaterial isoliert. Polymerase-Kettenreaktion (PCR), Sequenzierung und „Multiplex ligation-dependent probe amplification“ (MLPA) wurden zur Detektion der Fusionen verwendet.

Im Gefriermaterial konnten Fusionstranskripte in 38/48 Tumoren festgestellt werden wobei mittels Sequenzierung die KIAA1549-BRAF Fusionen 16-9 (24 Fälle), 15-9 (7 Fälle), 16-11 (6 Fälle) und 18-9 (1 Fall) bestätigt wurden. Die Detektion der Fusionen aus Formalin fixiertem Gewebe wurde bei 26 Proben durchgeführt. In keiner der untersuchten FFPE Gewebeproben konnte keine oder eine bestimmte Fusion mit Sicherheit bestimmt werden. Weiters wurde DNA von 35 Patienten isoliert und mittels MLPA auf die Tandemduplikation des Locus 7q34 analysiert. Vergleiche der Resultate aus der RT-PCR und der MLPA-Analyse zeigten, dass in 9 von 22 Fällen mit einem DQ zwischen 0,8 und 1,2 keine Fusion mittels RT-PCR gefunden wurde, während in den anderen 13 Fällen eine Fusion nachweisbar war. In allen Fällen bei denen der DQ zwischen 1,21 und 1,29 lag konnte mit der RT-PCR eine Fusion nachgewiesen werden. In einem von 6 Fällen bei denen der DQ zwischen 1,30 und 1,65 lag konnte keine Fusion nachgewiesen werden während bei den anderen 5 Fällen eine Fusion detektiert werden konnte.

Zusammenfassend lässt sich feststellen, dass wir keine klaren und überzeugenden Ergebnisse für das Vorliegen der Tandemduplikation erhalten haben sowohl im Gefriermaterial als auch im FFPE-Gewebe. Da die Paraffinblöcke dieser Studie mindestens ein Jahr bei Raumtemperatur gelagert wurden und auch die Zeit der Fixierung in Formalin nicht genau dokumentiert wurde, benötigen wir weitere Untersuchungen um den Einfluss von Lagerung und Fixationszeit auf die Qualität von RNS und DNS, die aus Paraffinblöcken isoliert werden, zu untersuchen.

List of abbreviations

α-KG α-ketoglutarate

°C degree Celsius

2-HG 2-hydroxyglutarate

ATRX αthalassemia/mental retardation syndrome X-linked CBTRUS Central Brain Tumor Registry of the United States

cDNA complementary DNA

CLCN6 chloride transport protein 6 CNS central nervous system

DA diffuse astrocytoma

ddH₂O double-distilled H₂O DEPC diethylpyrocarbonate DNA deoxyribonucleic acid

dNTP deoxyriboucleotide triphosphate

DQ dosage quotient

EORTC European Organization for Research and Treatment of Cancer ERK extracellular signal-regulated kinases

FFPE formalin-fixed paraffin-embedded FGFR1 fibroblast growth factor receptor 1 FISH fluorescence in situ hybridization FITC fluorescein isothiocyanate

FLAIR fluid-attenuated inversion recovery GAB1 GRB2-associated-binding protein 1 GDP guanosine diphosphate

GFAP glial fibrillary acid protein

GRB2 growth factor receptor-bound protein 2 GTP guanosine triphosphate

Gy Gray

IDH isocitrate dehydrogenase JPA juvenile pilocytic astrocytoma

LGG low-grade glioma

MACF1 microtubule-actin cross-linking factor 1 MAPK mitogen activated protein kinase MBP myelin basic protein

MEK mitogen-activated protein kinase kinase

MEKK MEK kinase

MGMT O(6)-methylguanine-DNA-methyltransferase MKRN1 makorin ring finger protein 1

MLPA multiplex ligation-dependent probe amplification MRI magnetic resonance imaging

mRNA messenger RNA

MRS magnetic resonance spectroscopy mTOR mammalian target of rapamycin

NADPH nicotinamide adenine dinucleotide phosphate NF1 neurofibromatosis 1

NSC neural stem cells

Olig2 oligodendrocyte transcription factor 2 OS overall survival

PA pilocytic astrocytoma PCR polymerase chain reaction PCV procarbazin-lomustin-vincristin PDGF platelet-derived growth factor PET positron emission tomography PFS progression-free survival PI3 phosphatidylinositide 3

RAF rapidly accelerated fibrosarcoma

RAS rat sarcoma

RIN RNA integrity number RNA ribonucleic acid RNF130 ring finger protein 130

rRNA ribosomal RNA

RTOG Radiation Therapy Oncology Group

RT-PCR reverse transcription-polymerase chain reaction SHP2 Src homology phosphotyrosyl phosphotase 2 SOS Son of Sevenless protein

SRGAP3 Slit-Robo-Rho GTPase-activating protein 3

TAE Tris-acetate-EDTA

TERT telomerase reverse transcriptase

TMZ temozolomide

US United States

UV ultraviolet

VEGFR vascular endothelial growth factor receptor WHO World Health Organization

Key terms

KIAA1549-BRAF Pilocytic astrocytoma MAPK pathway Low-grade glioma Molecular pathology

Table of content

1. I

... 1

1.1. Epidemiology of CNS tumors ...2

1.2. Low-grade gliomas ...4

1.2.1. Clinical symptoms...4

1.2.2. Molecular Pathology ...4

1.2.3. Treatment ...7

1.2.4. Monitoring...10

1.3. Pilocytic astrocytoma...11

1.3.1. Symptoms...11

1.3.2. Treatment ...11

1.3.3. Histopathologic classification ...12

1.3.4. Signaling pathways...12

1.3.5. Genomic alterations...14

1.3.6. Therapeutic approaches ...18

1.3.7. Detection of KIAA1549-BRAF fusion transcripts ...19

1.3.8. Prognostic importance and clinical relevance ...20

1.4. Aim of the study...21

1.5. Hypothesis ...21

1.6. Research questions...21

1.7. Framework conditions and origin of the samples ...21

2. M

M

... 22

2.1. Materials ...22

2.1.1. Kits ...22

2.1.2. Consumables...22

2.1.3. Chemicals...23

2.1.4. Equipment ...24

2.2. Methods...25

2.2.1. Frozen tissue ...25

2.2.2. DNA isolation...25

2.2.3. Measurement of DNA concentrations ...25

2.2.4. RNA isolation...25

2.2.5. RNA concentration...26

2.2.6. cDNA synthesis ...26

2.2.7. KIAA1549-BRAF RT-PCR amplification from frozen tissue ...27

2.2.9. Sequencing reaction ...29

2.2.10. Purification of the sequencing reactions...30

2.2.11. Capillary electrophoresis on ABI 3130 sequencer...30

2.2.12. Detection of BRAF V600E mutations ...31

2.2.13. FFPE tissue samples ...32

2.2.14. DNA isolation...32

2.2.15. Multiplex Ligation-dependent Probe Amplification (MLPA) ...33

2.2.16. RNA isolation...37

2.2.17. cDNA synthesis ...38

2.2.18. KIAA1549-BRAF RT-PCR amplification from FFPE tissues...39

3. R

... 46

3.1. Detection of KIAA1549-BRAF fusions in frozen tissue samples of pilocytic astrocytomas using RT-PCR...46

3.2. Sequencing analysis of KIAA1549-BRAF fusions as well as BRAF V600E mutations ...52

3.3. Correlation of the results of the KIAA1549-BRAF fusion transcript analysis and clinical data ...53

3.4. Testing of primer specificity for the detection of KIAA1549-BRAF fusions in FFPE samples of pilocytic astrocytomas using RT-PCR...54

3.5. Detection of KIAA1549-BRAF fusions in FFPE samples of pilocytic astrocytomas ...57

3.6. Using MLPA analysis for the detection of the KIAA1549-BRAF fusions in fresh frozen and FFPE tissue samples...63

4. D

... 66

R

... 70

L

... 74

L

... 75

A

... 77

1. Introduction

Central nervous system (CNS) tumors represent about 25 % of neoplastic disease in childhood and are the leading cause of cancer-related death in children and young adults

11. CNS neoplasms are classified based on histopathological characteristics into four grades by the World Health Organization (WHO) ¹⁴. Classification criteria are the origin of the cells and the grade based on the degree of differentiation and malignancy ²⁹. The criteria define if the tumor type belongs to either astrocytic or oligodendroglial groupings and the further classification occurs by grade and biological behavior in the absence of treatment ¹³. The grading is based on the characteristics of the tumor cells like mitotic index, nuclear atypia, vascular proliferation and necrosis ²⁹. The classification of CNS tumors is based on the concept that tumors can be classified according to microscopic similarities with different putative cells of origin and their presumed levels of differentiation

49. The characterization is dependent on the examination of hematoxylin and eosin- stained sections with a light microscope as well as on the immunohistochemical expression of proteins ⁴⁹. In 2014, guidelines for the incorporation of molecular findings into CNS tumor diagnosis were established ⁴⁹. Therefore, the diagnosis based on microscopy includes now molecular parameters for the CNS tumor classification and the current update of the WHO classification (2016 CNS WHO) changes the principle of diagnosis⁴⁹.

Grade I tumors have often well differentiated cells, are benign and can be cured ²⁹. The characteristics of grade II tumors are that they may follow long clinical courses and the early and diffuse infiltration of the surrounding brain makes them incurable by surgery ²⁹. Grade III tumors have increased anaplasia and proliferation and are more rapidly fatal whereas grade IV tumors show more features of malignancy like vascular proliferation and necrosis ²⁹. This type of tumor is generally lethal within 12 months²⁹. In general, low- grade gliomas (LGGs) are the most common central nervous system tumors in children and are classified as WHO grade I or II whereas WHO grade III-IV include high-grade tumors^1,11.

Tab. 1: Different subtypes of gliomas according to the WHO classification^1,13

Type Grade Examples

Low-grade glioma Grade I Pilocytic astrocytoma (PA) Grade II Pilomyxoid astrocytoma

Diffuse astrocytomas (DA) Pleiomorphic xanthoastrocytoma Oligodendroglioma

Oligoastrocytoma High-grade glioma Grade III Anaplastic astrocytoma

Anaplastic oligodendroglioma Grade IV Glioblastomas

Gliomas are neuroepithelial tumors that arise from the supporting glial cells of the CNS ¹⁴. They can be divided into histological subgroups based on the type of glial cell of origin²⁹. The subgroups are: astrocytomas (derived from astrocytes or their precursors), oligodendrogliomas (derived from oligodendrocytes or their precursors) and oligoastrocytomas (mixed lineage) ²⁹. The astrocytic tumors include for example diffuse astrocytomas, pilomyxoid astrocytomas, pleomorphic xanthoastrocytomas, subependymal giant cell astrocytomas, pilocytic astrocytomas, anaplastic astrocytomas and glioblastomas ¹⁴. The oligodendroglial tumors are oligodendrogliomas and oligoastrocytomas¹⁴.

Astrocytomas share the same cell of origin but the tumors have different characteristics including location, age and gender distribution, morphological features, growth potential, tendency for progression, invasiveness and clinical course ²⁹. They can be classified based on their degree of malignancy, for example pilocytic astrocytomas (WHO grade I), diffuse astrocytomas (WHO grade II), anaplastic astrocytomas (WHO grade III) and glioblastomas (WHO grade IV) (see also table 1) ²⁹. Pilocytic astrocytomas occur predominantly in children and are the most common CNS tumors in children and young adults ²⁹. PAs typically arise in the cerebellum, are slow growing and almost never show malignant progression ⁴. Furthermore, this type of tumor is surgically curable ⁴. Diffuse astrocytomas also occur mostly in children and young adults ⁴. They have a diffuse infiltrating nature and may progress to more malignant forms ²⁹. The characteristics of diffuse astrocytomas are the presence of nuclear atypia, a low mitotic rate and the absence of vascular proliferation or necrosis ¹. DAs show often isocitrate dehydrogenase (IDH) 1 or IDH2 mutations ⁴. Anaplastic astrocytomas are high-grade gliomas and often progress to glioblastoma whereas the glioblastoma is the most malignant CNS tumor²⁹. It can develop at any age but is relatively uncommon among children ^4,29.

Oligodendrogliomas show a peak of incidence in adults between 40 and 50 years but sometimes also children are affected ²⁹. Oligodendrogliomas (WHO grade II) and anaplastic oligodendrogliomas (WHO grade III) can be distinguished in two histological subtypes ²⁹. They can range from well-differentiated into high-malignant tumors ²⁹. Anaplastic oligodendrogliomas may develop from a pre-existing low-grade oligodendroglioma ²⁹. The diagnosis requires the status of IDH mutations and the 1p/19q status according to the new WHO classification of CNS tumors ⁴⁹. Also it is important to exclude pilocytic astrocytomas and dysembryoplastic neuroepithelial tumors at diagnosis

49.

Oligoastrocytomas are a diffuse infiltrating type of tumor and present a mixture of morphologic tumor cells from astrocytomas and oligodendrogliomas ²⁹. They can be divided into low-grade (WHO grade II) and high-grade (WHO grade III) oligoastrocytomas based on the most aggressive type of tumor cell that is present²⁹.

1.1. Epidemiology of CNS tumors

According to the Central Brain Tumor Registry of the United States (CBTRUS) statistical report, 356 858 incident CNS tumors were reported in the United States (US) between 2008 and 2012 ¹⁵. Overall, 42,1 % were diagnosed in males and 57,9 % in females ¹⁵. Thereof, 32,8 % (117 023) from the diagnosed tumors were of malign origin and 67,2 % (239 835) were of non-malign origin ¹⁵. Approximately, 55 % of the malign tumors occurred in males and 45 % in females ¹⁵. Further, 36 % of the non-malign tumors have

been detected in males and 64 % in females ¹⁵. The age-standardized incidence rate for 2008-2012 in the US is 21,97 per 100 000 population of all primary non-malignant and malignant CNS tumors ¹⁵. The incidence rates of malignant CNS tumor are higher in males than in females¹⁵.

The most common malignant CNS tumor is the glioblastoma with an incidence rate of 3,2 per 100 000 population ¹⁵. Furthermore, this statistical report showed that the incidence rate for tumors of the neuroepithelial tissue is higher in males with 7,79 per 100 000 population than in females with 5,6 per 100 000 population ¹⁵. Interestingly, the incidence rate of all brain and CNS tumors is lower for the race group American Indian/Alaskan Native (14,28 per 100 000 population) compared to whites (22,09 per 100 000 population), blacks (22,04 per 100 000 population) and Asian/Pacific Islanders (20,14 per 100 000 population) ¹⁵. The incidence rates in the Asian population are lower than those in Europe and North America and also in Europe differ the incidence rates between the countries ²⁸. Caucasians are more often affected than Africans or Asians and this outcome has also been observed in children ²⁸. It was also shown that the incidence rates for glioblastomas, astrocytomas and oligoastrocytic tumors are approximately 2 times greater in whites than in blacks, the incidence rate for oligodendroglial tumors is 3 times greater and for pilocytic astrocytomas it is also higher among whites than blacks ¹⁵. An ethic analysis of 8947 cases of primary CNS tumors at the Armed Forces Institute of Pathology, Washington, DC and similar CBTRUS data showed that gliomas were twice as frequent in whites as in blacks ²⁸.

The median age at diagnosis is 59 years for all CNS tumors ¹⁵. Whereas pilocytic astrocytomas usually have younger median ages at diagnosis and glioblastomas are primarily diagnosed at older ages (median age: 64,65 years)¹⁵. CNS tumors are the most common solid tumors in children and about 7 % of the reported CNS tumors during 2008- 2012 occurred in children and young adults (0-19 years) ¹⁵. Tumors of neuroepithelial tissue have incidence rates of 3,71 per 100 000 population and among these tumors the pilocytic astrocytoma with an incidence rate of 0,87 per 100 000 population is the highest

15. The mortality rate of primary CNS tumors is 4,32 per 100 000 population in the US and males show higher rates than females ¹⁵. More than 70 % of all CNS tumors are gliomas from astrocytic, oligodendroglial and ependymal origin ²⁸. The development of computerized tomography and magnetic resonance imaging (MRI) is leading to stable incidence rates of CNS tumors with a tendency of higher rates in more developed, industrialized countries ²⁸. This is also due to the differences in diagnostic practices and the access to adequate health care²⁹.

Environmental risk factors were investigated to show correlations between environmental exposure and an increased risk of CNS tumor formation ¹⁴. Typical risk factors for human tumor disease include genetic and environmental factors like smoking, diet, excessive alcohol intake, occupation and exposure to ultraviolet (UV) radiation ²⁹. But none of these is consistently correlated with the risk for gliomas ²⁹. Though, one factor showed a correlation with an increased risk of secondary CNS tumors: CNS exposure to therapeutic or high-dose radiation ²⁹. This established environmental cause of glioma manifests clinically only several years after exposure^14,29. Furthermore, people are often exposed to different endogenous and exogenous chemicals through their diet, personal habits and occupation like N-nitroso compounds, reactive oxygen and nitrogen species, several industrial used chemicals and polycyclic aromatic hydrocarbons ³⁰. Data from animal

studies and other studies support that some of these compounds are biological neurocarcinogens ³⁰.

Currently the association between cell phone use and glioma risk is a hot topic ^2,9. But since the widespread use is not so long ago, there has been limited opportunity to investigate the long-term health effects²⁸.

1.2. Low-grade gliomas

Low-grade gliomas include a group of primary and diffuse glial CNS tumors and the tumors consist of astrocytic and oligodendroglial lineage ^13,21. These tumors are normally slow growing but could also be associated with significant morbidity and mortality ¹³. LGGs often arise in young and healthy patients and have an indolent course with longer- term survival compared to high-grade gliomas ¹⁴. Factors that are associated with longer survival are younger age, good WHO status, the absence of neurological deficits and the absence of contrast enhancement on imaging ²¹. Also a significant predictor for the overall survival (OS) is the tumor size ²¹. The clinical signals and symptoms vary and the diagnosis is made through a combination of imaging, histopathology and molecular diagnostic methods ¹⁴. A pathological diagnosis can be achieved by means of surgery and it can also help to resect the tumor completely when possible ¹⁴. Preoperative functional magnetic resonance imaging, tractography as well as intraoperative neurophysiological monitoring are advances that allow surgeons to maximize resection¹⁴. Afterwards, the identification and classification of the tumor type is performed by staining the tissue sample with hematoxylin and eosin ¹⁴.

1.2.1. Clinical symptoms

The symptoms of low-grade gliomas are dependent on the location of the tumor but they often vary and are lead back to mass effect from invasion into the surrounding parenchyma or to obstructive hydrocephalus ^1,14. Tumors in the posterior fossa typically show acute signs and symptoms of increased intracranial pressure, obstructive hydrocephalus and cerebellar signs ¹. Low-grade gliomas of the optic pathway affect vision and a loss of acuity or field defects could occur ^1,3. If the tumor is located within the cerebral cortex, symptoms with focal neurological manifestations such as seizures or behavioral changes occur ¹. Seizures are the presenting symptom in up to 80 % of patients and are also associated with a temporal, frontal or parietal localization ^1,14. In general, the most common symptoms in cerebellar tumors are headache, vomiting, gait disturbance, neck stiffness and visual impairment ⁴⁴. Neurological findings at the initial examination are often papilledema, ataxia, nystagmus, dysmetria and diplopia ⁴⁴. However, patients may also be asymptomatic ¹⁴.

1.2.2. Molecular Pathology

The genetic characterization has become more and more important for the identification and classification of tumors and it is as well necessary for providing information about prognosis and/or expected response to treatment ¹⁴. A step forward in the glioma diagnostic was the discovery of the 1p/19q deletion in oligodendroglial tumors ⁹. However, it took more than a decade until the predictive and prognostic value was investigated and verified⁹.

1.2.2.1. 1p/19q

The deletion of selected regions on the chromosomes 1p and 19q is important for the diagnosis of low-grade gliomas ^9,14. Chromosome losses are often associated with a pericentromeric translocation of the chromosomes 1 and 19 ⁹. The translocation is unbalanced and the cells stay with one copy of the short arm of chromosome 1 and one copy of the long arm of chromosome 19⁹.

The 1p/19q status is prognostic highly relevant for patients with oligodendroglial and oligoastrocytic tumors ⁹. Patients with tumors that lack 1p and 19q had longer median survival times and longer progression-free survival (PFS) and better chemotherapy response ⁹. Radiotherapy alone and in combination with chemotherapy are the most well studied treatment strategies ⁹. Whereas in cases with 1p/19q codeletion is chemotherapy alone often suggested ³¹. In clinical trials were patients with a 1p/19q codeletion in gliomas treated with first-line chemotherapy (temozolomide (TMZ) or procarbazin- lomustin-vincristin (PCV)) and it was shown that there is a high chance of good clinical response ⁹. Lassman et al. retrospectively identified that the application of chemotherapy alone or in combination with radiotherapy shows no significant overall survival ³¹. But the European Organization for Research and Treatment of Cancer (EORTC) Brain Tumor Group Study 26951 showed that chemotherapy (PCV) in combination with radiotherapy has a significantly better overall survival ³². In patients with 1p/19q codeletion was the overall survival increased and a trend toward more benefit form adjuvant PCV was shown

32. Patients with noncodeleted 1p/19q are usually treated with first-line radiotherapy without combined chemotherapy⁹.

1.2.2.2. IDH1/2

Also isocitrate dehydrogenase 1 and 2 mutations occur in low-grade gliomas ¹⁴. Furthermore, the mutation of the codon 132 in the IDH1 gene is frequent in WHO grade II and III astrocytic and oligodendroglial tumors as well as in secondary glioblastomas ¹⁴. In high-grade gliomas is the association between IDH1/2 mutation and a favorable prognosis better established whereas the prognostic value in WHO grade II tumors is less clear ⁹. A substitution of the amino acid arginine to histidine at codon 132 of the IDH1 gene is the most common mutation in glial CNS tumors and can be used as a diagnostic biomarker ⁹. The IDH enzymes catalyze the decarboxylation of isocitrate to α- ketoglutarate (α-KG) and nicotinamide adenine dinucleotide phosphate (NADPH) ³³. Cancer-associated IDH mutations cause a loss of the normal IDH catalytic activity and gain the function to produce 2-hydroxyglutarate (2-HG) ³³. Tumor cells with IDH1/2 mutation are expected to have reduced α-ketoglutarate levels and increased 2- hydroxyglutarate levels ³³. The 2-HG can accumulate to high levels in tumors ³³. The 2- HG may act as a competitive inhibitor of α-KG by antagonizing the function of α-KG dependent enzymes ³³. These enzymes are involved in processes like demethylation of deoxyribonucleic acid (DNA) and histones and hydroxylation and degradation of the hypoxia inducible factor 1 α³³.

1.2.2.3. αthalassemia/mental retardation syndrome X-linked (ATRX)

Another biomarker is the ATRX protein ¹³. ATRX is a DNA helicase and a essential member of a multiprotein complex with a role in regulating chromatin remodeling, nucleosome assembly, telomere maintenance and incorporation of Histone H3.3 proteins

into telomeric regions of chromosomes ^{9, 13}. The ATRX loss is useful in the diagnosis of IDH mutant astrocytomas and may be used to differentiate these tumors from oligoastrocytomas and oligodendrogliomas ⁹. Further, the loss of ATRX occurs almost only in IDH mutant tumors and ATRX and IDH mutant anaplastic astrocytomas have a more favorable prognosis compared to tumors where only the IDH mutation occurs ⁹. A clinical trial showed that patients with astrocytomas and additional ATRX mutation have a survival benefit⁹.

1.2.2.4. O(6)-methylguanine-DNA-methyltransferase (MGMT)

The O(6)-methylguanine-DNA-methyltransferase is a DNA repair protein and plays also a role in the treatment-related prognosis ¹⁴. The enzyme converts the naturally occurring mutagenic O(6)-methylguanine back to guanine and therefore counteracts the chemotherapeutic effects of alkylating agents like temozolomide ⁹. The MGMT promoter methylation silences the gene and thus it is leading to reduced expression of the active enzyme ⁹. High levels of MGMT activity in tumors lead to resistance to alkylating agents whereas methylation of the MGMT promoter results in reduced MGMT repair activity ⁹. Patients with MGMT promoter methylation had a survival benefit when they were treated with TMZ and radiotherapy compared to patients who only received radiotherapy ^9,14. It was also shown that chemotherapy alone or together with radiotherapy had no survival benefit for patients with MGMT promoter-unmethylated tumors ⁹. The MGMT promoter methylation can also be used to stratify older glioblastoma patients for radiotherapy or chemotherapy ⁹. Trial results suggest that in older patients who suffer from glioblastoma the MGMT promoter methylation status should be tested before a decision about the treatment is made⁹.

1.2.2.5.

(TERT)

The telomerase reverse transcriptase gene is essential in maintaining telomere length.

The telomere length gets shorter with every cell division and the ability to maintain telomere length is a feature of neoplasia ⁹. Mutations in the TERT promoter result in increased activity in some human cancers as well as in many CNS tumors including glioblastomas, medulloblastomas and low-grade gliomas ^9,13. A large study with glioblastomas, astrocytomas and oligodendrogliomas showed that TERT promoter mutations occur in 74 % of the glioblastomas but only a few mutations were detected in WHO grade II and III astrocytomas ⁵⁰. So far, conflicting results with regard to the TERT mutation status and survival were obtained⁹. In a study by Killela et al. it was shown that patients with TERT promoter mutations had the poorest overall survival ⁵⁰. Patients without TERT or IDH1/2 mutation had a slightly better OS and patients with only IDH mutation had the best survival⁵⁰.

1.2.2.6. BRAF

Furthermore, BRAF gene mutations and gene rearrangements are present in low-grade glial and glioneuronal tumors ⁹. The BRAF V600E point mutation is common in pleomorphic xanthoastrocytomas, gangliogliomas and pilocytic astrocytomas but the prognostic value is less well established ^{9, 43}. The BRAF gene rearrangements and generation of fusion gene products is predominantly found in pilocytic astrocytomas ⁹.

1.2.3. Treatment 1.2.3.1. Surgery

More and more studies show that for the improvement of the overall survival a surgical resection of the tumor is better than observation alone ¹⁴. Furthermore, these studies show that the extent of resection has a positive impact on PFS and it has been reported that surgery improves seizure control ¹⁴. On the other hand management of low-grade gliomas in selected patients with minimal or medically controlled symptoms would favor watchful waiting^,since this approach did not worsen the patients’ quality of life nor has it a negative impact on the overall survival ²². Other studies show a trend towards improvement in survival with more extensive surgical resection ¹⁴. In a comparative population-based study with patients suffering from LGGs a survival benefit was observed

16. The patients were treated with resection of the tumor in contrast to diagnostic biopsy and watchful waiting ¹⁶. The surgical strategies in low-grade gliomas have been conflicting and are based on case series¹⁶. In selected patients clinicians support a wait- and-scan policy but on the other hand an association between survival and extent of resection was reported ¹⁶. In patients with minimal or medically controlled symptoms watchful waiting is used with the arguments based on data that such an approach did not worsen the patients’ quality of life ¹⁴. But retrospective surgical studies demonstrated significant overall survival benefit with surgical resection ¹⁴.

A number of developments help to improve the surgeons’ ability to maximize the tumor resection and spare healthy brain tissue. Functional magnetic resonance imaging and magnetic source imaging allow the surgeon to see functional brain areas like motor and language cortices ²³. The use of intraoperative MRI and magnetic resonance spectroscopy (MRS) can be used to evaluate the tumor resection during the surgery and identify the residual tumor more clearly¹⁴.

1.2.3.2. Radiotherapy

Completely resected low-grade gliomas usually do not need further treatment but in the case of an incomplete resection additional treatments are required and so radiotherapy could be used as another effective treatment⁶.

The utility of high-dose versus low-dose radiation and the costs versus benefits of early versus delayed radiotherapy were investigated in several clinical trials ¹⁴. 379 patients with LGGs were treated postoperatively with radiotherapy ¹⁴. Thereby, one group was treated with 45 gray (Gy) in 5 weeks and the other with 59,4 Gy in 6,6 weeks. There was no significant difference in the overall survival or progression-free survival and no dose- response relationship for radiotherapy in LGGs ¹⁴. Similar results were observed in a clinical trial from the North Central Cancer Treatment Group/Radiation Therapy Oncology Group (RTOG)/Eastern Cooperative Oncology Group ¹⁷. The survival and the toxicity were compared in patients who suffer from low-grade gliomas ¹⁷. 203 patients were randomized to a low-dose versus high-dose radiotherapy treatment group ¹⁷. No advantage of high-dose radiotherapy was observed but there was a trend toward improved survival at 2 and 5 years with low-dose therapy ¹⁷. Additionally, there was a higher incidence of radiation neurotoxicity observed in the high-dose radiotherapy group

17.

Also the timing of the radiotherapy was investigated¹⁸. Early versus delayed radiotherapy

the early radiotherapy group¹⁸. Patients from 24 centers across Europe were divided into either early radiotherapy or delayed radiotherapy ¹⁸. The study showed that patients who were treated with early radiotherapy after surgery had a significant improvement on the length of the period without progression ¹⁸. Though, there was no effect on the overall survival ¹⁸. It was also reported that early radiotherapy did not increase the risk of malignant transformation from low-grade gliomas to higher-grade tumors ¹⁸. Newer techniques allow a more precise radiation and improve the ability to target only the tumor and spare the healthy surrounding brain tissue and thus reduce radiation toxicity ¹⁴.

1.2.3.3. Chemotherapy

Temozolomide is used for the treatment of progressive and newly diagnosed malignant gliomas ¹⁹. In patients with high-grade gliomas chemotherapy is often used but the treatment of patients with low-grade gliomas remains a topic of investigation ¹⁴. In a phase II study the activity of TMZ was tested in patients with progressive low-grade gliomas¹⁹. 46 patients with LGGs have been treated and the median PFS was 22 months with a 6 months PFS of 98 % and a 12 months PFS of 76 %¹⁹.

The Radiation Therapy Oncology Group 9802 clinical trial compared patients with low- grade gliomas that were treated with radiation alone versus radiation followed by 6 weeks chemotherapy with procarbazine, lomustine and vincristine ²⁰. The study included 251 patients and there was a significant improvement in the PFS but not in the OS of the patients that received radiotherapy and PCV ²⁰. In another RTOG phase II study temozolomide-based chemotherapy in patients with high-risk low-grade gliomas was investigated ²⁴. The patients in this non-randomized multicenter study received oral temozolomide once daily on 42 days and underwent radiotherapy followed by postradiation temozolomide for up to 12 additional months²⁴. Primary outcome measures include overall survival at 3 years, progression-free survival, association of survival and PFS with MGMT methylation status, quality of life and neurocognitive function ²⁴. The 3- year overall survival rate was 73,1 % and thus higher than the overall survival of historical controls²⁵.

A phase III randomized multicenter study compared the PFS of patients with LGGs treated with radiotherapy versus temozolomide ²⁶. The patients were stratified according to participating center, chromosome 1p status (deleted versus normal versus undeterminable), contrast enhancement on MRI (yes versus no), age (< 40 years versus

≥ 40 years) and WHO (0 or 1 versus 2) status and randomized to treatment arm 1 or 2²⁶. Arm 1 included a radiotherapy group which underwent radiotherapy once daily, 5 days a week for a total of 28 fractions ²⁶. Arm 2 consisted of a chemotherapy group where the patients received oral temozolomide once daily on 21 days ²⁶. The treatment repeated every 28 days for up to 12 courses ²⁶. Outcome measures included PFS, OS and quality of life ²⁶. The results showed no difference in PFS and OS between the two groups ²⁷. Patients in the chemotherapy group with deleted chromosome 1p showed a trend towards poorer PFS²⁷, but an improvement in the overall survival²⁷.

Tab. 2: Summary of treatment modalities for low-grade gliomas

Treatment modality Summary

Observation Maybe in low-risk patients with minimal or no symptoms²²

Less favorable in patients with high-risk features²²

· Age≥40

· Astrocytic tumor histology

· Tumor size≥6 cm

· Tumor crossing midline

· Presence of neurologic deficits before surgery

Surgery Studies suggest survival benefit from early tumor resection^14,16.

Radiotherapy No dose-response relationship for high-dose versus low-dose radiotherapy and no difference in OS and PFS¹⁴.

Low-dose radiotherapy showed a trend towards improved survival at 2 and 5 years¹⁷.

Higher incidence of radiation neurotoxicity was observed with high-dose radiotherapy¹⁷.

Improvement in PFS but not in OS in patients with early radiotherapy¹⁸.

Early radiotherapy did not increase the risk of malignant transformation from LGG to higher-grade tumors¹⁸.

Chemotherapy Improved outcome in LGG patients treated with TMZ or PCV chemotherapy^19,20.

No difference in the OS and PFS between radiotherapy and TMZ chemotherapy²⁷.

Trend towards improvement in OS in 1p-deleted tumors treated with TMZ²⁷.

1.2.4. Monitoring

Imaging techniques are important to determine the tumor size and associated peritumoral edema ¹⁴. Low-grade gliomas appear on computer tomography scan as diffuse areas of low attenuation ¹⁴. The current imaging of choice is magnetic resonance imaging ¹⁴. This is done on the basis of T1-weighted MRI or T2/Fluid-Attenuated Inversion Recovery (FLAIR)-weighted MRI with or without (gadolinium) contrast enhancement ³⁴. LGGs often look homogeneous with low signal intensity on T1-weighted sequences and hyperintensity on T2-weighted and FLAIR-weighted MRI¹⁴.

The uptake of contrast enhancement in LGGs is minimal and they differ from WHO grade III and WHO grade IV gliomas ¹⁴. High-grade gliomas have higher tumor heterogeneity and a higher uptake of contrast enhancement ¹⁴. They also often demonstrate restricted diffusion on diffusion-weighted imaging magnetic resonance sequences and increased relative cerebral blood volume on perfusion-weighted MRI¹⁴. Newer techniques like MRS and positron emission tomography (PET) may improve the diagnostic potential but the histopathology remains the gold standard for diagnosis and grading ¹⁴. Magnetic resonance imaging is the basis for the primary diagnosis, therapy planning and during follow up ³⁴. The equipment is widely available and the costs are lower than for PET examinations ³⁴. It is possible to incorporate perfusion analyses into the daily practice because the contrast injection is already required and the added acquisition time is short

34. Also the use of diffusion-weighted imaging only needs a couple of minutes to the routine acquisition time³⁴.

T1-weighted MRI with gadolinium contrast enhancement has limitations especially for low-grade gliomas or after treatment ³⁴. This technique does not provide a specific measure of tumor size and activity³⁴. The changes in enhancement often do not correlate with the response and one example is pseudoregression where the increase in contrast uptake does not reflect the tumor progression and this can happen after radiotherapy with or without TMZ ³⁴. If the reverse effect of pseudoregression happens then there is a decrease in contrast enhancement that does not reflect tumor regression in patients treated with antiangiogenic agents ³⁴. Pseudoresponse is due to a normalization of abnormally permeable microvessels³⁴. This is mostly noted with antiangiogenic treatment which is leading to a rapid decrease of contrast enhancement of gadolinium T1-weighted MRI³⁴. But it does not reflect the real decrease in tumor activity or size³⁴.

It is suggested that the treatment outcome would be more reliable assessed using advanced imaging techniques including amino acid positron emission tomography, MRS and/or cerebral blood volume assessment with perfusion-weighted MRI ¹⁴. But these alternatives have to be validated in clinical trials to ensure standardization and for the implementation in clinical practice¹⁴.

Advanced imaging techniques need to be established for their potential to visualize the biological changes of the tumor, to detect proliferative activity, hypoxia, apoptosis and necrosis and tumor vasculature ³⁴. Many aspects of tumor metabolism like hypoxia, angiogenesis, invasiveness, turnover of glucose, amino acids and nucleosides and perfusion can be evaluated with modern imaging techniques ³⁴. Advanced techniques such as perfusion-weighted MRI, diffusion-weighted MRI, MRS and PET may show the detection of treatment-induced changes in tumor biology and physiology³⁴.

Perfusion-weighted MRI, diffusion-weighted MRI and MRS are able to characterize different pathophysiological aspects of CNS tumors and changes related to the treatment

34. Positron emission tomography analyses can show specific quantitative information on the metabolic status and allow localizing expression of enzymes or transporters by measurement of the respective enzyme or transporter substrates ³⁴. Also various molecular processes can be visualized³⁴.

1.3. Pilocytic astrocytoma

There are various histological subtypes of low-grade gliomas and the WHO grade I classified pilocytic astrocytoma is the most frequent CNS tumor in the pediatric population and extremely rare in adults^1,2.

Pilocytic astrocytomas can occur anywhere in the central nervous system but mostly in the posterior fossa in children ⁵². Other typical locations are the cerebellum, the supratentorial compartment, the optic pathway and hypothalamus, the brainstem and the spinal cord ³. The pilocytic astrocytoma is a relative benign and slow growing tumor ⁴. It almost never shows malignant progression and has a more favorable prognosis than most other gliomas ^4,5,10. In addition, PAs are not usually wide infiltrating and have a good prognosis with 10-year overall survival of around 90 % ^4,5. The outcomes tend to be very good with low rates of recurrence when the tumor is surgically accessible and located in the cerebellum and more superficial parts of the cerebrum ⁵². Tumors located in deeper midline structures like the brainstem and diencephalon are more difficult to achieve and to resect totally ⁵². So there are higher risks of recurrence and the need for treatment with adjuvant therapies is considered to be important ⁵². Although this type of tumor is associated with excellent overall survival, children can suffer morbidity from the tumor and the therapy ¹. All together, pediatric low-grade astrocytomas have a heterogeneous clinical course ranging from periods of growth arrest to continuous progression¹².

1.3.1. Symptoms

The symptoms caused by the development of the tumor can be unpredictable because of the slow growth and the identification is dependent on the localization of the tumor and if the patient is already able to communicate neurological changes and discomfort ³. Symptoms of cerebellar tumors are ataxia, cranial nerve defects and signs of increased intracranial pressure like headache, nausea and vomiting ³. If the tumor is present in the optic pathway, loss of visual acuity or field defects could appear and a location in the hypothalamus may result in endocrine syndromes such as diabetes mellitus ³.

1.3.2. Treatment

The treatment of choice is a complete surgical resection, which usually results in excellent long-term survival rates ^2,3. Patients with a total resection normally do not need further treatment but sometimes a complete resection is not possible due to the location of the tumor and the related neurological impairments ¹. Unfortunately, some tumors are located in the optic pathway, brainstem and spinal cord or are diffuse infiltrative and in these cases a total resection is usually not possible ^11,12. Under these circumstances the goal of surgery is a maximal resection without risking neurologic defects and for those tumors chemotherapy and radiotherapy are applied ^1,2. Patients with tumors without a complete surgical resection or which are located in the hypothalamic/chiasmatic region have less favorable progression-free and overall survival ³. Radiotherapy or

chemotherapy can be followed up in case of progression, recurrence or other symptoms

1. Radiotherapy is an effective treatment and was for many years the primary treatment for local tumor control ⁶. However, there were concerns about long-term effects and it is also associated with significant morbidities like neuroendocrine-cognitive deficits, vasculopathy and secondary tumors ^6,12. Therefore the application of low-dose chemotherapy is more favorable and used after the surgery to avoid radiotherapy, especially in young children^6,12.

Adjuvant chemotherapy and radiotherapy are effective but the overall survival beyond 15 years for unresectable tumors is poor¹¹.

1.3.3. Histopathologic classification

Pilocytic astrocytomas are macroscopically relative soft in texture, well-defined and cysts are common in the tumor tissue and around ³. Furthermore, calcium deposits and hemosiderin could be present and the cellularity of the tumor is low to moderate with compact densely fibrillated areas ³. The histological hallmarks are Rosenthal fibers, bipolar tumor cells, vascular proliferation and eosinophilic granular bodies ¹. These eosinophilic granular bodies are often near cystic areas and may be in cyst formation ¹. The bipolar tumor cells show glial fibrillary acid protein (GFAP) immunoreactivity and other positive immunohistochemical markers are oligodendroglial markers like the oligodendrocyte transcription factor 2 (Olig2), myelin basic protein (MBP) and platelet- derived growth factor (PDGF)^1,3.

Due to the variety of morphology and the same histopathological features with other gliomas of higher malignancy, the diagnosis of pilocytic astrocytomas can be challenging

4. However, the differential diagnosis of these tumors is still dependent on histopathological assessment ³. It is important to distinguish pilocytic astrocytomas from astrocytomas of higher malignancy grade because they may require a more aggressive therapy ⁴. Some patients receive an unnecessary aggressive therapy due to the challenging classification of low-grade gliomas and this could have been avoided with the availability of specific molecular markers ⁶.

1.3.4. Signaling pathways

1.3.4.1. Mitogen activated protein kinase (MAPK) pathway

The MAPK pathway is a signaling cascade transducing extracellular signals from the cell membrane to the nucleus ⁸. It mediates cell growth, regulates cell survival and senescence, gene expression, cell differentiation and apoptosis ^8,9. Activation of this pathway often occurs in glioblastomas, the most malignant astrocytic tumor in adults and takes place via different mechanisms⁸. The cell membrane receptors are most commonly affected whereas the cytosolic components are less frequently affected ⁸. In pediatric low- grade gliomas, like the pilocytic astrocytoma, a mechanism of MAPK pathway activation also was identified⁸.

The MAPK cascade consists of a three-kinase module and the included MAP kinases are regulated by phosphorylation cascades ⁵⁴. The first characterized MAPK cascade consists of RAF isoforms, mitogen-activated protein kinase kinase (MEK) 1/2 and extracellular signal-regulated kinases (ERK) 1/2 and is regulated by RAS ³⁹. The small G

protein RAS is located at the inner surface of the plasma membrane and RAF, MEK and ERK are cytoplasmic protein kinases⁹.

The activation of MAPK happens through two upstream protein kinases which are activated in series ⁵⁴. These protein kinases are members of the MAP/ERK kinase family and are dual enzymes that can phosphorylate hydroxyl side chains of serine/threonine and tyrosine residues⁵⁴.

The MAPK is activated by MEK which is activated by a MEK kinase (MEKK) ³⁹. MEKKs include RAF isoforms and these proteins phosphorylate MEK1 and MEK2 which in turn phosphorylate and activate ERK1 and ERK2 ⁵⁴. The three isoforms of the RAF family in mammals are composed of ARAF, BRAF and CRAF (RAF-1) ⁵⁴. CRAF is ubiquitous whereas the highest expression of BRAF occurs in neuronal tissue and testis and ARAF appears to function primarily in the urogenital tissue ⁵⁴. The genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS⁹. RAS is mutated in about 15

% of human cancers⁹.

The MAPK signaling is activated by binding of an extracellular ligand to transmembrane receptor tyrosine kinases under physiological conditions ⁹. Thus, ligand binding results in phosphorylation of specific intracellular tyrosine residues and creates binding sites for adapter proteins (GRB2, SOS, GAB1 and SHP2) ⁹. The receptor gets activated and recruits SOS which leads to binding, phosphorylation and an activation of the RAS proteins ⁸. Activated RAS has many substrates including RAF, BRAF, ARAF or RAF1 ⁸. The RAS protein in turn activates a RAF kinase which phosphorylates MEK, leading to activation of ERK and effector molecules such as nuclear transcription factors ⁸. This process results in activation of multiple cellular processes ⁸. The MAPK cascade has a wide range of effects like proliferation, survival and tumorigenesis but it can also trigger cell differentiation and senescence ⁵². This might explain why most gliomas with BRAF activation are low-grade gliomas and tend to remain it⁵².

Fig. 1: Schematic overview of the MAPK pathway⁸.

GRB 2 SOS

RAS

MEK ERK nuclear transcription factors cell differentiation

cell proliferation cell survival

RAF

The BRAF fusion proteins and the BRAF V600E mutation both activate BRAF signaling and act similar to the physiological action of RAF ⁹. The KIAA1549-BRAF protein is constitutively active and activates the downstream cascade of MEK and ERK^8,9. Thereby, the signaling cascade gets stimulated without an external signal and triggers intracellular growth-stimulating transcription factors ⁸. The downstream activation was confirmed by high levels of MEK and ERK in the presence of the BRAF fusion products⁸.

In Neurofibromatosis 1 (NF1) related pilocytic astrocytomas this pathway is activated through increased RAS signaling after unrestrained phosphorylation of RAS through the absence of neurofibromin ³⁵. The loss of neurofibromin increases the RAS activity and induces downstream activation of the MAPK pathway as well as the PI3K-Akt-mTOR- pathway ³⁵. In many sporadic PAs genetic alterations like BRAF fusions and fibroblast growth factor receptor 1 (FGFR1) mutations are responsible for the activation³⁵.

1.3.4.2. Mammalian target of rapamycin (mTOR) pathway

This pathway is critical in cell survival, cell growth and proliferation and was shown to be a driver of tumorigenesis ⁴⁵. The mammalian target of rapamycin is a mediator of neurofibromatosis 1 tumor growth and the mTOR-pathway is hyperactivated in NF1- associated pilocytic astrocytomas ^35,46. Neurofibromin, the NF1 protein, functions as a negative regulator of cell growth by inactivating the RAS proto-oncogene ⁴⁶. Studies showed that neurofibromin/RAS-mediated growth regulation operates through the mTOR- pathway⁴⁶.

It has been reported that oncogenic BRAF induce mTOR pathway activation in melanoma, thyroid and breast carcinomas ⁴⁷. Studies demonstrated that neurofibromin regulates murine astrocytes, neural stem cells (NSC) and glioma growth in a mTOR- dependent manner and it raises the possibility that NF1-associated and sporadic PAs share mTOR pathway activation as a mitogenic driver⁴⁷. Consistent with this prediction is that KIAA1549-BRAF expression in cerebellar NSCs resulted in increased phosphorylation of ribosomal S6 protein and S6 kinase⁴⁷.

NF1-deficient human and mouse cells exhibit increased mTOR pathway activation as evidenced by high levels of activity of the mTOR effector, ribosomal S6⁴⁶. With the use of activation specific phospho-S6 antibodies the increased mTOR activation in human and mouse NF1-associated tumors was shown⁴⁶.

1.3.5. Genomic alterations

Neurofibromatosis 1 or von Recklinghausen`s disease is an autosomal-dominant hereditary tumor-bearing disease and patients with NF1 have an increased risk for the development of pilocytic astrocytomas ^3,4. In children these tumors arise along the optic pathway⁴⁶. The incidence is worldwide 1 per 2500-3000 persons³⁵. Characteristics of the disease are café-au-lait spots, intertriginous freckling, Lisch nodules, neurofibromas and optic pathway gliomas³⁵.

Until 2008 the mutational inactivation of NF1 tumor suppressor genes in children was the only known genetic alteration that was associated with pilocytic astrocytomas ². Most of the patients inherit germline mutations of the NF1 gene ⁴. NF1 is caused by a mutation in the NF1 gene. This mutation is located on chromosome 17q11.2 ³⁵. The NF1 gene codes

for neurofibromin which is a cytoplasmatic, 2818 amino acids long protein and contains three differentially spliced exons ^2,35. Neurofibromin is widely expressed in neurons and astrocytes of the CNS ³⁵. This gene is a member of the RAS GTPase-activating protein family and acts as a negative regulator of the RAS-RAF-MEK-ERK-pathway ^1,4.

Neurofibromin acts as a tumor suppressor protein because it speeds up the conversion of the RAS proto-oncogen from its active, guanosine triphosphate (GTP)-bound form to its inactive guanosine diphosphate (GDP)-bound form ². In NF1-associated gliomas is a loss of this expression is noticed and this leads to increased RAS-GTP levels and hyperactivation of downstream RAS signaling pathways associated with increased cell growth ². The RAS activation leads to increased signaling through RAS downstream effector proteins in many cell types also including PI3 (phosphatidylinositide 3)-kinase and RAF/MEK².

The alterations lead to activation of the MAPK pathway and induce downstream activity of this pathway as well as the PI3K-Akt-mTOR pathway^4,35. The MAPK pathway is activated because of the increased RAS signaling after unrestrained phosphorylation of RAS through the absence of neurofibromin ³⁵. In addition, the mTOR pathway is critical in cell survival, cell growth and proliferation and it is hyperactivated in NF1 associated PAs³⁵. Upstream alterations in the MAPK pathway, primary in the tyrosine kinase receptor FGFR1 are leading to the growth cascade in pilocytic astrocytomas ³⁶. The FGFR1 amplification is also found in breast, ovary and lung cancer ³⁶. In pilocytic astrocytomas the described FGFR1 point mutations are described within a hotspot tyrosine kinase region ³⁶. The FGFR1 K546K and K656E mutations have been described and represent an alternative mechanism of the MAPK pathway activation to KIAA1549-BRAF fusions and BRAF mutations³⁷.

Another fusion gene, the FAM131B-BRAF fusion was found in 2011 ³⁸. It is composed of the 5`-part of the FAM131B gene and the 3`-part of the BRAF gene ³⁸. The breakpoint in this case was between exon 2 of the FAM131B gene and exon 9 of the BRAF gene ³⁸. Cin et al. detected three novel FAM131B-BRAF fusion variants and indicated activation of downstream effectors on the MAPK pathway ³⁸. It was also shown that the fusion gene results from an interstitial deletion at 7q34 rather than tandem duplication ³⁸. Subsequently, other fusion transcripts involving BRAF genes and other fusion partners like SRGAP3, MACF1, RNF130, CLCN6 and MKRN1 have been described¹.

In 2008 genetic defects in the BRAF gene were identified by Jones et al., which are responsible for constitutive activation of the MAPK pathway ¹⁰. This pathway drives the formation of the majority of low-grade gliomas through various mechanisms including BRAF mutations and KIAA1549-BRAF fusions and BRAF is a downstream effector in this pathway ^6,7. MAPK pathway activation can often be found in glioblastomas, the most frequent and malignant astrocytic tumor in adults and now it was identified in pediatric low-grade gliomas, above all in PAs⁸.

Tandem duplication at the locus 7q34 is the most common mechanism of MAPK pathway activation ⁷. The duplication is resulting in a fusion transcript between the 5`-end of the KIAA1549 gene and the 3`-end of the BRAF gene ².