KRISTIINA NORDFORS
Antioxidative Enzymes, Carbonic Anhydrases and Claudins in Pediatric Brain Tumors Prognostic and predictive value
ACADEMIC DISSERTATION To be presented, with the permission of the board of the School of Medicine of the University of Tampere, for public discussion in the Jarmo Visakorpi Auditorium, of the Arvo Building, Lääkärinkatu 1, Tampere, on October 8th, 2011, at 12 o’clock.
UNIVERSITY OF TAMPERE
ACADEMIC DISSERTATION University of Tampere, School of Medicine Pirkanmaa Hospital District, Laboratory Centre Finland
Supervised by Docent Hannu Haapasalo University of Tampere Finland Professor Ylermi Soini University of Eastern Finland Finland
Reviewed by Professor Riitta Herva University of Oulu Finland Docent Olli Lohi University of Tampere Finland
Distribution Bookshop TAJU P.O. Box 617 33014 University of Tampere Finland
Tel. +358 40 190 9800 Fax +358 3 3551 7685
[email protected] www.uta.fi/taju http://granum.uta.fi
Cover design by Mikko Reinikka
Acta Universitatis Tamperensis 1648 ISBN 978-951-44-8546-6 (print) ISSN-L 1455-1616 ISSN 1455-1616
Tampereen Yliopistopaino Oy – Juvenes Print Tampere 2011
Acta Electronica Universitatis Tamperensis 1109 ISBN 978-951-44-8547-3 (pdf ) ISSN 1456-954X http://acta.uta.fi
Kristiina, Arvo, and Lea at Luosto 1986
To my inspirators and all the children with brain tumor 3
CONTENTS LIST OF ORIGINAL PUBLICATIONS
7
ABBREVIATIONS
8
ABSTRACT
10
TIIVISTELMÄ
12
1
INTRODUCTION
14
2
REVIEW OF THE LITERATURE
16
2.1. Pediatric brain tumors
16
2.1.1. Pilocytic astrocytoma
17
2.1.2. Ependymoma
18
2.1.3. Medulloblastoma
20
2.1.4. Primitive neuroectodermal tumor
22
2.2. Antioxidant enzymes
24
2.2.1. Manganese superoxide dismutase
26
2.2.2. Glutamate cysteine ligase, catalytic and regulatory subunit
27
2.2.3. Thioredoxin and thioredoxin reductase
28
2.2.4. Peroxiredoxins
30
2.2.4.1. Peroxiredoxin I
31
2.2.4.2. Peroxiredoxin II
32
2.2.4.3. Peroxiredoxin III
32
2.2.4.4. Peroxiredoxin IV
33
2.2.4.5. Peroxiredoxin V
33
2.2.4.6. Peroxiredoxin VI
34
2.3. Carbonic anhydrases
34
2.3.1. Carbonic anhydrase II
35
4
2.3.2. Carbonic anhydrase IX
36
2.3.3. Carbonic anhydrase XII
37
2.4. Claudins
39
2.4.1. Tight junctions
39
2.4.2. The blood-brain barrier
39
2.4.3. Claudin expression in normal tissue
40
2.4.4. Claudin expression in neoplasms
41
2.4.5. Epithelial-mesenchymal transition
44
3
AIMS OF THE STUDY
45
4
MATERIALS AND METHODS
46
4.1. Patients
46
5
4.1.1. Pilocytic astrocytomas
46
4.1.2. Ependymomas
46
4.1.3. Medulloblastomas and PNETs
47
4.2. Tumor tissue samples
47
4.3. Immunohistochemistry
48
4.3.1. Antioxidative enzymes and peroxiredoxins
48
4.3.2. Carbonic anhydrases
49
4.3.3. Claudins
49
4.3.4. Other immunohistochemistry and TUNEL
50
4.4. Histopathological features
51
4.5. Statistical methods
51
4.6. Ethics
51
RESULTS
52
5.1. Immunohistochemical expression
52
5
5.1.1. Antioxidative enzymes
52
5.1.2. Peroxiredoxins I-VI
52
5.1.3. Carbonic anhydrases II, IX, and XII
53
5.1.4. Claudins 2-5, 7, and 10
53
5.2. Clinicopathological features
55
5.2.1. AOEs and Prxs in pilocytic astrocytomas
55
5.2.2. AOEs in ependymomas
56
5.2.3. CAs in medulloblastomas and PNETs
56
5.2.4. CLDNs in ependymomas
57
5.3. Prognosis
6
57
5.3.1. Patients with pilocytic astrocytoma
57
5.3.2. Patients with ependymoma
58
5.3.3. Patients with MB or PNET
58
DISCUSSION
60
6.1. Current state of pediatric brain tumors
60
6.2. AOEs in pediatric brain tumors
60
6.3. Carbonic anhydrases in pediatric brain tumors
62
6.4. Claudins in pediatric brain tumors
63
6.5. The relationship between claudins and carbonic anhydrases
64
6.6. Limitations of the study
65
6.7. Future prospects
65
7
SUMMARY AND CONCLUSIONS
68
8
ACKNOWLEDGEMENTS
70
9
REFERENCES
72
10
ORIGINAL COMMUNICATIONS I-IV
110
6
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications referred to in the text by their roman numerals.
I
Nordfors K, Haapasalo J, Helén P, Paetau A, Paljärvi L, Kalimo H, Kinnula VL, Soini Y and Haapasalo H (2007): Peroxiredoxins and antioxidant enzymes in pilocytic astrocytomas. Clin Neuropathol 26:210-218.
II Järvelä S*, Nordfors K*, Jansson M, Haapasalo J, Helén P, Paljärvi L, Kalimo H, Kinnula V, Soini Y and Haapasalo H (2008): Decreased expression of antioxidant enzymes is associated with aggressive features in ependymomas. J Neurooncol 90:283-291. (*shared first authorship).
III Nordfors K, Haapasalo J, Korja M, Niemelä A, Laine J, Parkkila AK, Pastorekova S, Pastorek J, Waheed A, Sly WS, Parkkila S and Haapasalo H (2010): The tumour-associated carbonic anhydrases CA II, CA IX and CA XII in a group of medulloblastomas and supratentorial primitive neuroectodermal tumours: an association of CA IX with poor prognosis. BMC Cancer 10:148.
IV Nordfors K, Haapasalo J, Sallinen P, Haapasalo H and Soini Y: Expression of claudins relates to tumour aggressivity, location and recurrence in ependymomas. (submitted)
Permission for the reproducing the original articles of this thesis has been given by the copywright owners.
7
ABBREVIATIONS AEC AIDS AML AOE AOP2 APC AXIN1 AXIN2 BBB Bcl-2 BRAF CA CAM CARPs CCTNNB1 cDNA CGNP CLDN C-MYC CNS CSF CT Cu/Zn-SOD CYS DNA D/N MB EC-SOD EMT -GC GEM GLCL GLCL-C GLCL-R GPX GSH GSSG GTR HER2 HIF HP iPNET LC/A MB MB MBEN MIB-1 MMP MN MnSOD
aminoethyl carbazole Aquired Immune Deficiency Syndrome acute myeloid leukemia antioxidative enzyme antioxidative protein 2 adenomatous polyposis coli axis inhibitor 1 axis inhibitor 2 blood-brain barrier B-cell lymphoma 2 v-raf murine sarcoma viral oncogene homolog B1 carbonic anhydrase cell adhesion molecule carbonic anhydrase-related protein cadherin- associated protein beta 1 complementary DNA cerebellar granule cell precursor claudin v-myc myelocytomatosis viral oncogene homolog central nervous system cerebro-spinal fluid computer tomography copper-zinc superoxide dismutase cysteine deoxyribonucleic acid desmoplasmic/nodular medulloblastoma extracellular superoxide dismutase epithelial-mesenchymal transition –glutamylcysteine genetically engineered mouse glutamate cysteine ligase gamma glutamyl cysteinyl synthetase catalytic subunit gamma glutamyl cysteinyl synthetase regulatory subunit glutatahione peroxidase glutathione oxidized glutathione gross-total resection Human Epidermal growth factor Receptor 2 hypoxia-inducible (transcription) factor Helicobacter pylori infratentorial primitive neuroectodermal tumor large cell/anaplastic medulloblastoma medulloblastoma medulloblastoma with extensive nodularity an antibody against Ki-67 matrix metalloproteinase carbonic anhydrase IX manganese superoxide dismutase
8
MRI mRNA NADPH NF1 NF-B NKEF-A NKEF-B N-MYC Nrf2 ORF6 PA PBS PG PKC PLA2 PNET PRC Prx PTCH1 PTEN RAS RCC RG ROS Shh SLUG SMOH SNAIL SOD SUFU sPNET TGR Tj TMA TMD TNF- TRAIL Trx TrxR TUNEL VEGF VEGFR vHL WHO ZEB1
magnetic resonance imaging messenger-ribonucleic acid nicotinamide adenine dinucleotide phosphate neurofibromatosis 1 nuclear factor kappa-light-chain-enhancer of activated B cells natural killer enhancing factor A natural killer enhancing factor B v-myc myelocytomatosis viral related oncogene, neuroblastoma derived nuclear factor 2 open reading frame 6 pilocytic astrocytoma phosphate-buffered saline proteoglycan protein kinase C phospholipase A2 primitive neuroectodermal tumor protein kinase C peroxiredoxin patched homolog 1 phosphatase and tensin homolog RAt Sarcoma renal cell carcinoma radial glia reactive oxygen species sonic hedgehog neural crest transcription factor, member of snail family smoothened homolog zinc finger transcription factor superoxide dismutase suppressor of fused homolog supratentorial primitive neuroectodermal tumor thioredoxin glutathione reductase tight junction tumor multitissue array transmembrane proteins with domain tumor necoris factor-alpha TNF-Related Apoptosis-Inducing Ligand thioredoxin thioredoxin reductase terminal deoxynucleotidyl transferase dUTP nick end labeling vascular endothelial growth factor VEGF receptor von Hippel-Lindau World Health Organization zinc finger E-box binding homeobox 1
9
ABSTRACT
Brain tumors are the second most common tumor type in children after leukemia. The outcome of the patients has become more favorable over the past few decades due to improved treatment modalities. Nowadays, the 5-year prognosis is from less than 60% to 90% depending on the tumor type. Nevertheless, the tumor itself and its treatment reduce quality of life, increase the risk of being handicapped, and raise expenses. Novel treatment modalities are under intense investigation, although a major breakthrough has yet to be discovered.
The aim of this thesis was to find new molecules to be used in the process of diagnosing and evaluating the predictivity, prognosis and follow-up of children with the most common pediatric brain tumors, including pilocytic astrocytomas, ependymomas, medulloblastomas (MBS), and primitive neuroectodermal tumors (PNETs). In this thesis, antioxidative enzymes were investigated in pilocytic astrocytomas and ependymomas, as well as peroxiredoxins in pilocytic astrocytomas. In addition, the role of carbonic anhydrases was studied in medulloblastomas and PNETs. Finally, claudins were analysed in ependymomas.
The first study analysed the antioxidative enzymes (AOEs), including manganese superoxide dismutase (MnSOD), gamma glutamyl cysteinyl synthetase catalytic and regulatory subunits (GLCL-C, GLCL-R), thioredoxin (Trx), thioredoxin reductase (TrxR) and peroxiredoxins (Prx) IVI in a series of 105 pilocytic astrocytomas. All of them were expressed in pilocytic astrocytomas, suggesting that oxidative damage and consequent defence take place during the progression of the tumors. AOEs correlated with the degenerative features and angiogenesis, possibly associating with reactive oxygen species-derived cellular damage. Moreover, the expression of the AOEs was associated with each other in terms of concurrent activation of the enzymes. With the exception of MnSOD, a strong expression of AOEs was generally associated with higher cell proliferation. Prx VI seemed to have a positive association with a longer recurrence-free interval.
The second study investigated the relationship between AOE (MnSOD, GLCL-C, GLCL-R, Trx, TrxR) expression and clinicopathological features in 67 ependymal tumors. Most of the tumors expressed AOEs. Lower GLCL-C and GLCL-R expression was associated with higher tumor grade. MnSOD, GLCL-C and TrxR expressions were significantly higher in tumors located in the spinal cord compared with those in the brain. Interestingly, decreased expression of Trx predicted worse
10
outcome for the patients. This finding may have clinical relevance when planning treatment modalities and follow-up for patients.
The aim of the third study was to analyse the expression of carbonic anhydrases (CAs) II, IX, and XII in a set of 39 medulloblastomas and PNETs. Interestingly, CA II was expressed in both the neovessel endothelium and tumour cell cytoplasm. CA IX was mainly expressed in the tumor cells located in perinecrotic areas. CA XII showed the most homogenous distribution within the tumors. Importantly, CA IX expression predicted poor prognosis in both univariate and multivariate analyses. CA IX has been previously found to be a promising target molecule for anticancer treatment in other tumors. The results suggest that this could also be the case for medulloblastomas and PNETs.
In the fourth study, expression of claudins (CLDNs) 2-5, 7, and 10 was investigated in a set of 61 ependymomas. According to the results, all CLDNs except for CLDN4 were expressed in these tumors. CLDN5 was related to more aggressive tumors compared with CLDN2 and 10. Tumors expressing these two claudins displayed a better degree of differentiation and a better prognosis. There were also differences in the expression of claudins associated with location of the tumor and between primary and recurrent tumors, CLDNs 3 and 5 were more often found in the cerebrum than in other sites and CLDN7 in primary tumors compared with recurrent ones. Evidently claudins influence the growth and differentiation in ependymomas.
In summary, the studied antioxidative enzymes, peroxiredoxins, carbonic anhydrases, and claudins were expressed in the most common pediatric brain tumors, including pilocytic astrocytomas, ependymomas, medulloblastomas, and PNETs. Prx VI was associated with longer recurrence-free interval in patients with pilocytic astrocytoma, whereas decreased Trx expression predicted worse prognosis of patients with ependymoma. CA IX correlated with worse outcome in patients with medulloblastoma or PNET. Claudins had no significant association with prognosis, nevertheless CLDN5 was related to more aggressive ependymomas.
11
TIIVISTELMÄ
Aivokasvaimet ovat lasten toiseksi yleisin kasvaintyyppi leukemian jälkeen. Erityisesti hoitojen kehityttyä lasten ennuste on parantunut selvästi viime vuosikymmenten aikana. Nykypäivänä 5vuotisennuste vaihtelee kasvaimesta riippuen alle 60%:sta 90%:iin. Tauti ja hoidot aiheuttavat kuitenkin elämänlaadun heikkenemistä, vammautumisia, sekä myös kustannuksia. Uusia hoitokeinoja tutkitaan jatkuvasti, mutta selviä läpimurtoja ei ole toistaiseksi saavutettu. Väitöskirjani tavoitteena oli etsiä uusia molekyylejä, joita voitaisiin hyödyntää lasten yleisimpien aivokasvainten diagnostiikassa, ennusteen arvioimisessa sekä seurannassa. Kirjassa läpikäydään antioksidatiivisten entsyymien esiintymistä pilosyyttisissä astrosytoomissa sekä ependymoomissa. Lisäksi
väitöskirjassani
käsitellään
peroksiredoksiineja
pilosyyttisissä
astrosytoomissa,
hiilihappoanhydraaseja medulloblastoomissa ja primitiivisissä neuroektodermaalisissa tuumoreissa (PNET:ssa), sekä klaudiineja ependymoomissa.
Ensimmäisessä osajulkaisussa tutkittiin antioksidatiivisten entsyymien (AOE; MnSOD, GLCL-C, GLCL-R, Trx, TrxR), sekä peroxiredoksiinien (Prx I-VI) esiintymistä 105 pilosyyttisessä astrosytoomassa. Kaikki ilmentyivät laajasti kyseisissä kasvaimissa. Tämä viittaa oksidatiivisen vaurion ja sen korjausmekanismien osallisuuteen pilosyyttisen astrosytooman kehittymisessä. Lisäksi AOE:t ilmentyivät kasvaimissa samanaikaisesti viitaten mahdolliseen yhteiseen aktivoitumiseen. Kaikilla AOE:lla oli yhteys korkeaan proliferaatioasteeseen, lukuun ottamatta MnSOD:ia. Prx VI oli tilastollisesti merkittävä tekijä arvioitaessa kasvaimen uusiutumista.
Toisessa osajulkaisussa tutkittiin AOE:n (MnSOD, GLCL-C, GLCL-R, Trx, TrxR) yhteyttä 67 ependymooman kliinispatologisiin muuttujiin. Useimmat kasvaimet ilmensivät entsyymejä. GLCLC:n ja GLCL-R:n vähäisyys oli yhteydessä korkeampaan gradukseen. MnSOD, GLCL-C ja TrxR värjäytyminen oli voimakkaampaa selkäytimen kuin aivojen ependymoomissa. Kiinnostava löytö oli Trx:n vähäisyyden yhteys huonoon ennusteeseen. Tällä havainnolla voi olla kliinistä merkitystä potilaiden hoidon ja seurannan suunnittelussa.
Kolmas osatyö käsitteli hiilihappoanhydraasien (CA) medulloblastoomassa
ja
PNET:ssa.
CA
II
esiintyi
II,
IX ja XII esiintymistä 39
uudisverisuonten
endoteelissä
sekä
sytoplasmassa, kun taas CA IX värjäytyi voimakkaimmin nekroottisten alueiden reunoilla. CA XII esiintyvyys oli tasaisempaa. CA IX oli huonon ennusteen merkki sekä yksimuuttuja, -että monimuuttujamalleissa. CA IX on osoittautunut lupaavaksi syövänhoidon kohdemolekyyliksi 12
muissa kasvaimissa ja osatyön perusteella näin voisi olla myös medulloblastoomien ja PNET:n kohdalla.
Neljännessä osajulkaisussa tutkittiin klaudiinien (CLDN 2-5,7 ja 10) esiintymistä 61 ependymoomassa. Lukuunottamatta CLDN4, kaikki klaudiinit esiintyivät ependymoomissa. CLDN5:llä oli yhteys kasvaimen aggressiivisuuteen, kun taas CLDN2:llä ja 10:llä oli trendi parempaan erilaistumiseen ja ennusteeseen. Klaudiinit ilmentyivät erilailla kasvaimen sijainnista riippuen; CLDN3 ja 5 värjäytyivät voimakkaammin isojen aivojen ependymoomissa. CLDN7 värjäytyvyys oli voimakkaampaa primaarikasvaimissa. Tuloksista voi päätellä, että klaudiinit vaikuttavat ependymoomien kasvuun ja erilaistumiseen.
Tutkitut antioksidatiiviset entsyymit, mukaanluettuna peroxiredoksiinit, sekä hiilihappoanhydraasit ja klaudiinit esiintyivät lasten yleisimmissä aivokasvaimissa (pilosyyttisissä astrosytoomissa, ependymoomissa, medulloblastoomissa sekä PNET:ssa). Prx VI oli yhteys pilosyyttisen astrosytooman uusiutumiseen, kun taas Trx:n väheneminen ennusti ependymooma-potilaiden huonompaa ennustetta. CA IX sen sijaan oli huonon ennusteen merkki medulloblastooma ja PNET –potilailla. Klaudiineilla ei ollut yhteyttä potilaiden ennusteeseen, mutta CLDN5 assosioitui ependymooman aggressiivisuuteen.
13
1
INTRODUCTION
Pediatric brain tumors are the most common solid tumors and the second most common tumors after leukemia in children. Although the prognosis has improved over the past few decades, children still have severe side effects from the treatment; they may be handicapped, and 5-year survival varies from less than 60% to 90% (Louis et al. 2007).
There are several types of pediatric brain tumor. Pilocytic astrocytoma (PA), is a grade I glial tumor arising mostly in the cerebellum of children (Ohgaki and Kleihues 2005). It is histologically benign but may present a clinical challenge to neurosurgeons due to its location. Nevertheless, the prognosis remains good and the 5-year survival rate is 80-90% (Watson et al. 2001, Mueller and Chang 2009).
Another tumor entity is grade I-III ependymoma, which originates from ependymal cells or their stem cells. The typical location is the 4th ventricle, though ependymomas may occur at any site along the ventricular system and in the spinal cord (Schiffer et al. 1991, Prayson 1999). The outcome of the patients depends mainly on age, tumor location, extent of resection, and histopathological grading (Jayawickreme et al. 1995, Ernestus et al. 1996, Horn et al. 1999, Louis et al. 2007). Children with intracranial ependymoma have a 50% 5-year progression-free survival rate (Robertson et al. 1998, Venkatramani et al. 2011).
The most common malignant brain tumor in children is grade IV medulloblastoma (MB). Embryonal MB locates in the vermis and 4th ventricle, and it may metastasize via cerebro-spinal fluid (Louis et al. 2007). The 5-year survival rate for patients with MB is 60-70%. The improvement compared to previous survival rate is mainly due to better treatment modalities (Ellison et al. 2003). Central nervous system (CNS) primitive neuroectodermal tumors (PNETs) are a group of tumors recently reclassified (Louis et al. 2007). PNET is a grade IV embryonal tumor with early onset and aggressive behavior. The prognosis is worse than with medulloblastoma (Geyer et al. 2005) and younger children have the worst outcome (Geyer et al. 1994).
The standard of care for brain tumors is neurosurgical gross total resection (GTR) and it is applied where possible. Adjuvant therapy, including chemo- and radiotherapy, is being used in selected cases (Mueller and Chang 2009). In addition, novel treatment modalities and prognosticators have been under intense investigation. Nevertheless, comprehensive and clinically valid tools have yet to 14
be developed. The blood-brain barrier (BBB) is a challenge for anticancer treatment.
Most
molecules are not capable of penetrating the BBB. In order to penetrate, the molecule should be electrically neutral, lipid-soluble, and small. The neural tissue is very sensitive to damage and this is another challenge for anticancer treatment.
Accurate knowledge of tumor biology is the basis for studying and diagnosing brain tumors. Genetic analyses give novel information and may lead to more individual treatments. This is unfortunately still not cost-effective for clinical use. The present study was conducted to establish new tools for clinical screening, diagnosis, as well as prognostic markers and methods for monitoring and predicting the outcome for patients with pediatric brain tumors. The study results may facilitate future research strategies for the field of brain tumors in children.
15
2
REVIEW OF THE LITERATURE
2.1. PEDIATRIC BRAIN TUMORS
Brain tumors are the second most common tumor type in children after leukemia. Typical pediatric type brain tumors are pilocytic astrocytomas, ependymomas, medulloblastomas and PNETs (Louis et al. 2007). Although most of the tumors can occur in any age group, the aforementioned tumors are typically seen in children or young adults (Figure 1) (Kieran et al. 2010). The biology of the tumors differs to some extent between age groups and thus, the following text concentrates on pediatric brain tumors.
Figure 1. Incidence of pilocytic astrocytoma, ependymoma, medulloblastoma, and PNET in different age groups. Modified from Kieran et al. (2010). 10 9
Incidence per 1 million
8 7 6 5
Pilocytic astrocytoma
4
Ependymoma
3
Medulloblastoma/PNET
2 1 0 0-14
15-19
20-34
Age (years)
16
35-44
2.1.1. Pilocytic astrocytoma Pilocytic astrocytoma (PA) is the most common astrocytoma of children, comprising 21% and 16% of all CNS tumors for age groups 0-14 years and 15-19 years, respectively (Central Brain Tumor Registry 2006). In children, the tumor arises mostly in the cerebellum (Ohgaki and Kleihues 2005), though other sites include optic chiasm, hypothalamus, thalamus, basal ganglia, cerebral hemispheres, brain steam and spinal cord (Louis et al. 2007). Although the tumor corresponds to World Health Organization (WHO) grade I and the 5-year overall survival is good, 80-90%, patients with pilocytic astrocytoma may have poor outcome owing to localisation (Watson et al. 2001, Mueller and Chang 2009) (Table 1). Patients can suffer from headache, visual loss, hydrocephalia, hemiparesis or may even die of the disease. Most frequently, pilocytic astrocytoma of patients less than 20 years old presents as clumsiness, worsening headache, nausea and vomiting.
The radiological diagnosis of pilocytic astrocytomas is made by computer tomography (CT) or magnetic resonance imging (MRI), in which it is present as a circumscribed and contrast enhancing tumor (Lee et al. 1989, Fulham et al. 1993). The tumor has typical cysts, which are an important detail when evaluating the tumor grade (Palma et al. 1983).
The histolopathology of pilocytic astrocytoma is heterogenous. The cells are bipolar piloid with Rosenthal fibers and multipolar with microcysts and granular bodies. Pilocytic astrocytomas have a low mitotic index, nuclei are hyperchromatic and pleomorphic. Although pilocytic astrocytomas often have vascular proliferation, the tumor is still of a benign type. Endothelial proliferation and necrosis, typically seen in high-grade astrocytomas, are rarely seen in PAs (Louis et al. 2007). In the literature, there are few examples of pilocytic astrocytoma undergoing malignant transformation, but the tumor should not be confused with glioblastoma because it is then an anaplastic pilocytic astrocytoma (Dirks et al. 1994, Tomlinson et al. 1994).
Pilocytic astrocytoma is associated with neurofibromatosis 1(NF1). Patients with NF1 usually have PA in the optic nerve and thus, about 30% of patients with optic nerve PAs have NF1. NF1associated tumors grow slowly or remain stable (Hoyt and Baghdassarian 1969). Although the cause of PA remains uncertain, people with NF1, Li-Fraumeni syndrome and prior radiation to the brain carry a higher risk of PA development (Mueller and Chang 2009).
Patients with PA undergo radical resection where possible. The extent of resection is strongly associated with survival (Laws et al. 1984). New operation techniques have enabled the most 17
eloquent brain areas accessible. Recurrences do occur and are often a reflection of cyst reformation. Nevertheless, recurrence-free survival is greater than 95% at 10 and 20 years in patients with radical resection (Watson et al. 2001).
If complete resection is possible, it is rare for patients to undergo additional adjuvant therapy, unless there is evidence of recurrence or progression from MRI (Table 1). Typical genetic alterations seen in diffusively infiltrating astrocytomas are rarely seen in PAs (Cheng et al. 2000) and thus, similar future treatment modalities can not be used. Previous studies show that there is trisomy of chromosome 5 and 7 or gain of 1q (Jones et al. 2006). In addition, several studies have shown a mitogen-activated protein kinase pathway activation in PAs and duplication of the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene locus (Pfister et al. 2008).
2.1.2. Ependymoma Ependymoma is mainly a tumor of children and young adults, though it may occur in all age groups (Central Brain Tumor Registry 2006). The age distribution depends on the histological type and location, younger patients having mostly an infratentorial tumour and 30-40 year olds mostly a spinal tumor (Waldron and Tihan 2003). Otherwise, the tumor may occur at any site in the CNS, but mostly in the fourth ventricle, in the spinal cord and the lateral and third ventricles (Schiffer et al. 1991, Prayson 1999). One third of ependymomas are localised supratentorially and two thirds infratentorially (Zacharoulis and Moreno 2009) (Table 1).
Most ependymomas are considered to be WHO histological grade II. Other ependymomas include anaplastic ependymoma (grade III) and rare entities: grade I myxopapillary ependymoma and subependymoma, grade I. Grade II ependymoma consists of four variants, such as cellular ependymoma (the most common), papillary, clear cell, and tanycytic ependymoma. The tumor cells are usually round or oval and may contain eosinophilic cytoplasmic granules and dense chromatin material. Grade III ependymoma has a higher proliferation and mitotic index, though distinction of grade II from grade III can be rather difficult (Louis et al. 2007) (Table 1).
Symptoms and signs are dependent on location. Typically the symptoms are caused by mechanical compression of the cerebral fluid circulation leading to hydrocephalus, headache, nausea, vomiting, dizziness and, with very young children, head enlargement (Duncan and Hoffman 1995).
18
The tumor is initially diagnosed with gadolinium-enhanced MRI, in which ependymoma is well circumscribed and shows varying degrees of contrast enhancement (Furie and Provenzale 1995). About half of the patients have multifocal calcification, and some of these tumors are hemorrhagic (Zacharoulis and Moreno 2009).
The molecular basis of ependymoma remains uncertain. Chromosome 22 mutations are the most frequent finding, and about 30% of cases have monosomy or translocation in this chromosome (Reni et al. 2007). Additionally, anaplastic ependymoma has an association with gain in chromosome 1q and this chromosomal change is also correlated to a worse outcome (Rand et al. 2008). Comparative genomic hybridisation has revealed a variable number of genetic imbalances. Added to this, abnormal Notch and Sonic Hedgehog (Shh) signaling have been reported in ependymomas (Modena et al. 2006). Similarly to other brain tumors, the pathogenesis of ependymomas may include neural cancer stem cells (Taylor et al. 2005).
Surgical resection is the standard of care for patients with ependymoma. Gross total resection (GTR) can be applied in about 40-60% of cases, and is obviously more common with a supratentorial location (Zacharoulis and Moreno 2009). Even children with a metastatic tumor benefit from GTR (Zacharoulis et al. 2008). Chemotherapy has only a minor role in the treatment of ependymomas. Evans et al. (1996) have shown in a randomised study that adding vincristine and lomustine after radiotherapy does not improve survival. Infants may be the only patient group who may benefit from chemotherapy. In contrast, focal radiotherapy is an important treatment modality for children with ependymoma. Approximately half of the patients will experience relapse, typically locally in the first two years, though late recurrences are also seen (Zacharoulis and Moreno 2009). The most common treatment for patients with a relapse is re-operation and this increases progression-free survival (Vinchon et al. 2005). Adjuvant chemotherapy has a modest effect on survival (Zacharoulis and Moreno 2009) whereas radiotherapy is beneficial for patients with relapsed ependymoma (Combs et al. 2006) (Table 1).
The prognosis of the patients relies mainly on four different clinical features. First, tumor location is the main feature determing patient survival. Posterior fossa tumors are usually more aggressive than supratentorial tumors. Ependymoma in the spinal cord tend to have late recurrences, but still have a better prognosis compared to cerebral neoplasms (Ernestus et al. 1996). Second, the extent of the resection is an independent prognosticator as well (Jayawickreme et al. 1995). Third, there are many histopathological features indicating patient outcome. Patients have a worse prognosis 19
when ependymoma shows anaplasia, e.g. tumor cells are less differentiated and show a high mitotic index and enhanced proliferation (Schiffer and Giordana 1998, Korshunov et al. 2004, Kurt et al. 2006). Fourth, children under three years of age seem to have a worse outcome compared to the elderly (Horn et al. 1999).
2.1.3. Medulloblastoma Medulloblastoma (MB) is a malignant, WHO grade IV brain tumor occuring mainly in children (Arseni and Ciurea 1981). The peak age is seven years, and 70% of the patients are less than 16 years of age (Roberts et al. 1991). There is a male predominance (65%). Medulloblastomas are usually located in the cerebellar vermis and the fourth ventricle. Older patients usually have desmoplastic/nodular subtypes, which locate mainly in the cerebellar hemipheres (Louis et al. 2007) (Table 1). In addition, desmoplastic MB occurs at a high frequency among infants (Ellison 2010).
According to the WHO classification MB is separated into classic tumor and four variants: desmoplasmic/nodular (D/N), MB with extensive nodularity (MBEN), anaplastic and large cell MB. Classic MB is the most common medulloblastoma. Large cell and anaplastic MB are aggressive tumors, whereas MBENs and D/N MBs in infants have better outcome than classic MB. (Louis et al. 2007) D/N and MBEN typically have nodules of differentiated neurocytic cells and internodular desmoplasia (Giangaspero et al. 1999). Large cell MB contains groups of uniform large cells with round nuclei and a single nucleus. Both large cell MB and anaplastic MB show a high mitotic activity and apoptosis. Anaplastic MB is dominated by nuclear pleomorphism (Eberhart et al. 2002). Many studies combine D/N MB and MBEN as desmoplastic tumors, and large cell and anaplastic MB as large cell/anaplastic (LC/A) tumors (Table 1). Classic medulloblastoma has monotous small cells and the nuclei may be either round or oval. Some tumors show rosettes and palisades (Ellison 2010).
The molecular pathology of MBs is under extensive research. There is evidence of abnormalities in Shh pathway through mutations in patched homolog 1 (PTCH1), smoothened homolog (SMOH) and suppressor of fused homolog (SUFU) genes in 25% of medulloblastomas. This works as a stimulator to cerebellar granule cell precursors (CGNPs) and Purkinje cells to release Shh during CNS development, a phenomenon which has been shown in several genetically engineered mouse (GEM) models. Another pathway is the Wnt pathway comprising 15% of MBs. In this pathway, there are mutations in cadherin- associated protein), beta 1 (CTNNB1), adenomatous polyposis coli. (APC), and axis inhibitor (AXIN)1/2. The pathway produces nuclear accumulation of -catenin, 20
which acts as a transcriptional activator (Ellison 2010). In addition to the two signaling pathways, there are two non-Shh/Wnt subgroups, which are associated with up-regulation of specific gene classes, but not with aberrant activation of signaling pathways (Northcott et al. 2011). The nonShh/Wnt tumors account for 60% of medulloblastomas (Ellison 2010). Isochromosome 17q (i17q) is the most frequent structural aberration in medulloblastomas, found in 30–40% of cases (Northcott et al. 2009). V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (N-MYC) gene amplification has also been identified in up to 10% of medulloblastoma specimens and, like vmyc myelocytomatosis viral oncogene homolog (C-MYC), is often found in tumors with large cell/anaplastic features (Aldosari et al. 2002).
Patients with medulloblastoma often have mechanical obstruction of cerebro-spinal fluid (CFS-) flow, and this is usually the reason for typical symptoms. These include increased intracranial pressure, headache, vomiting and nausea. Patients may also have excessive lethargy and ataxia. CTscans or MRI reveal a solid, intensely contrast-enhancing mass (Louis et al. 2007).
The prognosis of young patients has increased over the past few decades, and nowadays the 5-year survival rate is 60-70%. This improvement is mainly due to better treatment modalities. The most important treatment is surgical resection, but perioperative chemo- and radiation therapy are also applied (Ellison et al. 2003). MBs are very radiosensitive tumors, so children over three years of age have radiation therapy. Side effects of the treatment have led to a reduction in the amount of radiation (Deutsch et al. 1996). The problem is that lowering the radiation dose without adding chemotherapy leads to a worse outcome and thus, chemotherapy is a standard choice for children with MB (Mueller and Chang 2009) (Table 1). The patients are divided into low and high risk (age 75% of tumor cells showing positivity. In addition to the researcher, two experienced pathologists analyzed the staining results, and a consensus meeting was held in the case of a disagreement.
4.3.2. Carbonic anhydrases Parkkila et al. (1993) have produced and characterised rabbit antiserum against human CA II. The previously described monoclonal antibody M75, recognising the N-terminal domain of human CA IX, was used in the CA IX immunostaining procedure (Pastorekova et al. 1992, Chrastina et al. 2003). Rabbit anti-human CA XII antiserum against the secretory form of CA XII has been previously characterised (Karhumaa et al. 2000) and was used in this study. The detailed staining protocol is described in study III.
The staining reactivities for CA II, CA IX and CA XII were scored from multitissue- blocks on a scale from 0 to 3 as follows: 0 = no reaction, 1 = weak reaction (< 10% positive cells), 2 = moderate reaction (10-30% positive cells), 3 = strong reaction (>30% positive cells). Due to the sample size, staining results were categorised into two groups: negative staining was considered as CA-negative and weak, moderate and strong staining were considered as CA-positive.
4.3.3. Claudins The primary antibodies for the detection of claudins 2-5, 7, and 10 in formalin-fixed paraffinembedded tissues were purchased from Zymed Laboratories Inc (South San Francisco, CA). The antibodies included polyclonal rabbit anti-claudin 2 (clone JAY.8), polyclonal rabbit anti-claudin 3 (clone Z23.JM), monoclonal mouse anti-claudin 4 (clone 3E2C1), monoclonal mouse anti-claudin 5 (clone 4C3C2), polyclonal rabbit anti-claudin 7 (clone ZMD.241) and polyclonal anti-rabbit claudin 10 (Ca N:o 38-8400).
Immunostaining of claudins was carried out as follows. The microarray sections were first heated in a microwave oven in 10 mM citrate buffer (pH 6.0) for 10 minutes. After a 60-minute incubation with the primary antibody (dilution 1:50 for anti-claudin 1, 3, 4, 5 and 7, 1:100 for claudin 10), a biotinylated secondary anti-rabbit or anti-mouse antibody and Histostain-SP kit (Zymed Laboratoris Inc) were used. The bound antibodies were demonstrated with diaminobenzidine as a chromogen. 49
The sections were then lightly counterstained with haematoxylin and mounted with Eukitt (Kindler, Freiburg, Germany). Positive controls included non-neoplastic kidney, breast, skin and liver samples. Non-immune rabbit or mouse serum were used as substitutes for the primary antibodies to act as the negative control.
Two neuropathologists analyzed the staining results, and a consensus meeting was held in the case of a disagreement. Membrane bound immunoreactivity was considered significant, although cytoplasmic expression was also detected to be present. The immunoexpression of claudins was assessed as follows; negative = < 5% of cells positive, weak = 5-25 % of cells positive, moderate = 25-75 % of cells positive, strong = 75-100 % of cells positive. Due to the sample size, the tumors were recorded as CLDN-negative or CLDN-positive (from weak to strong staining) for statistical analyses.
4.3.4. Other immunohistochemistry and TUNEL Analysis of cell proliferation was carried out using a mouse monoclonal antibody MIB-1 (Ki-67 antigen, dilution 1:40, Immunotech, S.A. Marseille, France). The tissue sections were counterstained with methyl green. The proliferative activity was reported as a percentage of nuclei with positive immunoreaction. The analysis of Ki-67/MIB-1 positive nuclei in tissues was evaluated quantitatively using a computer-assisted image analysis system (CAS-200 TM Software). Immunohistochemical analysis for p53 status was performed as described earlier (Haapasalo et al. 2003). Apoptosis was detected using ApopTagTM In Situ Apoptosis Detection Kits (Oncor Inc., Gaithersburg, MD) as described previously (Haapasalo et al. 1999).
The mouse monoclonal TWIST (ab50887, Abcam, Cambridge, UK) and ZEB1 (clone 416A7H10, GenWay, San Diego, CA, USA) antibodies at a 1:500 dilution with the microarray sections were incubated overnight at 4°C. The slides were then stained using a standard avidin-biotin-enhanced immunoperoxidase technique (ABC Vectastain Elite Kit, Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine tetrahydrocloride (DAP, in phosphate-buffered saline) (Sigma, St. Louis, MO, USA) was used as chromogen. The sections were counterstained with Mayer's haematoxylin, washed, dehydrated, cleared and mounted with Depex (BDH, Poole, UK). Ovarian tumor tissue, known to be positive for TWIST and ZEB1 expression, was used as a positive control. Nuclear immunoreactivity for TWIST and ZEB1 was considered significant. Tumors with widespread staining (over 5% positivity) were recorded as TWIST/ZEB1-positive.
50
4.4. Histopathological features The presence of the following histological features was recorded for further statistical analyses in case of ependymomas: atypia, endothelial proliferation, necrosis, mitosis, hypercellularity, and pseudorosettes, true rosettes, and papillarity. In pilocytic astrocytomas and ependymomas, calcification and myxoid change were recorded.
4.5. Statistical methods All statistical analyses were performed using SPSS for Windows 11.0 (Chicago, IL). The significance of the associations was defined using the chi-square test, the Mann-Whitney test and the Kruskal-Wallis test. A log-rank test, Kaplan-Meier curves and Cox multivariate regression analysis were used in the survival analysis.
4.6. Ethics The study designs for all the original publications were approved by the Ethics committee of Tampere University Hospital and the National Authority for Medicolegal Affairs.
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5
RESULTS
5.1. Immunohistochemical expression
5.1.1. Antioxidative enzymes Antioxidative enzymes, including MnSOD, Trx, TrxR, GLCL-C, and GLCL-R, were studied in 105 pilocytic astrocytomas (96 primary and 9 recurrent tumors) and in 67 ependymomas (46 primary and 21 recurrent tumors). Pilocytic astrocytomas showed mainly cytoplasmic AOE immunostaining pattern.
In primary ependymomas, AOE immunoreactivity was as follows. 87% showed immunoreactivity for MnSOD. Positive immunoreactivity for GLCL-C was found in 74% of the cases and 89% for GLCL-R. Trx immunoreactivity was seen in 72%. 54% of primary tumors were TrxR immunoreactive (Table 6).
The correlations between AOEs in ependymomas were as follows (unpublished data): negative MnSOD expression was associated with TrxR negativity, (p = 0.017, chi-square test). Similarly, GLCL-C negativity correlated with negative expressions of GLCL-R, Trx, and TrxR (p = 0.002, p = 0.023, p < 0.001, chi-square test, respectively). GLCL-R was more often negative when there was negative TrxR expression (p = 0.005, chi-square test). As expected, Trx and TrxR were expressed simultaneously (p = 0.003, chi-square test).
5.1.2. Peroxiredoxins I-VI Peroxiredoxins (Prxs) were analysed in 105 pilocytic astrocytomas. In the tumor cells the immunostaining was cytoplasmic, but Prxs I, II, IV, V and VI also showed variable nuclear positivity. For Prx I, 98 % of cases were positive). All tumors were Prx II positive and also Prx III. Most (92%) of all tumors expressed positivity for Prx IV, (13 % strongly, 49 % moderately, 30 % weakly) and 87% were positive for Prx V. Prx VI expression was found in 97 %, of all tumors (Table 6).
In the further statistical analysis, the tumors were recorded Prx-negative (no and weak immunostaining) or Prx-positive (moderate and strong immunopositivity). Co-expression was found between different Prxs as follows. Prx I was associated with Prx II, Prx IV, and Prx VI (p < 0.001, p = 0.010, p < 0.001, chi-square test, respectively). Prx II was associated with Prx IV and Prx 52
VI (p = 0.002, p < 0.001, chi-square test, respectively). Similarly, Prx III was associated with Prx IV (p = 0.004, chi-square test). Prx IV was associated with all other Prxs (Prx V, p = 0.007; Prx VI, p = 0.010, chi-square test, respectively).
5.1.3. Carbonic anhydrases II, IX, and XII Carbonic anhydrases were investigated in 39 medulloblastomas and PNETs. CA II was found in the endothelium of neovessels and the cytoplasm of MB/PNET cells. Endothelial CA II expression was detected in 49% of all tumors, whereas, cytoplasmic CA II was found in 73% of the cases. CA IX and CA XII were less frequently expressed than CA II in the tumor samples: CA IX positivity was found in 23% of tumors, and 11% of the tumors were positive for CA XII (Table 6). Similar to previous studies, CA IX expression was linked to necrosis, while CA XII was more homogenously stained. In further analysis, the tumors were recoded as CA-negative and CA-positive (weak, moderate and strong staining combined). The co-expression of CA II, IX and XII in the subgroups of MBs and PNETs or in the total tumor material did not reach statistical significance (chi-square test).
5.1.4. Claudins 2-5, 7, and 10 Claudins were analysed in a group of 61 ependymoma samples. CLDNs were rather widely expressed, except for CLDN4 which was not expressed in our tumor material. CLDN2 -positivity was found in forty-two percent of all), and 23% of the tumors were positive for CLDN3. For CLDN5, 26% of ependymomas showed positive immunostaining). Most (81%) of the tumors expressed CLDN7, and 11% of cases were positive for CLDN10 (Table 6).
There was an association between negative CLDN3 and negative CLDN5 (p = 0.010, chi-square test). None of the other claudins had correlations with each other.
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Table 6. Expression of peroxiredoxins, antioxidative enzymes, claudins, and carbonic anhydrases in pediatric brain tumors.
Intensity of expression Pilocytic astrocytomas Prx I Prx II Prx III Prx IV Prx V Prx VI Ependymomas MnSOD GLCL-C GLCL-R Trx TrxR CLDN2 CLDN3 CLDN5 CLDN7 CLDN10 Medulloblastomas/PNETs CA II endothelial CA II cytoplasmic CA IX CA XII
negative
weak
moderate
strong
2% 0% 0% 82% 13% 3%
3% 76% 27% 30% 35% 15%
11% 20% 47% 49% 23% 37%
84% 4% 26% 13% 29% 45%
13% 26% 11% 28% 46% 58% 77% 74% 19% 89%
46% 30.5% 41% 35% 17% 14% 9% 5% 45% 7%
30% 17.5% 33% 33% 26% 16% 7% 5% 31% 4%
11% 26% 15% 4% 11% 12% 7% 16% 5% 0%
51% 27% 77% 89%
6% 24% 7% 5%
11% 38% 13% 3%
32% 11% 3% 3%
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5.2. Clinicopathological features Various clinicopathological features were tested in statistical analysis. The features were divided as patient dependent, including patient age and sex, or tumor dependent (location, and molecular pathological features).
5.2.1. AOEs and Prxs in pilocytic astrocytomas The pilocytic astrocytomas of the older patients had more intense staining for MnSOD and GLCLC (p = 0.022 and p = 0.010, respectively, Mann-Whitney test). In addition, recurrent tumors had a significantly lower MnSOD staining pattern (p = 0.019, Mann-Whitney test). No such differences were seen as regards Prxs. Additionally, there was no association between location of tumors and enzyme expression (chi-square test).
Expression of Prx II, Prx III, Prx IV, GLCL-C and TrxR were associated with vascular pathology in pilocytic astrocytomas. Positive Prx II and Prx III expressions had an association with higher endothelial proliferation (p = 0.046, p = 0.023, chi-square test, respectively). In addition, cells with less immunopositivity for GLCL-C also had less vascular hyalinisation (p = 0.013, chi-square test), and similarly lower levels of TrxR had less perivascular lymphocytes (p = 0.006, chi-square test). We analysed AOEs’ relationship with degenerative features and found that Prx I, Prx II and TrxR had significant correlations: Prx I, Prx II, and TrxR were more positive in tumors with cystic pattern (p < 0.001, p = 0.002, p = 0.027, chi-square test, respectively). Expression of GLCL-R and TrxR correlated significantly with an aggressive tumor growth pattern. Positivity for TrxR was associated with a higher atypia rate (p = 0.004, chi-square test), whereas tumors with negative staining pattern for GLCL-R were less necrotic (p = 0.008, chi-square test).
Proliferation index (Ki-67/MIB-1) was also studied in pilocytic astrocytomas. We found that Prx VI and TrxR positive tumors had a higher proliferation rate (Prx VI: negative tumors, 1.8 ± 3.1 (mean±sd), positive tumors 3.0 ± 3.1, p = 0.037; TrxR: negative tumors, 2.2 ± 3.1, positive tumors, 3.5 ± 3.0, p = 0.028, Mann-Whitney test, respectively). However, MnSOD -positivity was associated with lower proliferative activity (negative tumors, 3.3 ± 3.2, positive tumous 2.2 ± 2.9, p = 0.039, Mann-Whitney test). AOE -status and p53 immunopositivity or terminal deoxynucleotidyl transferase dUTP nick end labeling (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), apoptosis) positivity (Mann-Whitney test) were not associated with AOEs.
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5.2.2. AOEs in ependymomas In ependymomas, GLCL-C and GLCL-R expression was linked to tumor aggressivity in such a way that the expression decreased significantly as the grade increased (p = 0.047 and 0.049, chi-square test). When the recurrent tumors were included in the analysis, GLCL-C expression was even more significantly different (p = 0.026, chi-square test).
When tumor site (spinal vs. brain) and AOE expression were compared, tumors were divided into AOE-negative and AOE-positive groups; negative and faint staining was considered as a negative group and moderate and strong staining as a positive group. In the total tumor material, all AOEs were even more intensely stained in spinal tumors than in the intracranial tumors. This difference was statistically significant for MnSOD, GLCL-C and TrxR expression (p = 0.044, 0.046 and 0.004, respectively, chi-square test).
We did not find associations between AOEs and tumor proliferation rate (Ki-67/MIB-1), nor with p53. In contrast, B-cell lymphoma 2 (Bcl-2) –positivity correlated significantly with MnSOD, GLCL-C, Trx and TrxR expression (p = 0.049, p = 0.037, p = 0.024 and p = 0.022, respectively, chi-square test). Though p53 had no significant association to other clinicopathological features, higher proliferation correlated to higher tumor grade (p = 0.033, Kruskal-Wallis test). In addition, lower grade tumors were linked to Bcl-2 positivity (p < 0.001, chi-square test), higher patient age (p = 0.029, Kruskal-Wallis test) and spinal tumor location (p < 0.001, chi-square test).
5.2.3. CAs in medulloblastomas and PNETs CA II, IX and XII expression were also analysed in corcondace with various clinical features and molecular markers. Proliferation (Ki-67/MIB-1), apoptosis (chi-square and Mann-Whitney test) or expression of Bcl-2, p53 or c-erbB-2 were not associated with CAs in any of the groups except for the correlation between positive c-erbB-2 and positive CA IX expression in PNETs (p = 0.047, chisquare test). Interestingly, the tumors of young patients had more CA XII-positivity (total material p < 0.001, MBs p < 0.001, chi-square test). CA IX was also positivitely associated with female gender (total material p = 0.048, MBs p = 0.023, chi-square test). There was no significant difference in the expression of CAs between primary and recurrent tumors in any of the groups (chi-square test). Moreover, there were no correlation between tumor type (MBs/PNETs) and CA intensity (chi-square test).
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5.2.4. CLDNs in ependymomas The association between claudins and several clinicopathological features were analysed in ependymomas. We found that different claudins had particular effects on tumor behavior. When tumor grade and claudins were compared, we found that tumors with high grade were often CLDN5 immunopositive (p = 0.049, chi-square test), whereas CLDN10 positive ependymomas were more often low grade tumors (p = 0.039, chi-square test). Ependymomas with different locations also had different CLDN expression. CLDN5 and CLDN3 were more often found in the cerebrum than in the cerebellum or the spinal cord (p = 0.036, p = 0.007, chi-square test, respectively). When primary and recurrent tumors were compared with their claudin expression, only CLDN7 had significant association, as CLDN7 immunoreactivity was more often found in primary than in recurred ependymomas (p = 0.041, chi-square test).
Typical histopathological features of ependymomas were also linked to claudin expression. This was the case especially with CLDN5, which was more often positively expressed in tumors with increased nuclear atypia, endothelial proliferation, mitotic rate and hypercellularity (p = 0.007, p = 0.018, p = 0.041, p = 0.010, respectively, chi-square test). CLDN5 positive tumors also showed higher Ki-67/MIB-1 cell proliferation rate than CLDN5 negative tumors (p = 0.015, Mann-Whitney test). The immunoexpression of the other claudins was not associated with histological features or cell proliferation index of ependymomas.
When EMT associated transcription factors ZEB1 and TWIST were analysed in ependymomas we found that tumors positive for ZEB1 were often negative for CLDN2 (p = 0.031, chi-square test). Negative expression of TWIST was also associated with negative expression of CLDN5 and CLDN10 (p = 0.013, p = 0.017, chi-square test, respectively). Neither ZEB1 nor TWIST was associated with tumor grade, cell proliferation rate, or histological features.
5.3. Prognosis
5.3.1. Patients with pilocytic astrocytoma All 96 patients with primary pilocytic astrocytomas were included in the survival analysis. None of the studied AOEs or Prxs had an association with overall survival, whereas tumors immunopositive for Prx VI seemed to be associated with significantly better recurrence-free survival when compared with immunonegative tumors (p = 0.032, log-rank test) (Table 7). However, when patient age, Ki-67/MIB-1, location (cerebellum versus other location) and Prx VI –immunopositivity were 57
included in multivariate analysis, none of these factors independently predicted recurrence-free survival (Cox multivariate analysis).
5.3.2. Patients with ependymoma Patients with primary ependymoma (N = 46 in study II, and N = 44 in study IV) were included in the survival analysis. Among the studied AOEs, only Trx had statistical significance for patient survival; tumors that did not express Trx had worse survival in follow-up than patients whose tumors were Trx-positive (p = 0.045, log-rank test). Curiously, we found that with adults (age >19 years), Trx acted as a prognosticator of better outcome and all whose tumors expressed Trx were alive at the end of the follow-up period (p = 0.011, log-rank test). Younger patients did not have a similar effect. In Cox multivariate analysis, Trx was the only independent prognosticator (Odds ratio 7.30, 95% confidence interval 0.93-55.56, p = 0.059), whereas WHO grade, location (spinal vs. brain) and patient age (child vs. adult) were not included in the Cox model (Table 7).
When prognosis of the patients with primary ependymoma (N = 44) was analysed with a log rank test, we found that CLDNs were not associated with patient prognosis. Nevertheless, it seems that patients whose tumors express CLDN2, 5, or 10 may have better clinical outcome than those with CLDN3 or 7 positivity. Interestingly, all the patients with CLDN10 positive tumors or CLDN7 negative tumors were alive at the end of follow-up. TWIST and ZEB1 had no effect on patient outcome.
5.3.3. Patients with MB or PNET All 35 patients with primary MB/PNET were included in the survival analysis. CA IX-positivity was a marker of worse outcome in patients with MB/PNET (all tumors p = 0.041, MBs p = 0.030, PNETs p = n.s.; log-rank test). In addition, patients with medulloblastoma had worse prognosis when their tumors showed CA XII positivity (p = 0.010, log-rank test). When the prognostic value of CAs was tested in the multivariate analysis, we included the following prognostic indicators: patient age, Ki-67/MIB-1 proliferation index, apoptosis index and expression of p53, c-erbB-2 and Bcl-2. In addition, the histopathological type (MB vs. supratentorial PNET), CA II, CA IX and CA XII were used in the analysis. Interestingly in our material, only expression of CA IX (odds ratio 4.31; 95% confidence interval (CI) 1.31 - 14.11; p = 0.016) and the apoptosis index (odds ratio 3.29; 95% CI 1.05 - 10.31, p = 0.041) were independent prognostic factors (Table 7). The expression of neither CA II nor CA XII showed a significant association with survival.
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Table 7. Association between the studied enzymes and the prognosis of the patients with pediatric brain tumors. Pilocytic astrocytoma Prx VI+ recurrences Ependymoma Trx- prognosis Medulloblastoma/PNET CA IX+ prognosis CA XII+ prognosis (medulloblastoma) Apoptosis+ prognosis +/- = positive/negative immunostainig = better prognosis = less recurrences/worse prognosis
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6
DISCUSSION
6.1. Current state of pediatric brain tumors Pediatric cancers are still the main cause of death in children aged 1-14 years in the UK and Finland (Gatta et al. 2005, Statistics Finland 2003-2009). Brain tumors are the second largest tumor group in children after leukemia and the current 5-year survival rate for patients with brain tumor is approximately 60-70% in Nordic countries (Gatta et al. 2005), including Finland (Pokhrel and Hakulinen 2009).
The evolving brain is vulnerable. Even though patients survive, the brain tumor itself and its treatments can cause side effects. The majority of children with a brain tumor live an active life with only minor disabilities. Unfortunately, about a half of the patients do have neurological motor disabilities, and even more patients have an abnormal neurological status. Supratentorial tumors, tumor reoperations, shunt revisions and chemotherapy increase the risk of these problems (Lannering et al. 1990, Ilveskoski et al. 1996, Reimers et al. 2003). There is only a limited scope for improvement with conventional chemotherapy and thus, there is an urgent need for novel, therapeutic agents for these patients.
6.2. AOEs in pediatric brain tumors Our aim was to study the expression of different AOEs in pilocytic astrocytomas and ependymomas. In addition, we analysed the expression of related Prxs in pilocytic astrocytomas.
AOEs and Prxs were widely expressed in pilocytic astrocytomas. According to our study, Prxs and other AOEs were commonly expressed simultaneously. This suggests that the neoplastic tissue suffers from a constant oxidative load, leading to simultaneous up-regulation of these enzymes. Coexpression and a relationship between Prxs have also been observed in previous studies of other neoplasms (Kinnula et al. 2002, Karihtala et al. 2003). An increased oxidative load may lead to degenerative features and cellular destruction in tumor tissue. Prx enzymes and other antioxidant enzymes were usually associated with an elevated proliferation rate, but MnSOD seemed to depress proliferative activity. This is in line with its putative tumor suppressor function, which is further supported by the fact that in the recurrences the expression of MnSOD was significantly decreased. Isolated over-expression of MnSOD decreases the growth, proliferation and invasion of cultured malignant cells (Bravard et al. 1992, St Clair et al. 1992, Yan et al. 1996, Zhong et al. 1997, reviewed by Kinnula and Crapo 2004). Some AOEs have a role as tumor suppressors, and thus, we 60
included proliferation index (Ki-67/MIB-1), the apoptosis regulating Bcl-2 and p53 in the studied variables.
Although there was no association between apoptosis rate and AOEs, histopathologically degenerative and regressive features were often associated with Prx expression. Cystic degeneration with Rosenthal fibres (often seen in pilocytic astrocytomas) and calcification implies cell death, which may be a sign of previous hypoxic periods. Elevated levels of AOEs in pilocytic astrocytomas with such degenerative features indicate the tendency of these tumors to suffer from oxidative stress and increased defence against ROS- mediated tissue damage. Prx II, Prx III, Prx IV, GLCL-c and TrxR were associated with vascular pathology, e.g. endothelial proliferation as a sign of angiogenesis. Hypoxia and ROS induce HIF-alpha, which stimulates angiogenic factors such as VEGF (Kinnula and Crapo 2004). In diffuse astrocytomas it is most probably hypoxia that induces the expression of VEGF in tumor cells. This binds to VEGF receptors (VEGFRs) in endothelial cells, leading to endothelial vascular proliferation. In pilocytic astrocytomas endothelial vascular proliferation is also a common feature and could be a consequence of ROS and AOE induction, but without such a sinister implication as in diffuse astrocytomas.
Furthermore, many AOEs, such as MnSOD and Prxs, increase chemo- and radio-resistance of tumor cells. Contrary to diffuse astrocytomas, pilocytic astrocytomas are treated by surgery alone. This might partly explain the differences in AOEs/Prxs and association with tumor behavior. Even though Prx VI was associated with higher cell proliferation, it seemed to be associated with significantly better recurrence-free survival. This might be due to an association between Prx VI expression and young patient age.
The majority of 67 ependymomas expressed MnSOD, GLCL-C, GLCL-R, Trx and TrxR. However, the intensity of immunoreactivity had a decreasing trend as the tumor grade increased. This is the opposite phenomenon to diffusively infiltrating astrocytomas or oligodendrogliomas (Haapasalo et al. 2003, Järvelä et al. 2006). Lack of antioxidant protection would render tumor cells more vulnerable to DNA damage, which during the development and growth of higher-grade ependymomas might be expressed as higher aneuploidy and higher malignancy.
Furthermore, AOE expression (for MnSOD, GLCL-C and TrxR) depended on the location of the tumor, as spinal tumors showed more intense staining patterns than tumors of the brain. The reason for this remains obscure, but may indicate either biologic differences between the tumors of these 61
two sites or alternatively, there may be some fundamental differences in the oxidative milieu between different compartments of the central nervous system. Furthermore, grade I myxopapillary ependymomas differ from grade II and III tumors in their typical conus- cauda filum terminale location which makes it possible to resect the tumor radically.
Positive AOE (except for GLCL-R) expression had correlation with a well-known inhibitor of apoptosis, Bcl-2. ROS induce apoptosis and AOEs counteract apoptosis and cell damage by neutralizing ROS. It seems interesting that those AOEs that affect apoptotic activity have upstream regulatory steps to that of Bcl-2. One theoretical explanation would be that when oxidative stress is below a critical threshold, AOEs are expressed to protect the cell from irreversible damage and immediate apoptosis is not appropriate. Finally, we discovered that there is an association between higher Trx expression and longer survival, as discussed later.
6.3. Carbonic anhydrases in pediatric brain tumors We found that several MBs and supratentorial PNETs in a group of 39 tumor samples express the CA isozymes CA II, CA IX and CA XII. The expression of CA II, CA IX and CA XII in the normal nervous system has been investigated in several previous studies. The localization of CA II is well documented in the normal human oligodendrocytes (Kumpulainen et al. 1983). Based on our previous studies, CA IX is not present in the normal human brain, except for choroids plexus (Haapasalo et al. 2006). CA XII mRNA expression has been shown to be very weak in the human brain by RT-PCR analysis (Haapasalo et al. 2008). In the mouse, immunohistochemistry has shown that CA XII is in the choroid plexus (Kallio et al. 2006). A recent study by Chiche et al. (2009) provided clear evidence that both CA IX and CA XII are functionally involved in tumor growth, including in renal cell cancer. In vivo experiments showed that CA9 gene silencing alone led to a 40% reduction in xenograft tumor volume, and the silencing of both CA9 and CA12 resulted in an 85% reduction in tumor volume. In medulloblastomas and PNETs we found that CA IX was found in the perinecrotic areas of the tumors whenever necrosis was present. Similar findings have been reported in astrocytomas (Haapasalo et al. 2006). Because necrosis is an uncommon feature and is not considered to be a significant prognostic factor in MBs, the induction of CA IX in MBs/PNETs may also involve hypoxia-independent mechanisms. Interestingly, CA II was, once again, found in the endothelium of neovessels. Thus, CA II may play an important functional role in tumor metabolism. The expression of CA XII in the tumor cells was associated with patient age, as previously reported for the expression of CA XII in patients with diffuse astrocytomas (Haapasalo et al. 2008). These findings reflect the fact that patient age is a significant factor that contributes to 62
carcinogenesis by several mechanisms, including CA expression, and that tumor phenotypes are different depending on the age of the patient. The expression of CA IX and apoptotic activity were associated with a poor prognosis, as discussed later. Although in the total tumor material CA II and CA XII did not reach statistical significance for use as prognostic indicators, CA II had a similar trend to that of CA IX. Furthermore, CA XII showed a significant correlation with survival in MBs.
6.4. Claudins in pediatric brain tumors In our material, claudins 2,3,5,7, and 10 were widely expressed in 61 ependymomas. CLDN4 was not expressed in our material. CLDNs seemed to be even more highly expressed than in the previous study (Hewitt et al. 2006). We found differences associated with location of the tumor and between primary and recurrent tumors in the expression of claudins. CLDNs 3 and 5 were more often found in tumors of the cerebrum than in the cerebellum or the spinal cord. Such regional variation in their expression may originate from the expression pattern of parental ependymal cells in the liquor space which may vary as has been shown in other tissues (Rahner et al. 2001, KiuchiSaishin et al. 2002). In addition, primary tumors showed more intense CLDN7 staining patterns compared with recurrent ones. Our results showed claudin 7 downregulation in the progression of ependymoma, but curiously, all patients with CLDN7 negative tumors survived compared to the positive group. CLDN 7 is a member of the family which is abundantly present in epithelial tumors (Soini 2005). However, depending on tumor type, claudin 7 expression may be over- or underexpressed. Downregulation in malignant melanoma, head and neck cancer and in breast cancer is reasonable considering the disruption of tight junctions leading to loss of cohesion and invasiveness (Krämer et al. 2000, Kominsky et al. 2003). Interestingly, CLDN7 is upregulated in hepatocellular carcinoma, ovarian epithelial cell carcinoma, prostate carcinoma or renal chromophobe carcinoma (Singh et al. 2010).
CLDN5 immunoreactivity in ependymomas associated with increased nuclear atypia, endothelial proliferation, mitotic rate and hypercellularity, higher proliferation, and tumor grade. Our study indicates that CLDN5 tends to be related to more aggressive tumors. In contrast, CLDN2 and 10 tend to display a better degree of differentiation and a better prognosis. Claudin 2 tended to be expressed in ependymomas with a better prognosis, even though no significant association was found. Claudin 2 is commonly present in epithelial tumors and, as with claudins 7 and 10, its presence has been associated with leakiness of the tight junctions (Soini 2005, Krause et al. 2008). Our study showed claudin 10 to be expressed in 11 % of cases, and expression tended to be located
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in low grade tumors with a better prognosis. Evidently claudins influence the growth of and differentiation in ependymomas.
The expression of claudins is partly regulated by epithelial-mesenchymal transition (EMT) associated transcription factors, such as snail1, slug, ZEB1 or TWIST (Martinez-Estrada et al. 2006). An inverse association between ZEB1 and claudins 1 and 2, and TWIST and claudin 5 has been shown in lung carcinoma (Merikallio et al. 2011). ZEB1 was inversely associated with claudin 2 in ependymoma, whereas claudins 5 and 10 showed a positive correlation with TWIST. Indeed, ependymomas contained a high frequency of ZEB1 expression compared to lung carcinomas, for instance (Merikallio et al. 2011). This may be due to the importance of ZEB1 in neural development. In mice, activation of ZEB1 mRNA expression is found in cells of the periventricular area (Yen et al. 2001). Thus, ZEB1 might serve other functions in ependymoma, not necessarily related to invasion type features like EMT which is actually a phenomenon related to epithelial tumors. TWIST expression was found in ependymomas with a lesser frequency. In glioblastoma cells, TWIST expression has been found to be related to a more aggressive behaviour and mesenchymal change (Mikheeva et al. 2010). Such a discrepancy may be due to the special nature of the ependymal cells and neoplasias which do not straightforwardly correspond to epithelial tumors.
6.5. The relationship between claudins and carbonic anhydrases Previous studies show that both claudins and carbonic anhydrases participate in the formation of tight junctions and the BBB. CA II is necessary for human vasculogenesis and formation of the BBB (Kida et al. 2006). Like claudins, CA IX is a cell adhesion molecule (CAM) (Závada et al. 2000) and seems to play a role in intercellular adhesion with its proteoglycan-like region (Svastová et al. 2003). CA IX can perturb E-cadherin-mediated cell-cell adhesion via interaction with β– catenin and potentially contribute to tumor invasion (Svastová et al. 2003).
Furthermore, CA IX is known to be hypoxia-inducible (Wykoff et al. 2000). Hypoxia has also been shown to regulate the barrier function of neural blood vessels by reducing the expression of claudin 5 in endothelial cells (Koto et al. 2007). This could indigate that claudins and carbonic anhydrases may be connected in multiple ways. AOEs and related Prxs are induced in hyperoxic conditions and protect cells from ROS. (Halliwell 1991, Rabilloud et al. 2002). Hypoxia is known to be one of the causes of treatment failure and poor outcome in a variety of adult malignancies by increasing resistance to radiotherapy and to cytotoxic agents (Teicher 1994, Brown et al. 2006). In addition, 64
hypoxia increases invasion, angiogenesis and metastasis (Tatum et al. 2006). Resistance to druginduced apoptosis in hypoxia has been found in pediatric tumors, e.g. neuroblastoma (Das et al. 2005, Hussein et al. 2006). Tyrosine kinase inhibitor, AZD 2171, has been studied initially in children with tumors including medulloblastoma, and one third of the patients showed growth delay of the tumor (Maris et al. 2008). Interestingly, CA IX inhibitors have been under intense investigation and promising results have been published in tumors like breast cancer (Robertson et al. 2004). In children, possible side-effects may be more severe and the exact biology of hypoxia and its clinical relevance in childhood tumors is still unclear. Thus, further studies will be needed before novel agents concerning hypoxia can be introduced into pediatric oncology.
6.6. Limitations of the study Small sample size is probably the major limitation of the study. Fortunately, children with brain tumors are not very common in Finland. This has necessitated certain procedures in our study, including recording different diagnostic and prognostic markers in statistical analyses. Additionally, there were disparate treatment modalities in the different hospitals, heterogeneous age variation and a rather long time interval between the first and last patients brain tumor operations.
Technical limitations included complex staining methods with numerous laboratory steps. It would be of interest to stain the tumors with different antibodies and verify the staining results of the thesis. Although staining protocols and TMA system have been widely and repeatedly used, one could utilize different laboratory steps and thus study the repeatability.
Despite small sample size, the thesis reached many statistically significant results concerning essential features of brain tumors, such as tumor behavior, diagnostics, and prognosis. In order to assess the clinical value of our findings, larger clinical trials with patients receiving standardised brain tumor treatments are required. This may, however, be a challenge since treatments are expected to develop when new data based on genetic profiling of individual patients is available.
6.7. Future prospects It is clear that brain tumors of children are rare, and thus collaboration is needed between reseachers. Others have found interesting and novel possible diagnostic and prognostic tools for patients with pilocytic astrocytoma, ependymoma, medulloblastoma, or PNET. The main question is which tool is clinically valid and cost-effective for clinical use.
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According to our study, Prx VI is a marker of recurrence-free survival in pilocytic astrocytomas. This is probably a result of the fact that Prx VI over-expression in pilocytic astrocytomas without the bias produced by radio- or chemotherapy decreases ROS activity and in this way might abrogate genomic instability and tumor progression. The tumor is histologically benign but the clinical outcome may sometimes be poor and thus, Prx VI negativity could be used to define patients with higher risk and could indicate that these patients should have a closer follow-up. There are only a few prognosticators found for patients with pilocytic astrocytoma (Haapasalo et al. 1999). In earlier studies, univariate statistical analyses found that partial resection, older patient age, and histology (Haapasalo et al. 1999), especially pilomyxoid tumor variant, (Fernandez et al. 2003) were associated with a worse prognosis.
According to our study, decreased Trx-expression in ependymomas is associated with worse outcome. Interestingly, a similar association between high AOE expression and better prognosis has been shown in breast cancer where peroxiredoxins III and IV expression was associated with better survival (Karihtala et al. 2003). Furthermore, in oligodendroglial or diffuse astrocytic brain tumors, high Trx was associated with a worse prognosis of the patients (Haapasalo et al. 2003, Järvelä et al. 2006), whereas in ependymomas it was associated with an improved prognosis. Such a phenomenon could also be ascribed to the protective function of Trx and other AOEs against oxidant damage which, in the case of AOE expressing ependymomas, would mean a higher genetic stability and less aggressive behavior. In addition, there are differences between the treatment of different tumor types and this might be one reason for the opposite relationship between AOEs and tumor behavior. Accordingly, AOEs have been shown to have an effect on anti-cancer drug resistance (Tew 1994, Kinnula and Crapo 2004). The stem cell/progenitor cell origin of ependymal cells is also different from that of astrocytes and oligodendrocytes (Linskey and Gilbert 1995), which might be one reason for differences, as well. Embryonic radial glia (RG) are neural progenitor cells that are likely to be the source of ependymomas independent of patient age. Astroglial cells with functional and molecular characteristics of RG persist in the supraventricular zone of the lateral ventricles and possibly the spinal cord, suggesting that some RG give rise to adult neural stem cells (Merkle et al. 2004, Barry and McDermott 2005). RG-derived stem cells may be the cells of origin of adult ependymomas.
In our study on medulloblastomas and PNETs, we found that CA II was, once again, found in the endothelium of neovessels. Thus, CA II may play an important functional role in tumor metabolism. CA II has been found to be a target molecule for dendritic cell therapy in melanoma patients 66
(Yoshiura et al. 2005). Further studies are, therefore, clearly warranted to evaluate the role of CA II as a possible therapeutic target not only in melanoma but also in other forms of cancer, including MBs/PNETs.
CA IX was found to be of prognostic value in medulloblastomas in our material. Similar findings have been reported previously in other tumors as well. The clear division of CA IX expression in normal and neoplastic cells makes CA IX a promising diagnostic and prognostic tool in various tumors. CA IX is associated with higher grade, necrosis, and worse outcome in breast tumors (Chia et al. 2001, Wykoff et al. 2001). A similar effect is seen in head and neck cancer (Beasley et al. 2001, Koukourakis et al. 2001), in bladder cancer (Hoskin et al. 2003), in cervix carcinoma (Loncaster et al. 2001), and in in RCC (Bui et al. 2003 and 2004). CA IX seems to have several inductors. It has also been shown that higher CA IX expression is associated with a more favourable overall survival in some tumors, such as RCC and in acute myeloid leukemia (AML). In RCC, the CA IX induction is associated with VHL-mutation and not with hypoxia as in most brain tumors (Patard et al. 2008). In AML the association is thought to be involved with immune system and Tcell response (Greiner et al. 2006).
Claudins did not reach statistically significant correlations with patient prognosis, but there were interesting trends between CLDNs and outcome of patients with ependymoma. Previous studies have shown that CLDNs have potential as prognosticators as well. Due to high specificity of CLDN expression in cancer, it has been suggested that claudins may represent useful molecular markers for many different cancers, such as CLDN3 in ovarian cancer (Lu et al. 2004) and CLDN10 as an independant prognosticator for recurrence of hepatocellular carcinoma (Cheung et al. 2005). CLDN5 expression is high in brain cancer in vascular endothelial cells, and thus may represent a target for antiangiogenic therapy (Hewitt et al. 2006). In addition, Nitta et al. (2003) have shown that knockout of CLDN5 results in a selective increase in paracellular permeability of small molecules, and thus makes CLDN5 a possible target for the development of drugs for this purpose.
In the future, children diagnosed with brain tumor will be more accurately stratified based on a combination of clinical variables and molecular profiles. Improved risk stratification will enable individualised therapies, which could be a combination of conventional treatment modalities and novel, targeted therapeutic approaches. These changes will hopefully result in improved survival without detriment to the quality of life.
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7
SUMMARY AND CONCLUSIONS
1. Altogether eleven different antioxidative enzymes were studied in pilocytic astrocytomas. All AOEs (MnSOD, GLCL-C, GLCL-R, Trx, TrxR, and Prx I-VI) were expressed in pilocytic astrocytomas suggesting that oxidative damage and consequent defence take place during the progression of the tumors. AOEs correlated with degenerative features and angiogenesis, possibly associating with reactive oxygen species derived cellular damage. Moreover, the expression of the AOEs was associated with each other in terms of concurrent activation of the enzymes. With the exception of manganese superoxide dismutase (MnSOD), a strong expression of AOEs was generally associated with higher cell proliferation. Prx VI seemed to have a positive association with a longer recurrence-free interval.
2. When five AOEs (MnSOD, GLCL-C, GLCL-R, Trx, TrxR) were studied in ependymomas, some AOEs seemed to be associated with tumor grade and location in ependymomas. Lower GLCL-C and GLCL-R expression was associated with higher tumor grade. MnSOD, GLCLC and TrxR expressions were significantly higher in tumors located in the spinal cord compared to those in the brain. Interestingly, decreased expression of Trx predicted worse outcome for the patients. This finding may have clinical relevance when planning the treatment modalities and follow-up for the patients.
3. Carbonic anhydrases II, IX, and XII were widely expressed in medulloblastomas and PNETs. CA XII was associated with young patient age. Interestingly, CA IX was found to be of prognostic importance in medulloblastomas and PNETs. Previous studies have shown that CA IX is an attractive target molecule for anticancer treatment. Additional studies will be needed to analyse whether CA IX could also be used in practice in the case of medulloblastomas and PNETs.
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4. Claudins 2, 3, 5, 7, and 10 were widely expressed in ependymomas. CLDN5 tends to be related to more aggressive tumors compared with CLDN2 and 10, which tend to display a better degree of differentiation and a better prognosis. There were also differences associated location of the tumor and between primary and recurrent tumors in the expression of claudins; CLDNs 3 and 5 were more often found in the cerebrum than in other sites and CLDN7 in primary tumors compared with recurrent ones. Evidently claudins influence the growth of and differentiation in ependymomas.
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8
ACKNOWLEDGEMENTS
The present study was carried out at the Department of Pathology, University Hospital of Tampere. My deep gratitude goes to the entire staff working there and the professor of the department, Timo Paavonen, M.D., Ph.D.
I am greatly indebt to my supervisors, Docent Hannu Haapasalo, M.D., Ph.D. for his consistent encouragement in the field of science, and Professor Ylermi Soini, M.D., Ph.D., the most efficient and reliable researcher. They both have introduced me to scientific thinking and working. Many thanks for your support and patience.
I thank Professor Seppo Parkkila, M.D., Ph.D. and Docent Anna-Kaisa Parkkila, M.D., Ph.D. for their work as the members of the supervisory committee and collaborators. They have both given me wise advice and new ideas.
I appreciate the time, constructive criticism and suggestions given by Professor Riitta Herva, M.D., Ph.D, and Docent Olli Lohi, M.D., Ph.D, the official referees of this manuscript.
I wish to thank my collaborators, Professors Hannu Kalimo, M.D., Ph.D, Vuokko Kinnula, M.D., Ph.D, Silvia Pastorekova, M.D., Ph.D, Jaromir Pastorek, M.D., Ph.D, Abdul Waheed, M.D., Ph.D, and William S. Sly, M.D., Ph.D. for their valuable co-work. I sincerely thank Docents Leo Paljärvi, M.D., Ph.D, Pauli Helén, M.D., Ph.D, Jukka Laine, M.D., Ph.D, and Anders Paetau, M.D., Ph.D for their expertise. I would like to thank also Sally Järvelä, M.D., Ph.D, Pauli Sallinen, M.D., Ph.D, Miia Jansson M.D., and Anssi Niemelä, M.D. Especially I acknowledge Miikka Korja, M.D., Ph.D, who has taught me that only the sky is the limit.
Special thanks to Mrs Reija Randen, Mrs Eila Pohjola, Ms Aija Parkkinen, Mrs Aulikki Lehmunen, Mrs Riitta Koivisto, Mr Jorma Lampinen, Mrs Päivi Koukkula and Mr Manu Tuovinen for skillful, technical assistance. I also thank Docent Mikko Arola, M.D., Ph.D., for his expertise on pediatric oncology in the third manuscript. I thank Nick Bolton, Ph.D. for revising the first study.Prakash Oommen, M.D. and Mike Nelson, Ph.D. are acknowledged for their help in revising the language of the second manuscript. The Proofreading247 Team is acknowledged for revising this thesis.
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My parents Liisa and Heimo I would like to thank for their love. They have encouraged me to study medicine and kept my feet on the ground when needed. I am grateful to my sisters Hannele and Katri and their families for support, kindness and being interested in my work. I warmly thank all my friends for great moments and relaxing company.
This study was financially supported by the Nona and Kullervo Väre Foundation, the Finnish Medical Society, Competitive Research Funding of the Tampere Medical Research Fund of Tampere University Hospital (Grants 9L067, 9J013 and 9M010), by the Irja Karvonen Cancer Trust, by the Orion-Farmos Research Foundation, the EVO foundation of the Northern Savo district, the Finnish Cancer Society, the anti-tuberculous association of Finland, EU 6th Framework programme (DeZnIT), and the Kevo funding of Kuopio University Hospital. They are all acknowledged.
And the last and the dearest thanks go to my husband, Joonas. Never have I met such a person, who is so enthusiastic about research. Joonas has pushed me forward when needed and supported me every step of the way. There are no words to describe the gratitude. Thank you for your patience and love.
Tampere, August 2011
Kristiina Nordfors
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Peroxiredoxins and antioxidant enzymes in pilocytic astrocytomas Original ©2007 Dustri-Verlag Dr. K. Feistle ISSN 0722-5091
K. Nordfors1,2, J. Haapasalo1,2, P. Helén3, A. Paetau4, L. Paljärvi1, H. Kalimo4,5, V.L. Kinnula6, Y. Soini7 and H. Haapasalo1 1Department
of Pathology, Center for Laboratory Medicine, Tampere University Hospital, Tampere, 2Faculty of Medicine, University of Turku, 3Unit of Neurosurgery, Tampere University Hospital, Tampere, 4Department of Pathology, Helsinki University Hospital, Helsinki, Finland, 5Department of Neuropathology, University of Uppsala, Sweden, 6Department of Medicine, Division of Pulmonary Medicine, University of Helsinki, 7Department of Pathology, Oulu University Hospital, Oulu, Finland Peroxiredoxins in pilocytic astrocytomas
Key words antioxidant – antioxidative enzymes – peroxiredoxins – pilocytic astrocytoma – prognosis
Received August 13, 2006; accepted in revised form January 9, 2007 Correspondence to K. Nordfors, MB Department of Pathology, Center for Laboratory Medicine, Tampere University Hospital, P.O. Box 2000, FI-33521 Tampere, Finland
[email protected].
Abstract. Objective: Peroxiredoxins are antioxidant enzymes (AOEs), which are redox-regulated thiol proteins with potential effects on the growth, invasion and drug resistance of neoplastic cells. In this study, their biology and clinical significance were examined in pilocytic astrocytomas (PAs). Material and methods: The expression of peroxiredoxins (Prx I-VI) was investigated in 105 PAs by the means of immunohistochemistry and compared with the expression of selected other antioxidant enzymes, cell proliferation, angiogenesis, apoptosis, p53, histopathology and patient survival. Results: Peroxiredoxins were strongly expressed in general suggesting that oxidative damage and consequent defense takes place during the progression of pilocytic astrocytomas. In agreement with this hypothesis, several other AOEs correlated with the degenerative features and angiogenesis possibly associated with reactive oxygen species-derived cellular damage. Moreover, the expression of the AOEs was associated with each other indicating a concurrent activation of the enzymes. With the exception of manganese superoxide dismutase (MnSOD), a strong expression of AOEs was generally associated with higher cell proliferation. Prx VI seemed to have a positive association with a longer recurrence-free interval while other AOEs had no association with patient survival. Many AOEs, such as MnSOD, induce chemo- and radioresistance and are highly elevated in aggressive malignancies. PAs lack this confounding factor, and these tumors are treated only by surgery. Conclusions: Taken together, the results of this study on pilocytic astrocytomas suggest that the levels of Prxs and other AOEs and their related thiol proteins are generally strongly expressed in these tumors. At least Prx VI can contribute to tumor behavior which can make it a potential prognostic factor.
Introduction Antioxidant enzymes (AOEs) regulate the cellular redox state and constitute the major cellular protection factors against reactive oxygen species (ROS). These (e.g. O2–, H2O2, OH–) are harmful, since they cause oxidative damage to cellular proteins, lipids and genetic material. Low levels of ROS also modulate cell proliferation and apoptosis, and activate/induce the synthesis of growth factors [Kinnula et al. 2004]. Superoxide dismutases (SODs), glutathione-related enzyme systems such as glutamate cysteine ligase and catalase are the major antioxidant enzymes in mammalian cells. In addition to these, other major regulators of the cellular redox state include the thioredoxin (Trx) and peroxiredoxin (Prx) systems. These enzymes are also found in malignant tumors, inducing resistance of tumor cells to cytotoxic drugs and radiation [Kinnula and Crapo 2004], but possibly also independently contributing to tumor growth and invasion. Prxs occur in a wide variety of organisms from prokaryotes to mammals. They are present in various cellular compartments and reduce peroxides to the corresponding alcohol (or water), just like the other peroxidases. However, the Prx family differs in some respects from other groups of peroxidases and other antioxidant enzymes. Peroxiredoxins act both as a cosubstrates and peroxidases. Each Prx has a unique function and also interacts with other family members. They are expressed in different cell compartments in-
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cluding the cytosol, nucleus and mitochondria and also extracellularly [Fujii and Ikeda 2002]. Prx I and II are cytosolic proteins, Prx III is present in mitochondria and Prx IV in lysosomes, endoplasmic reticulum and extracellularly. Prx V is located in peroxisomes and mitochondria, and because of its location it is thought to have a more important role in protection against ROS than other Prxs. Prx VI is found at high concentrations, especially in the lung [Kinnula et al. 2002a]. Prxs are also expressed in the brain. In normal brain tissue, Prx I is expressed primarily in astrocytes and Prx II in neurons [Sarafian et al. 1999]. There are changes of Prx II expression, e.g. in Parkinson’s and Alzheimer’s diseases and in Down’s syndrome [Basso et al. 2004, Kim et al. 2001, Krapfenbauer et al. 2003]. Prx III has been found to protect hippocampal neurons from excitotoxic injury in vivo [Hattori et al. 2003]. Our previous study suggested that manganese SOD (MnSOD), thioredoxin (Trx), thioredoxin reductase (TrxR) and the catalytic (GLCL-c) and regulatory (GLCL-r) subunits of glutamate cysteine ligase (g-glutamylcysteinesynthetase) play an important role in the pathogenesis of diffusely infiltrating astrocytomas [Haapasalo et al. 2003]. In that paper, we compared the expression of these enzymes in a large series of diffuse astrocytomas and in a small series of pilocytic astrocytomas. In the present study, we principally investigate the expression of Prx I – VI in pilocytic astrocytomas in central nervous system. Pilocytic astrocytomas are one of the most common tumors in children, and in contrast to diffuse supratentorial astrocytomas in adults, they are often located in the cerebellum. They also have a totally different molecular genetic background. Our principal aim was to study the biology of peroxiredoxins in pilocytic astrocytomas, and our hypothesis was that they have a role in the pathogenesis of benign tumors. This has not been previously thoroughly examined. In addition, pilocytic astrocytomas offer a model without the bias produced by radiation or chemotherapy. We evaluated Prxs in association with age, survival, histopathological and molecular pathological features, including other antioxidant enzymes, in a series of 105 pilocytic astrocytomas.
Methods Immunohistochemistry of Prxs and other AOEs The immunostaining procedure was as follows. 4 m thick sections were cut from the microarray blocks, which were then deparaffinized in xylene and rehydrated in a descending ethanol series. In order to enhance immunoreactivity, the sections were incubated in 10 mM citrate buffer (pH 6.0), boiled in a microwave oven for 2 min at 850 W, and after that for 8 min at 350 W. Endogenous peroxidase activity was eliminated by incubation in 0.1% hydrogen peroxide in absolute methanol for 10 min. The polyclonal anti-Prx-antibodies were a gift from Dr. Kang (The Center for Cell Signalling Research and Division of Molecular Sciences, Ewha Womans University, Seoul, South Korea). Dilutions of the primary antibodies were 1 : 1,500 for Prx I, 1 : 1,000 for Prx II, 1 : 500 for Prx III, 1 : 1,000 for Prx IV and 1 : 2,000 for Prxs V and VI. Antibodies against other AOEs were as follows: polyclonal rabbit anti-human antibody to MnSOD (a gift from Professor J.D. Crapo, National Jewish Medical Center, Denver, CO, USA, dilution 1 : 1,000), rabbit polyclonal anti-human antibodies to GLCL-c and GLCL-r (a gift from Dr. Kavanagh, University of Washington, Seattle, WA, USA, dilution 1 : 1,000 for both), an affinity-purified goat-polyclonal human Trx antibody (American Diagnostica, Greenwich, CT, USA, dilution 1 : 200) and antibody to TrxR (a gift from Dr. Arne Holmgren, Karolinska Institutet, Stockholm, Sweden, dilution 1 : 1,000), which was the gammaglobulin fraction of a polyclonal rabbit anti-rat antibody directed against cytosolic TrxR in rat liver [Mustacich and Powis 2000, Nakamura et al. 1996]. Immunostaining of Prxs I – VI, MnSOD, GLCL-c, GLCL-r and TrxR was carried out using Histostain-Plus Kits (Zymed Laboratories Inc., South San Francisco, CA, USA) and the chromogen was aminoethyl carbazole (AEC) (Zymed Laboratories Inc.). For Trx, a biotinylated secondary anti-goat antibody was applied, followed by avidin-biotin peroxidase complex (both from Dakopatts, Glostrup, Denmark). Color was developed using 3,3’-diaminobenzidine, and the sections were lightly counterstained with hematoxylin and
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mounted in Eukitt (Kindler, Freiburg, Germany). Replacement of the primary antibody with phosphate-buffered saline (PBS) at pH 7.2, or with goat IgG immunoglobulin isotype (Zymed Laboratories, Inc., San Francisco, CA, USA) was carried for negative controls. The intensity of the immunostainings with all the antibodies was evaluated by dividing the staining reaction in 3 groups: 1 = weak staining intensity, 2 = moderate staining intensity, 3 = strong staining intensity. The quantity of the immunostaining was evaluated as follows: 0 = no positive immunostaining, 1 = < 25% of tumor cells showing positivity, 2 = 25 – 50% of tumor cells showing positivity, 3 = > 50% of tumor cells showing positivity. A combined score for the immunostaining, based on both qualitative and quantitative immunostaining was composed by adding both the qualitative and quantitative score which was then divided in 4 main groups: – = no immunostaining; score 0, + = weak immunostaining; scores 1 – 2, ++ = moderate immunostaining, scores 3 – 4, +++ = strong immunostaining, scores 5 – 6.
Proliferation index, p53 and apoptosis Ki-67MIB-1 and p53 were immunostained by using a standard streptavidin-biotin immunoperoxidase technique and scored as previously described [Haapasalo et al. 1999]. Apoptosis was detected by using ApopTagTM In Situ Apoptosis Detection Kits (Oncor Inc., Gaithersburg, MD, USA) as described previously [Haapasalo et al. 1999]. The microscope-based image analysis system (CAS200 Software, Becton Dickinson and Co., San Jose, CA, USA), equipped with 2 cameras for images of immunopositive (brown) and immunonegative (green) areas in nuclei, computed the proliferation indices from whole sections and the Ki-67MIB-1 labelling index (LI) [Haapasalo et al. 1999, Sallinen et al. 1994]. Tissues with unequivocal p53 staining of neoplastic nuclei (> 1/10 high power fields) were regarded as immunopositive [Haapasalo et al. 1993]. Apoptotic index was scored as percentages of ApopTag-immunoreactive cancer cell nuclei [Haapasalo et al. 1999].
Statistical analysis SPSS for Windows software (Chicago, IL, USA) was used for statistical analysis. The significance of associations was determined using Chi-square and Mann-Whitney tests. Kaplan-Meier curves and the log rank test were used in survival analysis.
Material The tumor specimens had been fixed in 4% phosphate-buffered formaldehyde, after which they had been processed into paraffin blocks. First, all HE-stained slides of the tumors were evaluated by two neuropathologists (HH together with HK, AP or LP), and histopathological typing and grading were carried out according to WHO criteria [WHO 2000]. Two neuropathologists (HH and HK) evaluated the presence or absence of the major histological features of pilocytic astrocytomas: atypia, necrosis, endothelial proliferation, vascular hyalinization, perivascular lymphocytes, cystic pattern, granular bodies and calcification. One neuropathologist (HH) pinpointed one histologically representative tumor region in each pilocytic astrocytoma. From this tumor region a sample was included in multi-tissue microarray blocks representing 105 astrocytic tumors [Kononen et al. 1998]. The microarray blocks were constructed with a custom-built instrument (Beecher Instruments, Silver Spring, MD, USA). The diameter of the tissue core in the microarray block was 600 mm.
Patients Brain tumor samples were obtained from 105 patients (49 females and 56 males) operated on because of pilocytic astrocytomas at the University Hospitals of Tampere, Helsinki, Kuopio and Turku, Finland, during 1980 – 1999. Age varied from newborn to 66 (median = 10, Mean ± SD = 15 ± 15) years. None of the patients received preoperative radiation and/or chemotherapy. The topography of the tumors was as follows: 85% were in the cerebellum, 5% in the cerebrum, 3% in the brainstem and spinal cord, and 7% in cra-
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Table 1. Peroxiredoxin expression in pilocytic astrocytomas (96 primaries and 9 residives). 0 = negative, 1,2,3 = weak, moderate and strong immunopositive, respectively (p = n.s., c2 test). Prx I Expression
Prx II
Prx III
Prx IV
Prx V
Prx VI
Prim
Resid
Prim
Resid
Prim
Resid
Prim
Resid
Prim
Resid
Prim
Resid
0
1
0
0
0
0
0
7
0
12
0
1
1
1
2
1
3
0
26
2
29
1
35
1
14
2
2
9
2
20
1
47
1
45
5
20
3
36
2
3
81
6
70
8
20
6
10
3
25
5
43
4
Figure 1. A: Prx I staining showing moderate cytoplasmic positivity in pilocytic astrocytoma with microcystic degeneration (× 630). B: Strong cytoplasmic Prx II positivity in densely packed astrocytic cells (× 630). C: Strong, dot-like Prx III cytoplasmic immunopositivity probably due to the mitochondrial location (× 630). D: MnSOD staining showing strong cytoplasmic immunopositivity (× 630).
nial nerves (mostly the optic nerve). There were 96 patients with primary tumors available and 9 patients with recurrences. Follow-up time of primary tumors varied from 0.5 – 19.5 (Mean ± SD = 7.0 ± 4.1) years.
Results The distribution of immunostaining (no immunostaining (0), weakly (1), moderately (2), strongly (3) positive) of Prxs in 96 primary and 9 recurrent tumors is shown in Table 1. In an area adjacent to tumor moderate expression of MnSOD was seen. Also weak
positivity for Prx II was observed while other AOEs were negative. In tumor cells the immunostaining was cytoplasmic, but Prxs I, II, IV, V and VI also showed variable nuclear positivity. Most (98%) of all tumors expressed positivity for Prx I (84% strongly, 11% moderately and 3% weakly). All tumors showed positivity for Prx II (4% strongly, 20% moderately and 76% weakly) and also for Prx III (26% strongly, 47% moderately, 27% weakly). For Prx IV, 92% of cases were positive (13% strongly, 49% moderately, 30% weakly) and 87% were positive for Prx V (29% strongly, 23% moderately and 35% weakly). Strong Prx VI expression was found in 45%, moder-
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Peroxiredoxins in pilocytic astrocytomas
Figure 2. The distribution of Prx immunostaining in 105 pilocytic astrocytomas. 0 = no immunostaining present; 1 = weak immunostaining; 2 = moderate immunostaining; 3 = strong immunostaining.
Figure 3. Kaplan-Meier curve showing recurrencefree survival in relation to Prx VI immunostaining in pilocytic astrocytomas (p = 0.032, log-rank test).
ate expression in 37% and weak expression in 15% of all tumors. The immunoreactivity patterns of Prxs in pilocytic astrocytomas are shown in Figure 1A,B,C and in Figure 2. An example of MnSOD immunoreactivity in a pilocytic astrocytoma is shown in Figure 1D. The expression patterns of Prxs, considered in the following analyses as either negative (no and weak immunostaining) or positive (moderate and strong immunopositivity) were next correlated with each other. When compared with each other there were associations between Prx I expression and that of Prx II (p < 0.001), Prx IV (p = 0.010) and Prx VI (p < 0.001, c2 test). In addition to Prx I, Prx II
was associated with Prx IV (p = 0.002) and Prx VI (p < 0.001). Similarly, Prx III correlated with Prx IV (p = 0.004). Prx IV correlated with all other Prxs (Prx V, p = 0.007; Prx VI, p = 0.010). Expression of Prx II, III, IV, GLCL-c and TrxR was associated with vascular pathology (endothelial proliferation, vascular hyalinization and perivascular lymphocytes), whereas Prx I, II and TrxR correlated with degenerative features (cystic pattern, granular bodies, calcification) (Table 2). Expression of GLCL-r and TrxR correlated significantly with an aggressive tumor growth pattern (atypia and necrosis) (Table 2). Proliferation by MIB-1 was greater in Prx VI- and TrxR-positive tumors (Prx VI: negative tumors, 1.8 ± 3.1 (Mean ± SD), positive tumors 3.0 ± 3.1, p = 0.037, TrxR: negative tumors, 2.2 ± 3.1, positive tumors, 3.5 ± 3.0, p = 0.028, Mann-Whitney test). However, MnSOD positivity was associated with lower proliferative activity (negative tumors, 3.3 ± 3.2, positive tumors 2.2 ± 2.9, p = 0.039, Mann-Whitney test). There was no correlation between AOE status and p53 immunopositivity or TUNEL (apoptosis) positivity (Mann-Whitney test). When clinical features were compared with the expression of Prx and other AOEs, MnSOD and GLCL-c positivity increased significantly with increasing patient age (p = 0.022 and p = 0.010, respectively, MannWhitney test). In addition, the expression of MnSOD was significantly lower in recurrences (p = 0.019, Mann-Whitney test). No such differences were seen as regards Prxs. Additionally, there was no association between the location of the tumors and the enzyme expression (c2 test). All 96 patients with primary pilocytic astrocytomas were included in survival analysis. Infrequent enzyme negativity of Prx I and II did not allow the possibility of survival analysis as regards these enzymes. Tumors immunopositive for Prx VI seemed to be associated with significantly better recurrence-free survival when compared with immunonegative tumors (p = 0.032, log-rank test) (Figure 3). However, when patient age, Ki-67 (MIB-1), location (cerebellum versus other location) and Prx VI immunopositivity were included to multivariate analysis, none of these factors predicted independently recurrence-free sur-
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Table 2. Peroxiredoxins and other antioxidant enzymes by histology in pilocytic astrocytomas (N=65, p = p-value, c2). neg = enzyme-negative; pos = enzyme-positive.
Prx I neg
pos
–
0
5
+
1
59
–
1
60
+
0
4
Prx II p
neg
pos
0
5
2
58
2
59
0
4
2
20
0
43
1
41
1
22
2
53
0
10
1
2
1
61
0
2
1
22
1
53
1
10
Prx III p
neg pos
Prx IV p
neg
pos
3
2
26
33
28
32
1
3
12
9
17
26
19
23
10
12
28
26
1
9
2
1
27
34
0
1
13
10
23
30
6
5
Prx V p
neg
pos
34
13
13
4
45
15
2
2
30
10
17
7
38
14
9
3
43
15
4
2
4
4
43
13
6
3
11
4
42
15
5
2
Prx V p
neg pos
p
atypia
n.s.
n.s.
2
3
16
44
18
43
0
4
10
12
8
35
10
32
8
15
16
39
2
8
1
2
17
45
1
1
10
13
13
41
5
6
n.s.
n.s.
n.s.
2
3
10
51
11
51
1
3
4
18
8
36
10
33
2
21
12
44
0
10
1
2
11
52
0
2
5
19
11
44
1
10
n.s.
necrosis
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
endothelial proliferation –
1
20
+
0
44
n.s.
0.046
0.023
n.s.
n.s.
n.s.
vascular hyalinization –
1
42
+
0
22
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
perivascular lymphocytes –
1
54
+
0
10
–
1
2
+
0
62
–
0
5
+
0
74
–
1
53
+
0
11
n.s.
n.s.
n.s.
0.015
n.s.
n.s.
cystic pattern
0.000
0.002
n.s.
n.s.
n.s.
n.s.
granular bodies
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
calcification
n.s.
n.s.
n.s.
vival (Cox multivariate analysis). In overall survival analysis none of the enzymes was of prognostic significance.
Discussion There is compelling evidence that ROS are linked to the pathogenesis and behavior of human malignancies [Kinnula and Crapo 2004, Kinnula et al. 2004]. A major hypothesis explaining the importance of oxidants and imbalance of the cellular redox state is an altered pro-oxidant intracellular environment that facilitates mutations and/or inactivation of tumor suppressor genes and activates on-
n.s.
n.s.
n.s.
cogenes. This leads to changes in cell growth and apoptosis, and finally the development of neoplasia [Burdon 1995, Osada and Takahashi 2002]. For example, it has been shown that the cellular redox state regulates several pathways important in carcinogenesis, including cMyc-, p53- and FAS-mediated apoptosis and ras-mediated epidermal growth factor (EGF) receptor-dependent angiogenesis [Casanova et al. 2002, Kasahara et al. 1997, Suhara et al. 1998]. It has also been shown that H2O2 produced in EGF-stimulated cells governs both the accumulation of PI 3,4,5triphosphate and activation of protein kinase Akt. It also appears that Prx II participates in growth factor signalling by removing H2O2
NP-6036 / 22.03.2007
7
Peroxiredoxins in pilocytic astrocytomas
[Kwon et al. 2004]. Several AOEs, including Prxs, also influence tumor resistance by inducing chemo- and radio resistance of tumor cells [Chung et al. 2001]. Normal tissue is protected against ROS by AOEs, including Prxs. We and others have shown that Prx proteins are expressed in neoplasms, e.g. in follicular thyroid carcinoma [Chung et al. 1999], malignant mesothelioma [Kinnula et al. 2002b], lung [Lehtonen et al. 2004] and breast cancer [Karihtala et al. 2003]. However, the expression of Prx enzymes has not previously been studied in pilocytic astrocytomas. These tumors usually have, if properly treated, a benign clinical course showing many histological features of degeneration/regression in addition to proliferative activity [Haapasalo et al. 1999], and in their treatment no radiation or chemotherapy is used. Consequently, influence of antioxidant enzymes on tumor behavior can be studied in these tumors without the bias produced by such treatments. According to our data, peroxiredoxin expression is a common phenomenon in pilocytic astrocytomas. Prx enzymes and other antioxidant enzymes are closely associated with tumor cell proliferation, immunopositive tumors usually having a greater proliferation rate. Although there was no association between apoptosis rate and AOEs, histopathologically degenerative and regressive features were often associated with Prx expression. The same was true for vascular pathology, e.g. endothelial proliferation as a sign of angiogenesis. Some AOEs were associated with histologically aggressive tumor features. Even though Prx VI was associated with higher cell proliferation, it seemed to be associated with significantly better recurrence-free survival. This might be due to association between Prx VI expression and young patient age. In the present study of pilocytic astrocytomas, Prxs and other AOEs were commonly expressed simultaneously. This suggests that the neoplastic tissue suffers from a constant oxidative load, leading to simultaneous upregulation of these enzymes. Co-expression and a relationship between Prxs have also been observed in previous studies of other neoplasms, indicating that such a phenomenon is common in tumors [Karihtala et al. 2003, Kinnula et al. 2002b]. An increased oxidative load may lead to degenerative features and cellular destruction in tumor tissue.
Some degenerative features, especially a cystic pattern, were closely associated with the expression Prx I, Prx II and TrxR. Cystic degeneration with Rosenthal fibres and calcification (often seen in pilocytic astrocytomas) implies cell death, which may be a sign of previous hypoxic periods. Elevated levels of AOEs in pilocytic astrocytomas with such degenerative features indicate the tendency of these tumors to suffer from oxidative stress and increased defence against ROS-mediated tissue damage. We also found a significant association between AOE expression (Prx II, III, IV, GLCL-c and TrxR) and vascular pathology, e.g. endothelial vascular proliferation. Hypoxia and ROS induce HIF-a, which stimulates angiogenic factors such as vascular endothelial growth factor (VEGF) [Kinnula and Crapo 2004]. In diffuse astrocytomas it is most probably hypoxia that induces the expression of VEGF in tumor cells. This binds to VEGF receptors (VEGFRs) in endothelial cells, leading to endothelial vascular proliferation. In pilocytic astrocytomas endothelial vascular proliferation is also a common feature and could be a consequence of ROS and AOE induction, but without such a sinister implication as in diffuse astrocytomas. In the present study, with 105 tumors, Prx VI and TrxR were significantly associated with higher proliferation indices. MnSOD, a putative tumor suppressor in the group of AOEs, has been widely investigated in malignant cells. Isolated overexpression of MnSOD decreases the growth, proliferation and invasion of cultured malignant cells [Bravard et al. 1992, Li et al. 1995, St Clair et al. 1992, Yan et al. 1996, Zhong et al. 1997] (reviewed by [Kinnula and Crapo 2004]). Interestingly, in the present study, MnSOD was overexpressed in those pilocytic astrocytomas that had a significantly decreased proliferation rate. This is in line with its putative tumor suppressor function, which is further supported by the fact that in the recurrences the expression of MnSOD was significantly decreased. In contrast to this, in our previous study, MnSOD positivity was associated with a significantly higher proliferation rate in 380 diffusely infiltrating astrocytomas [Haapasalo et al. 2003]. In that study, we concluded that down-regulation of MnSOD might be an event in the progression of diffusely infiltrat-
NP-6036 / 22.03.2007
8
Nordfors, Haapasalo, Helén et al.
ing astrocytomas. This might be a result of the loss of chromosome region 6q, the locus of the MnSOD gene, which frequently takes place in diffuse astrocytomas [Sallinen et al. 1997]. Such genetic changes do not occur in pilocytic astrocytomas, which generally have either a normal karyotype or only a few major cytogenetic aberrations. Many AOEs, such as MnSOD and Prxs, increase chemo- and radio resistance of tumor cells. Thus, in this context, therapy may also influence tumor behavior. Pilocytic astrocytomas are treated by surgery alone, while diffuse astrocytomas are also treated by means of radiotherapy and occasionally by means of chemotherapy. In diffuse astrocytomas the expression of AOEs may thus, in the late progression of the disease, induce more aggressive behavior. This phenomenon could also partly explain the fact that diffuse astrocytomas expressing MnSOD or Trx are associated with worse prognosis than pilocytic astrocytomas expressing the same molecules. In contrast, Prx VI was linked to significantly better recurrence-free survival in pilocytic astrocytomas. This is probably a result of the fact that Prx VI overexpression in pilocytic astrocytomas without the bias produced by radio- or chemotherapy decreases ROS activity and in this way might abrogate genomic instability and tumor progression. Prx VI could be used as a prognostic marker when planning therapy or follow-up for patients with pilocytic astrocytomas. Previously, only a few factors have been shown to be of prognostic significance in this disease [Haapasalo et al. 1999]. In earlier studies univariate statistical analyses revealed that partial resection, older patient age and histology [Haapasalo et al. 1999], especially pilomyxoid tumor variant [Fernandez et al. 2003] were associated with a worse prognosis. Overall, the role of antioxidant enzymes especially MnSOD and peroxiredoxins in human tumors is poorly understood and partly controversial. Our study suggests that several AOEs and Prxs, at least Prx VI probably play a role in tumor growth/invasion and, thus, in prognostication. However, an immunohistochemical study cannot give any suggestion about the functional activity or possible overoxidation of these proteins. In addition, the prognostic value of Prx VI needs to be evaluated with a large number of recurrent pilocytic
astrocytomas, perhaps with more thorough MRI and clinical analysis to differentiate the recurrent and residual tumors.
Conclusion In conclusion, our study shows that peroxiredoxins and other antioxidant enzymes are expressed in pilocytic astrocytomas and may modulate their behavior. Co-expression of several antioxidant enzymes was common in these tumors. Antioxidant enzymes had a connection with angiogenesis and cell proliferation. They were usually associated with an elevated proliferation rate, but the putative tumor suppressor MnSOD depressed proliferative activity. Most interestingly, degeneration and regressive features were associated with AOEs but seemed not to be associated with p53-mediated apoptotic activity. Elevated levels of AOEs in pilocytic astrocytomas with degenerative features indicate the tendency of these tumors to suffer from oxidative stress and increased defence against ROS-mediated tissue damage.
Acknowledgments We thank Mrs Reija Randen, Mrs Riitta Koivisto, Mr Jorma Lampinen, Mrs Päivi Koukkula and Mr Manu Tuovinen for skillful technical assistance. This work was supported by grants from the Cancer Society of Finland and the Medical Research fund of Tampere University Hospital.
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tumor suppressor PTEN in cells stimulated with peptide growth factors. Proc Natl Acad Sci. 2004; 101: 16419-16424. Lehtonen ST, Svensk AM, Soini Y, Paakko P, Hirvikoski P, Kang SW, Saily M, Kinnula VL. Peroxiredoxins, a novel protein family in lung cancer. Int J Cancer. 2004; 111: 514-521. Li JJ, Oberley LW, St Clair DK, Ridnour LA, Oberley TD. Phenotypic changes induced in human breast cancer cells by overexpression of manganese-containing superoxide dismutase. Oncogene. 1995; 10: 1989-2000. Mustacich D, Powis G. Thioredoxin reductase. Biochem J. 2000; 346: 1-8. Nakamura M, Watanabe T, Klangby U, Asker C, Winman K, Yonekawa Y, Kleinhues P, Ohgaki O’Brien ML, Tew KD. Glutathione and related enzymes in multidrug resistance. Eur J Cancer. 1996; 32: 967-978. Osada H, Takahashi T. Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer. Oncogene. 2002; 21: 7421-7434. Sallinen PK, Haapasalo HK, Visakorpi T, Helen PT, Rantala IS, Isola JJ, Helin HJ. Relation of Ki-67 (MIB-1), PCNA and S-phase fraction with patient survival in formalin-fixed, paraffin-embedded astrocytoma material. J Pathol. 1994; 174: 275-282. Sallinen SL, Sallinen P, Haapasalo H, Kononen J, Karhu R, Helen P, Isola J. Accumulation of genetic changes is associated with poor prognosis in Grade II astrocytomas. Am J Pathol. 1997; 151: 1799-1807. Sarafian TA, Verity MA, Vinters HV, Shih CC, Shi L, Ji XD, Dong L, Shau H. Differential expression of peroxiredoxin subtypes in human brain cell types. J Neurosci Res. 1999; 56: 206-212. St Clair DK, Wan XS, Oberley TD, Muse KE, St Clair WH. Suppression of radiation-induced neoplastic transformation by overexpression of mitochondrial superoxide dismutase. Mol Carcinog. 1992; 6: 238-242. Suhara T, Fukuo K, Sugimoto T, Morimoto S, Nakahashi T, Hata S, Shimizu M, Ogihara T. Hydrogen peroxide induces upregulation of Fas in human endothelial cells. J Immunol. 1998; 160: 4042-4047. Yan T, Oberley LW, Zhong W, St Clair DK. Manganese-containing superoxide dismutase overexpression causes phenotypic reversion in SV40-transformed human lung fibroblasts. Cancer Res. 1996; 56: 2864-2871. Zhong W, Oberley LW, Oberley TD, St Clair DK. Suppression of the malignant phenotype of human glioma cells by overexpression of manganese superoxide dismutase. Oncogene. 1997; 14: 481-490.
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Nordfors et al. BMC Cancer 2010, 10:148 http://www.biomedcentral.com/1471-2407/10/148
Open Access
RESEARCH ARTICLE
The tumour-associated carbonic anhydrases CA II, CA IX and CA XII in a group of medulloblastomas and supratentorial primitive neuroectodermal tumours: an association of CA IX with poor prognosis Research article
Kristiina Nordfors*1, Joonas Haapasalo1, Miikka Korja2,3, Anssi Niemelä1, Jukka Laine4, Anna-Kaisa Parkkila5, Silvia Pastorekova6, Jaromir Pastorek6, Abdul Waheed7, William S Sly7, Seppo Parkkila8 and Hannu Haapasalo1
Abstract Background: Medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumours (PNETs) are the most common highly aggressive paediatric brain tumours. In spite of extensive research on these tumours, there are only few known biomarkers or therapeutic target proteins, and the prognosis of patients with these tumours remains poor. Our aim was to investigate whether carbonic anhydrases (CAs), enzymes commonly overexpressed in various tumours including glioblastomas and oligodendrogliomas, are present in MBs and PNETs, and whether their expression can be correlated with patient prognosis. Methods: We determined the expression of the tumour-associated carbonic anhydrases CA II, CA IX and CA XII in a series of MB/PNET specimens (n = 39) using immunohistochemistry. Results: Endothelial CA II, cytoplasmic CA II, CA IX and CA XII were expressed in 49%, 73%, 23% and 11% of the tumours, respectively. CA II was detected in the neovessel endothelium and the tumour cell cytoplasm. CA IX was mainly expressed in the tumour cells located in perinecrotic areas. CA XII showed the most homogenous distribution within the tumours. Importantly, CA IX expression predicted poor prognosis in both univariate (p = 0.041) and multivariate analyses (p = 0.016). Conclusions: We suggest that CA IX should be considered a potential prognostic and therapeutic target in MBs and PNETs. Background Medulloblastomas (MBs) and primitive neuroectodermal tumours (PNETs) are classified as embryonal tumours of the central nervous system (CNS) and histologically correspond to WHO grade IV [1]. One viewpoint postulates that these tumours show a common ontogeny, arising from related progenitor cells that have the potential for divergent neuroepithelial differentation. However, in recent years molecular genetic analyses have demonstrated different genetic profiles for these tumours [1]. It * Correspondence:
[email protected] 1
Department of Pathology, Tampere University Hospital, Tampere, Finland
Full list of author information is available at the end of the article
has been proposed that MBs originate from the neoplastic transformation of granule cell precursors in the cerebellum via deregulation of molecular pathways involved in normal cerebellar development [2,3]. Correspondingly, PNETs arise in the cerebral hemispheres, brain stem or spinal cord. The neuroepithelial tumour cells of a PNET may be undifferentiated or poorly differentiated. In addition, the tumour cells may have aberrant differentiations, including neuronal, astrocytic and ependymal lines. MB is the most common childhood malignant tumour of the central nervous system and accounts for 12-25% of all paediatric CNS tumours. It is very rare in adults, accounting for only 0.5-1% of brain tumours [4]. The
© 2010 Nordfors et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
BioMed Central Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nordfors et al. BMC Cancer 2010, 10:148 http://www.biomedcentral.com/1471-2407/10/148
main defective cell signalling pathways involved in the development of MB include the Hedgehog and Wnt pathways, but the exact molecular mechanisms contributing to tumourigenesis in both MB and PNET are still poorly understood [5]. MBs are sensitive to chemotherapy and radiation, but surgical resection continues to be the most effective treatment [6,7]. Patients with PNET undergo a similar treatment process to patients with MB [8]. There has been a marked improvement in the 5-year survival rate in MB patients, as the survival percentage has improved from 2-30% in the 1970s to 60-70% currently [1,4]. Unfortunately, the current clinical staging does not effectively identify the patients whose tumours will be resistant to chemotherapy and radiation. To individualise therapies and minimise side-effects of aggressive treatments, we need to overcome the major challenge of identifying the high- and low-risk patients. While the prognosis for patients with MB has improved, children with PNET have an even worse prognosis than patients with MB. Currently, the 5-year survival rate for patients with PNET is 24-38% [9,10]. The carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyse the reversible hydration of carbon dioxide (CO2 + H2O ? HCO3- + H+), and, thus, participate in the maintenance of pH homeostasis in the body [11-14]. The mammalian α-CA gene family encodes at least thirteen enzymatically active isoforms with different structural and catalytic properties, and twelve of these are expressed in human tissues [15]. CA isozymes II, IX and XII have been associated with neoplastic processes, and they are potential histological and prognostic biomarkers of certain tumours, including diffuse astrocytomas [12,16,17]. CA II is the most widely distributed member of the CA gene family, being present in virtually every human tissue and organ. It is catalytically one of the most efficient enzymes known [18]. It is present to some extent in malignant cells, and, interestingly, it has been recently shown to be ectopically expressed in the endothelial cells of tumour neovessels [17,19]. Transmembrane enzyme, CA IX, was first recognised as a novel tumour-associated antigen expressed in several types of human carcinomas as well as in normal gastrointestinal tissue [12,20,21]. It has been functionally linked to cell adhesion, differentiation, proliferation and oncogenic processes [12,22], and its enzymatic activity is comparable to CA II [23]. Another transmembrane CA isozyme, CA XII, was first found in normal kidney tissue and renal cell carcinoma [24,25]. Later studies have shown that it is expressed in several other tumours, but also in some normal organs such as the colon and uterus [26,27]. CA IX and XII seem to be regulated by similar mechanisms, as transcription of these isozymes is induced in
Page 2 of 10
tumours under hypoxic conditions through hypoxia inducible factor-1 alpha (HIF-1α)-mediated pathways [28]. Even though very little is known about the regulation of CA II expression, it is unlikely that HIF-1α is involved. High expression of CA II, IX and XII in certain tumours, such as astrocytomas and oligodendrogliomas [16,17,29,30], has suggested that these enzymes may functionally participate in the invasion process, which is facilitated by acidification of the extracellular space [31]. In favour of this hypothesis, it has been shown in vitro that CA inhibitors can reduce the invasion capacity and proliferation of cancer cells [32-34]. To our knowledge, this is the first study to assess the expression of tumour-associated CAs in MBs and PNETs. Here we evaluate the expression of CA II, IX and XII in association with the patient age, survival and molecular pathologic features such as apoptosis and expression of cerbB2, MIB-1 and bcl-2.
Methods Study material
Brain tumour samples were obtained from 35 patients (15 females and 20 males) with either MBs or supratentorial PNETs who were operated on at the University Hospitals of Tampere and Turku, Finland, from1989-2005. The term supratentorial PNET is used as a synonym for CNS PNET, not otherwise specified [1]. MBs were observed in 28 patients and supratentorial PNETs in 7 patients. In addition, there were four patients with a recurrent tumour (two MBs: recurrence after 8 and 29 months in the cerebellum; two supratentorial PNETs: recurrences after 9 and 71 months in the brain stem and left frontal lobe, respectively). Taken together, our material included 39 surgical tumour samples. The age of the patients varied from newborn to 68 years (median = 7.4, mean ± SD = 14.4 ± 17.2), Table 1. In the early nineties eight-drugs-in-one -protocol and later vincristine, lomustine and prednisolon were widely used also for MB and PNETs [35]. Later the treatment in older children (over three years of age) started with radiation therapy with weekly vincristin doses (craniospinal dose 36 Gy and total tumor dose 54-55 Gy), and after irradiation a chemotherapy protocol using cisplatin, CCNU and vincristine was applied [36]. The later protocol is still in use. Children under three years of age have been treated with multidrug chemotherapy protocols from Childrens Cancer Group (USA) or German HITSKK-group generally without radiation therapy. Of the 35 patients, 4 received preoperative chemotherapy and/or radiation therapy, and these four patients all had a recurrent tumour. The tumours were radically resected if possible, and most patients were also treated with postoperative chemo- and/or radiotherapy as follows: three patients received surgery only, five patients were
Nordfors et al. BMC Cancer 2010, 10:148 http://www.biomedcentral.com/1471-2407/10/148
Page 3 of 10
Table 1: Patient characteristics in different tumour subtypes and the correlation between them. MB
PNET
All primary tumours
p-value
15.0 ± 17.3
11.9 ± 17.8
14.4 ± 17.2
0.343*
Females
10
5
15
Male
18
2
20
Surgery only
3
2
3
Surgery +radiation
3
1
4
Surgery+chemotherapy
3
0
5
Surgery+radiation+chemo
19
4
23
Age (mean, years) Sex
0.088**
Therapy
0.540**
* Mann-Whitney test ** chi-square test
post-operatively treated with chemotherapy, four underwent radiotherapy and twenty-three patients received both postoperative chemotherapy and radiotherapy, Table 1. The overall survival was known for 35 patients, and 17 patients were alive and 18 patients dead at the end of the follow-up period. The 5-year survival for our patients was 46% in the total tumour material, 39% in MBs and 71% in PNETs. All the material was gathered from surgical operations. For immunohistochemistry, the brain tumour specimens were fixed immediately in 4% phosphate-buffered formaldehyde and processed into paraffin blocks. Haematoxylin and eosin-stained slides of the tumours were evaluated by two experienced neuropathologists, and the histopathological typing and grading were carried out according to WHO criteria [1]. Following the typing and grading of the specimens, a neuropathologist (HH) pinpointed one histologically representative area from each tumour with a high cellular proliferation index (as assessed by Ki-67 (MIB-1) staining) [37], and this area was then inserted into a multitissue block. The blocks were constructed with a custom-built instrument (Beecher Instruments, Silver Spring, MD) and the diameter of the tissue cores was 2 mm. Immunohistochemistry
The monoclonal antibody M75, recognising the N-terminal domain of human CA IX, has been described previously [20,21]. The rabbit anti-human CA XII antiserum against the secretory form of CA XII has been characterised by Karhumaa et al. [27]. Rabbit antiserum against human CA II has also been produced and characterised previously [38]. Normal rabbit serum (NRS) was used for control staining.
Immunohistochemical staining for CA II, CA IX and CA XII were performed using an automated immunostaining system with the Power Vision+ Poly-HRP IHC Kit reagents (ImmunoVision Technologies, Burlingame, CA). Briefly, the sections were: (a) rinsed in a wash buffer; (b) treated with 3% H2O2 in ddH2O for 5 min and rinsed in a wash buffer; (c) blocked with the Universal IHC Blocking/Diluent for 30 min and rinsed in a wash buffer; (d) incubated for 30 min with the rabbit anti-human CA II serum, rabbit-anti human CA XII serum, monoclonal M75 antibody or NRS diluted 1:2000 (rabbit sera) or 1:1000 (M75) in Universal IHC Blocking/Diluent; (e) rinsed in a wash buffer for 5 min three times; (f ) incubated in Poly-HRP-conjugated anti-rabbit/mouse IgG for 30 min and rinsed in a wash buffer for 5 min three times; (g) incubated in a DAB (3,3' diaminobenzidine tetrahydrochloride) solution (one drop DAB solution A and one drop DAB solution B with 1 ml ddH2O) for 6 min; (h) rinsed with ddH2O; (i) treated with CuSO4 for 5 min to enhance the signal and (j) rinsed with ddH2O. All procedures were carried out at room temperature. The sections were finally examined and photographed with a Zeiss Axioskop 40 microscope (Carl Zeiss; Göttingen, Germany). The staining reactivities for CA II, CA IX and CA XII were scored from the multitissue- blocks on a scale from 0 to 3 as follows: 0, no reaction; 1, weak reaction (< 10% positive cells); 2, moderate reaction (10-30% positive cells); 3, strong reaction (>30% positive cells). Due to the sample size, the staining results were categorised into two groups: negative staining was considered as CA-negative and weak, moderate and strong staining were considered as CA-positive.
Nordfors et al. BMC Cancer 2010, 10:148 http://www.biomedcentral.com/1471-2407/10/148
The section preparation, immunostaining and analysis of apoptosis (TUNEL-labelling) [39] and the expression of c-erbB-2, p53 [40] and bcl-2 [41] were done as previously described. Statistical analysis
All statistical analyses were performed using SPSS 15.0 for Windows (Chicago, IL). The significance of the associations was defined using the chi-square test, the MannWhitney test and the Kruskal-Wallis test. A log rank test, Kaplan-Meier curves and Cox multivariate regression analysis were used in the survival analysis. Ethics
The study design was approved by the Ethics committee of Tampere University Hospital and the National Authority for Medicolegal Affairs.
Results Immunohistochemical staining of CA II, CA IX and CA XII in tumour specimens is shown in Figure 1. CA II showed two distinct staining patterns: the endothelium of neovessels and the cytoplasm of MB/PNET cells. Of all tumours, 49% (n = 18, 12 MBs/6 PNETs) stained positively for CA II in the tumour endothelium (32% strong, 11% moderate and 6% weak staining). Positive cytoplasmic CA II staining in tumour cells was found in 73% (n = 27, 20 MBs/7 PNETs) of the cases (11% strong, 38% moderate and 24% weak staining). CA IX and CA XII were less frequently expressed than CA II in the tumour samples: 23% (n = 9, 8 MBs/1 PNET) of the tumours were positive for CA IX (3% strong, 13% moderate and 7% weak staining), and only 11% (n = 4, 3 MBs/1 PNET) of the tumours were positive for CA XII (3% strong, 3% moderate and 5% weak staining). The CA IX-specific antibody stained perinecrotic areas in most of the tumours in which necrosis was visible. The CA XII was more homogenously distributed than CA IX, consistent with the results obtained previously in other tumours such as ovarian tumours [42]. Since MBs/PNETs are rare tumours and the availability of the specimens was limited to 39, the positively stained tumours (scores 1-3) were pooled for most statistical analyses, including the studies on patient survival. There was no significant correlation in the co-expression of CA II, IX and XII in the subgroups of MBs and PNETs nor did we found a correlation in the group of all tumours (chi-square test). We also compared CA II, IX and XII expression with various clinical features and molecular markers (Table 2). The expression of the CAs did not correlate with proliferation (MIB-1), apoptosis (chi-square and Mann-Whitney test) or expression of bcl-2, p53 or cerbB-2 in any of the groups except for the correlation between c-erbB-2 and CA IX in PNETs (p = 0.047, chi-
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square test). Interestingly, CA XII-positive staining correlated with younger patient age (total material p < 0.001, MBs p < 0.001, chi-square test, Table 2). Added to this, CA IX positivity associated with female gender (total material p = 0.048, MBs p = 0.023, Table 2, chi-square test). There was no significant difference in the expression of CAs between the primary and recurrent tumours in any of the groups (chi-square test). Moreover, there were no correlation between the tumour type (MBs/ PNETs) and CA intensity (chi-square test). All 35 patients with primary MB/PNET were included in the survival analysis (Figure 2). The patients with a CA IX-positive MB/PNET had a worse prognosis than those who had a CA IX-negative tumour (all tumours p = 0.041, MBs p = 0.030, PNETs p = n.s.; log-rank test; Figure 2). We also found a correlation between survival and CA XII staining in patients with MB. The patients with CA XIIpositive tumours showed significantly worse prognosis (p = 0.010, log-rank test). There was no significant difference in survival time between the histological subgroups (p = 0.463, log-rank test). Of the prognostic indicators used for MBs in the current WHO classification, (2007) the following variables were included into the Cox multivariate survival analysis: patient age, MIB-1 proliferation index, apoptosis index and expression of p53, c-erbB-2 and bcl-2. In addition, the histopathological group (MB vs. supratentorial PNET), CA II, CA IX and CA XII were used in the analysis. These variables were grouped as presented in Table 2. In the Cox analysis, only expression of CA IX (odds ratio 4.31; 95% confidence interval (CI) 1.31 - 14.11; p = 0.016) and the apoptosis index (odds ratio 3.29; 95% CI 1.05 - 10.31, p = 0.041) were independent prognostic factors. The expression of either CA II and CA XII failed to show any significant association with survival.
Discussion In the present study, we demonstrate that several MBs and supratentorial PNETs express the CA isozymes CA II, CA IX and CA XII. According to the univariate survival analysis, expression of CA IX was found to be associated with poor prognosis. Most importantly, the Cox multivariate analysis, which included patient age, tumour cell proliferation, apoptosis rate and several other molecular factors, demonstrated that CA IX expression and apoptotic activity were the only independent prognostic factors. The expression of CA XII in the tumour cells was associated with patient age, as previously reported for the expression of CA XII in patients with diffuse astrocytomas [29]. These findings reflect the fact that patient age is a significant factor that contributes to carcinogenesis by several mechanisms and that tumour phenotypes are different depending on the age of the patient.
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Figure 1 Representative immunostaining of CA enzymes in MBs. Panel A shows no immunoreaction for CA IX, whereas the tumour in panel B is strongly positive. Panel C demonstrates CA XII-positive immunoreactivity in tumour cells. In panel D, CA II-positive immunostaining is confined to the endothelium of small blood vessels (arrows). All magnifications ×400.
The expression of CA II, CA IX and CA XII in the normal nervous system has been investigated in several previous studies. The localization of CA II is well documented in the normal human oligodendrocytes [43]. Based on our previous studies, CA IX is not present in the normal human brain [16]. RT-PCR analysis has shown very weak CA XII mRNA expression in the human brain [29], and in mouse, immunohistochemical staining has located CA XII to the choroid plexus [44]. CA IX has several functions in tumour progression. It has been proposed to have a capacity to modulate E-cadherin-mediated cell adhesion, thus leading to a more aggressive phenotype of malignant cells. In intercellular junctions, CA IX may be linked to the E-cadherin/βcatenin complex, because CA IX co-immunoprecipitated with β-catenin in cultured MDCK cells, a kidney cell line [45]. It is also noteworthy that β-catenin is mutated in some sporadic cases of MB [46]. The presence of CA IX
in the E-cadherin/β-catenin complex might contribute by an unknown mechanism to increased invasion and spread of tumour cells. Indeed, embryonal tumours differ from other brain tumours by their tendency to metastasise. In addition, our previous findings in diffusely infiltrating astrocytomas are also in line with the suggested role of CAs in the invasion process. Typically, CA IX-positive astrocytic tumours are highly invasive tumours with an extremely poor prognosis [16]. A recent study by Chiche et al. [47] provided clear evidence that both CA IX and CA XII are functionally involved in tumour growth. In vivo experiments showed that CA9 gene silencing alone led to a 40% reduction in xenograft tumour volume, and the silencing of both CA9 and CA12 resulted in an 85% reduction in tumour volume. In this study, CA IX was found in the perinecrotic areas of the tumours whenever necrosis was present. A similar hypoxia-associated pattern of CA IX expression has been
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Table 2: Association of endothelial and cytoplasmic CA II, CA IX and CA XII immunostaining with clinicopathologic variables in medulloblastomas (MB) and primitive neuroectodermal tumours (PNET). endothelial CA II positivity N Primary tumors
cytoplasmic CA II positivity N
CA IX positivity N
CA XII positivity N
All
MB
PNET
All
MB
PNET
All
MB
PNET
All
MB
PNET
33
27
6
33
27
6
35
28
7
34
27
7
< 3 years
5
2
3
5
3
2
1
1
0
4*
3+
1
> 3 years
12
10
2
18
15
3
7
6
1
0
0
0
-female
6
3
3
10
7
3
1**
0++
1
1
0
1
-male
11
9
2
13
11
2
7
7
0
3
3
0
Age
Gender
Localization -cerebellum
12
18
7
3
-cerebrum
5
5
1
1
Total material MIB-1
36
28
8
36
28
8
37
29
8
36
28
8
-below median
9
6
3
13
10
3
5
4
1
1
1
0
-above median
9
6
3
13
9
4
4
4
0
2
1
1
Apoptosis
37
29
8
37
29
8
39
30
9
38
29
9
-below median
8
5
3
15
11
4
5
5
0
1
1
0
-above median
10
7
3
12
9
3
4
3
1
3
2
1
Bcl-2
37
29
8
37
29
8
39
30
9
38
29
9
-negative
6
4
2
13
9
4
3
2
1
1
0
1
-positive
12
8
4
14
11
3
6
6
0
3
3
0
P53
37
29
8
37
29
8
39
30
9
38
29
9
-negative
15
6
1
23
9
1
7
4
0
3
0
0
-positive
3
6
5
4
11
6
2
4
1
1
3
1
ErbB2
37
29
8
37
29
8
39
30
9
38
29
9
-negative
8
10
5
11
10
5
6
7
0Ψ
0
2
1
-positive
10
2
1
16
2
2
3
1
1
4
1
0
The total number of tumours analysed in each category is in bold. * p < 0.001, chi-square test ** p = 0.048, chi-square test + p < 0.001, chi-square test ++ p = 0.023, chi-square test Ψ p = 0.047, chi-square test
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previously detected in astrocytic tumours [16]. Hypoxia triggers architectural and phenotypic rearrangements of tumour tissue, resulting in the development of necrotic areas surrounded by zones of surviving hypoxic cells. Importantly, these cells often become the most aggressive tumour cells [48], in which CA IX expression is induced by HIF1-α-regulated pathway [28]. Because necrosis is an uncommon feature and is not considered to be a significant prognostic factor in MBs, the induction of CA IX in MBs/PNETs may also involve hypoxia-independent mechanisms. Similarly, in previous studies on gliomas, CA IX expression was seen in tumour cells located in close proximity to the blood vessels [49], and it has been shown that acidosis induces CA IX independently of pericellular hypoxia in glioblastoma cell lines [31]. Based on the previous studies, it has become clear that although hypoxia is the key factor for CA IX induction, there may be other important factors involved. Our text already pointed out that tumor cell acidosis seems to contribute to the expression level [31]. There are several studies where CA IX expression has been correlated to pimonidazole accumulation. The results have shown slightly conflicting results, which may reflect to biological variation between different tumor types and dynamics of tumor hypoxia. However, most results give support for the idea that CA IX follows the pattern of pimonidazole binding [50,51]. We have previously studied the expression of tumourassociated CAs in other types of brain tumours. Endothelial CA II was expressed in the neovessels of astrocytic tumours, in which it was associated with poor prognosis [17]. In addition, we have shown that both CA IX and CA XII are independent prognostic factors in glial tumours [16,29,30]. Based on these findings, CAs may play a central role in the pathogenesis of malignant brain tumours and may represent potential biomarkers for histopathological diagnosis of brain tumours. Although in the total tumour material CA II and CA XII did not reach statistical significance in for use as prognostic indicators, CA II had a similar trend to that of CA IX. Furthermore, CA XII showed a significant correlation with survival in MBs. As discussed above, these differences may be partly explained by regulatory mechanisms. It has been also shown that higher CA IX expression is associated with a more favourable overall survival in some tumours, such as in renal cell carcinoma (RCC) and in acute myeloid leukemia (AML). In RCC the CA IX induction is associated with VHL-mutation and not with hypoxia as in brain tumours [52]. In AML the association has been discussed to be involved with immune system and T-cell response [53]. Hypoxia-induced CA XII is less frequently expressed in MBs/PNETs than in gliomas. Interestingly, CA II was, once again, found in the endothelium of
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neovessels and, thus, may play an important functional role in tumour metabolism. In melanoma patients, endothelial CA II represents a major target antigen in dendritic cell therapy [19]. Further studies are, therefore, clearly warranted to evaluate the role of CA II as a possible therapeutic target not only in melanoma but also in other forms of cancer, including MBs/PNETs. CA IX-specific inhibitors would represent ideal candidate molecules for cancer therapy, because CA IX is highly expressed in several cancers while it shows a very limited distribution in normal tissues [22]. Design of isozyme-specific inhibitor has proved to be a great challenge, because the CA active site is quite similar in all active alpha CA isoforms. The recently published crystal structure of CA IX was certainly a major breakthrough that will help to design novel inhibitors with higher specificity [54]. According to our results, apoptosis was another independent prognostic factor in MBs/PNETs, although its role seems to be controversial. On one hand, as we show in this study, a higher apoptotic index is associated with better prognosis [55]. On the other hand, previous studies in which the degree of apoptosis was categorised as 'focal', 'diffuse' or 'extensive' demonstrated a correlation only between survival and focal apoptosis [6]. In addition, it is known that bcl-2 is an inhibitor of apoptosis. In our tissue samples, however, immunoreactivity for bcl-2 did not correlate with better prognosis; although similar results have been reported by others [56]. In our study, apoptosis was an independent prognostic indicator of MBs/PNETs. However, the study material was rather limited due to the fact that MBs and PNETs are rare tumours, and studies will be needed to clarify the association between apoptosis and survival. Added to this, the time period in which the patients were treated, was rather long and treatment protocols varied. In the future, children diagnosed with MB/PNET will be more accurately stratified based on a combination of clinical variables and molecular profiles. Improved risk stratification will enable individualised therapies, which could be a combination of conventional treatment modalities and novel, targeted therapeutic approaches. These changes will hopefully result in improved survival without a detriment in the quality of life. Several molecular alterations have already been identified in MBs, many of which appear to have prognostic significance.
Conclusions Based on our results, CA IX seems to be a promising prognostic marker that should be tested in a larger cohort of MB/PNET patients. The expression of CA IX in some MBs/PNETs suggests that it could be considered a potential therapeutic target, similar to other tumours including
Nordfors et al. BMC Cancer 2010, 10:148 http://www.biomedcentral.com/1471-2407/10/148
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A
B
C
D
Figure 2 Kaplan-Meier curves showing overall survival of patients with MB or PNET categorised by: A. tumour cell-associated CA II, B. endothelial CA II, C. CA IX (p = 0.041; log-rank test), and D. CA XII immunostaining results.
acute myeloid leukemia [53] and renal cancer [57]. Furthermore, CA IX could be used for in vivo imaging and as a target molecule for CA inhibitors [12,22]. Competing interests The authors declare that they have no competing interests. Authors' contributions KN as the corresponding author gathered the clinical data of the patients, made the statistical analyses, and drafted the first version of the manuscript. JH also participated in the collection of the clinical information, helped with the statistical analyses, and contributed to the writing of the first version of the manuscript. HH was the main organizer of the study. MK provided the clinical knowledge needed and chose the patients for the study at the University Hospital of Turku. AN, JL and HH performed essential microscopic analyses. AP and SPar provided further knowledge on CAs and were in charge of the microscopic analyses on CAs in MBs and PNETs. SPas and JP provided the antibodies against human CA IX, and the antibodies against human CA XII were from the laboratory of AW and WSS. All authors read and approved the final manuscript. Acknowledgements We thank Mikko Arola, MD, PhD for his support in gathering detailed oncological treatment protocols. We also thank Aulikki Lehmus and Reija Randen for their skilful technical assistance. This work was supported by grants from the Cancer Society of Finland, EU 6th Framework programme (DeZnIT), Medical Research Fund of Tampere University Hospital, The Finnish Medical Foundation and The Nona and Kullervo Väre Foundation.
Author Details 1Department of Pathology, Tampere University Hospital, Tampere, Finland, 2Department of Neurosurgery, Helsinki University Central Hospital, Helsinki, Finland, 3Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland, 4Department of Pathology, Turku University Hospital, Turku, Finland, 5Department of Neurology, Tampere University Hospital, Tampere, Finland, 6Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic, 7Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA and 8Institute of Medical Technology and School of Medicine, University of Tampere and Tampere University Hospital, Tampere, Finland Received: 9 November 2009 Accepted: 18 April 2010 Published: 18 April 2010 © This BMC 2010 is article Cancer an Nordfors Open is2010, available Access et10:148 al; licensee from: articlehttp://www.biomedcentral.com/1471-2407/10/148 distributed BioMed Central underLtd. the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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