The Etiology and Progression of Brain Tumors
Condition: Brain Tumor; Glioma; Meningioma
MeSH terms: Brain Neoplasms; Meningioma
Study type: Observational

Summary: 
      The main goal of the study is to present a framework, which integrates DNA, RNA and tissue
      data to identify and prioritize genetic events that represent clinically relevant new
      therapeutic targets and prognostic biomarkers for different kinds of brain tumors. The
      investigators study the regulation of neoplastic cell growth by oncogenes, tumor-suppressor
      and other cancer related genes using modern molecular genetic methods, such as
      chromogenic-in-situ hybridization, comparative genomic hybridization (CGH), array-CGH, cDNA
      microarray etc. In these studies the investigators utilize disease-specific tissue
      microarrays (TMA) which the investigators have constructed since 1999. Until now up to 3000
      different brain tumours have been sampled to our TMA:s. These permit high-volume simultaneous
      analysis of molecular targets at the DNA, mRNA and protein levels. Research group has also
      focused its interest on the neoplastic development of gliomas, particularly on their
      hereditary and environmental factors.
    

Detailed description: 
      Aims of the study:

        1. To collect adequate brain cancer tissue material for high throughput morphological,
           protein, RNA and DNA analyses.

        2. To combine tissue-related results of these analyses with clinical data to examine the
           potential of the new biomarkers to assess diagnosis, prognosis and heredity of brain
           tumors.

      Materials and Methods

      Paraffin-embedded tumor material

      Paraffin blocks of more than 4,000 brain tumours have been collected into the Laboratory of
      Pathology at Tampere University Hospital (Department of Pathology in Fimlab Laboratory). They
      have been used primarily for purposes of clinical diagnosis, but once patient or authority
      consent has been obtained the surplus of the material can be used for research purposes. A
      neuropathologist has marked the most representative region of each tumour in a tissue slide.
      Using these markings, tumour tissue regions are then biopsied into the tissue microarray
      block (Micro-Array Technology, Beecher Instruments, Inc.). Up to 1,000 histological samples
      can be collected into one tissue microarray block, which can be cut into 200 tissue sections.
      These sections can be used in various kinds of analysis (immunohistochemistry, fluorescence
      and chromogene in situ hybridisation and other histological standings). Among the advantages
      of the method are its high capacity, potential for automation, limited damage to the original
      tissue block and optimised circumstances for molecular biological analyses. We produced the
      first Finnish TMA block in 1999. Until now up to 3000 different brain tumors have been
      sampled to our TMA:s of which 2000 gliomas and meningiomas are used for this study. The
      following provides examples of projects in which the method is used. Similar strategies are
      used in the present study:

        1. We have studied the relationship between carbonic anhydrases and brain tumours. Carbonic
           anhydrase IX (CA IX) is a hypoxia-induced enzyme that is associated with tumorigenesis.
           CA IX immunopositivity was found in 80% of 362 astrocytic gliomas collected in tissue
           microarray blocks. According to multifactorial survival analysis, CA IX intensity was a
           significant and independent prognostic factor. CA IX is a possible candidate for
           targeted therapy.

        2. By using the cDNA-microarray method it is possible to analyse the expression of several
           cancer genes in one hybridisation. The cDNA-CGH microarray method makes simultaneous use
           of the cDNA-microarray method and comparative genomic hybridisation. This method allows
           us to study the relationship between gene copy number and the expression of the
           particular amplified gene, and also to define, at high resolution, the genes in cell
           lines that are amplified and over- expressed. The analysis gives a more focused picture
           of the relationship between changes in gene copy number and their expression in
           different kinds of tumours. With these methods we have found several new cancer gene
           candidates that may be involved in the pathogenesis of neuroblastomas. To ensure the
           role of these genes we have built neuroblastoma tissue micro array blocks and performed
           FISH and IHC analyses using these TMAs. We succeeded in characterising an amplification
           region which occurred in almost half of the tumour samples of neuroblastoma patients and
           which was significantly associated with patient survival.

           Fresh tissue and frozen tissue microarrays

           Our team at the Department of Pathology has been taking steps to prepare for molecular
           biological and genetic studies that require fresh tissue archives. To this end we have
           systematically collected and recorded brain tumour tissue for modern methods of cancer
           diagnosis and research. The processing, storage and archiving of brain tumour tissue
           from the neurosurgical operation theatres at

           Tampere University Hospital are undertaken centrally at the Department of Pathology
           frozen tissue laboratory. This work has been ongoing since 1992 and by now more than
           1,000 fresh tumour samples have been collected. The sample material is used for exact
           tumour diagnosis in the phase of primary diagnosis (e.g. genetic 1p19q LOH analysis of
           oligodendrogliomas). Our novel frozen tissue application serves several new molecular
           pathological methods. We have developed a completely new method based on the snap frozen
           technique and produced dozens of samples from different brain localisations (frozen
           brain array). The following provides one example of a project in which frozen tissue
           method was used. Similar principles are to be used in the present study:

        3. We have developed a new method which facilitates the differential diagnosis of brain
           tumours during operation. Using the Ultrarapid Ki-67 staining method, the proliferation
           marker Ki-67 can be analysed intraoperatively with the snap frozen technique and light
           microscopy within 10 - 15 minutes. We were able to ascertain the applicability of the
           method using frozen tissue sections of gliomas that had been previously collected into
           our tissue bank. On the basis of their proliferation indices, the gliomas could be
           divided into different malignancy grades and prognosis groups. This highly specific
           diagnostic method can be applied for example in situations where therapeutic drugs are
           placed into an intracranial operation field.

           Extracted RNA and DNA

           Our laboratory is well equipped and prepared for RNA and DNA studies. Following
           extraction, RNA and DNA are archived and kept at -700 C for later use. Example of
           projects that use this method:

        4. Linkage analysis offers a powerful tool for localising genes that predispose to familial
           diseases, provided that there is a sufficient number of families with the disease
           concerned. For linkage analysis, the polymorphic regions of the genome are investigated
           using markers of chromosomal regions that have been passed on from the family's founder
           parents to all members with gliomas in the family. We have collected blood and DNA
           samples from very rare glioma families (with a total of 183 members) for purposes of
           linkage analysis. On the basis of genome-wide linkage analysis, we found a new
           chromosome locus that was significantly associated with the familial glioma. Deep
           sequencing of the samples of familial glioma patients is the following step of our
           study.

      Data collection

      Our neuro-oncological material at the Tampere University Hospital Department of Pathology
      comprises 5,000 tissue samples (4,000 paraffin and 1,000 frozen tissue samples). This is too
      large a dataset to be managed by individual researchers in their own databases. Our aim
      therefore is to develop a new integrated research data system for the effective management of
      the tissue material that has been collected over the past 30 years, including a detailed
      register on all the samples. The register will also include digital photo material from TMA
      and other histological slides as well as virtual microscopy slides. The relevant clinical
      data of the patients (e.g. at least three year follow-up of glioma and meningioma patients
      operated during 1983 - 2009) is combined with the tissue data with the permission of Finnish
      authorities and the Tampere University Hospital.

      The project observes the Helsinki Declaration, current Finnish legislation and the principles
      of data protection, laid down by Tampere University Hospital. This study is retrospective and
      purely observational. The assignment of the medical intervention is not at the discretion of
      the investigator. The collection of samples for research purposes required each individual
      patient's informed consent in the familial glioma study. The archiving of diagnostic material
      in a tissue bank involves no ethical problems. Only excess material from diagnostic samples
      is used for research purposes, either with the patient's informed consent or with the
      permission of the relevant Finnish authorities (the National Authority for Medicolegal
      Affairs of Finland). The Ethical Committee of Tampere University Hospital has given
      permission for our project (R07042). The familial glioma study is conducted under a separate
      permit (Ministry of Social Affairs and Health , Diary number 127/08/95). Research permits
      have also been obtained on the basis of the Finnish tissue law (Valvira: Diary number
      7796/05.01.00.06/2011).
    

Observational model: Cohort
Time perspective: Retrospective
Sampling method: Non-Probability Sample
Study population: 
        Glioma and meningioma patients
      
Biospecimen description: 
      Paraffin-embedded and frozen brain tumor tissue
    
Keywords: Brain tumor; Glioma; Meningioma; Diagnosis; Prognosis; Heredity