3601_e09_p237-251 2/19/02 9:36 AM Page 237 Part III Genetics and Epidemiology of Primary Tumors 3601_e09_p237-251 2/19/02 9:36 AM Page 238 3601_e09_p237-251 2/19/02 9:36 AM Page 239 9 Genetic and Molecular Basis of Primary Central Nervous System Tumors C. DAVID JAMES, JUSTIN S. SMITH, AND ROBERT B. JENKINS Our understanding of the genetic etiology of nervous velopment. Many of the karyotypic abnormalities de- system tumors has advanced considerably during the scribed in the literature are based on the study of last decade. During this time, investigators studying glioblastoma multiforme, the most common and ma- genetic alterations in these tumors were able to make lignant central nervous system tumor. Anomalies that several successful transitions from cytogenetic ob- occur frequently in glioblastoma include gain of chro- servations to the identification of specific genes that mosome 7, loss of chromosomes 10 and 22, and are targeted for mutation. The new information as- structural alterations of chromosomes 1p, 9p, 11p, sociated with the results of their investigations will 12q, and 13q (Bigner et al., 1984). In addition, dou- benefit patients with nervous system cancer in at least ble-minute chromosomes are often observed in these two ways. One benefit involves the predictive value as- tumors; their presence suggests the occurrence of sociated with the identification of specific gene alter- gene amplification (Bigner et al., 1987). Alterations ations, that is, it is clear that certain mutations are associated with other types of glial tumors include consistently associated with specific clinical behav- loss of chromosomal arms 1p and 19q in tumors with iors. A second benefit concerns the ability of molec- oligodendroglial differentiation (Ransom et al., ular genetics to provide insights into the fundamen- 1992) and loss of chromosome 22 in ependymomas tal mechanisms associated with tumor development (Ransom et al., 1992; Weremovicz et al., 1992). and, in so doing, provide information about potential Whereas the most frequent chromosomal anomalies therapeutic targets. In the near future, it seems likely in gliomas involve numerical deviations, medulloblas- that concepts that have evolved from this area of study tomas exhibit predominantly structural chromosomal will allow for the application of individualized treat- abnormalities that often involve chromosomes 1, 3, 6, ments that will extend the length and quality of life 10, 17, and 20 (Bigner et al., 1988; Biegel et al., 1989); for people afflicted with nervous system cancer. among these, isochromosome 17q, with associated 17p loss, appears to be the most frequent. With regard to mesodermal tumors, loss of chromosome 22 occurs MOLECULAR AND CYTOGENETIC frequently in meningiomas and has also been reported METHODS in a significant proportion of schwannomas (Zang, 1982; Stenman et al., 1991). Cytogenetics Cytogenetic studies provided the earliest clues con- Fluorescence In Situ Hybridization cerning the genomic locations of genes whose alter- In the past few years, molecular techniques have been ations are associated with nervous system tumor de- combined with conventional cytogenetic methods to 239 3601_e09_p237-251 2/19/02 9:36 AM Page 240 240 GENETICS AND EPIDEMIOLOGY OF PRIMARY TUMORS develop new procedures for identifying chromosomal mor DNA shows either a gain or loss of genetic ma- alterations in brain tumors. The resulting molecular terial, there is either an increase or decrease in green cytogenetic procedures have not only helped to make fluorescence, respectively, at points on the normal infrequently used archival material amenable to ge- chromosomes where the gene alteration has occurred netic analysis, but have also provided information (Color Fig. 9–2). leading to the identification of novel gene alterations. This technique has provided information beyond The first of these to be discussed is referred to as that available through conventional cytogenetics for a FISH (fluorescence in situ hybridization). Although variety of brain tumors (Kim et al., 1995; Reardon et FISH was initially applied to the study of chromosome al., 1997; Weber et al., 1997) and has suggested sev- structure in 1986 by Pinkel et al., the widespread use eral chromosomal locations of genetic alterations for of this technique in a clinical setting has not been which there are no established mutation targets. Find- achieved until recently. ings obtained through the application of this tech- The FISH method involves the fluorescent labeling, nique have provided an effective springboard from either directly or indirectly (e.g., biotin labeling fol- which to launch positional cloning projects and/or lowed by fluorescence-labeled avidin detection), of from which to examine databases containing the relatively large segments of cloned human DNA. The chromosomal locations for thousands of genes so that cloned DNA segments, each of which has been pre- candidate sequences associated with specific chro- viously determined to contain known genes from spe- mosomal alterations can be examined. cific chromosomal regions, can be hybridized to ei- ther isolated metaphase chromosomes or to intact Molecular Genetics interphase nuclei. In many instances the probes can be used to find their target sequence in cells that have Linkage Analysis been embedded and preserved in paraffin (Color Fig. Linkage analysis, as studied with molecular genetic 9–1). By labeling different probes with different flu- methods, relies on subtle DNA sequence variations orochromes it is possible to examine multiple chro- (called polymorphisms) between chromosome ho- mosomes for alterations, and, in fact, there is a de- mologues that allow one to “track” the segregation rivative of FISH known as spectral karyotyping (SKY) pattern of a disease-predisposing locus (gene) (Schrock et al., 1997) in which 23 chromosome-spe- through multiple generations of an affected family cific probes, each labeled with a different fluo- (White and Lalouel, 1988). In the study of such fam- rochrome or combination of fluorochromes, are si- ilies, the chromosomal proximity of a DNA marker multaneously hybridized to metaphase preparations. (probe) to a cancer-predisposing gene is indicated Although yet to be extensively applied to the study of by the consistency of the marker’s co-segregation with brain tumors, this technique may prove useful for the the occurrence of cancer within the family. This ap- analysis of complex karyotypes that are typical of proach has been useful in identifying and/or associ- many nervous system malignancies. ating tumor suppressor genes (TSGs) such as TP53, NF2, and VHL with their respective cancer syndromes: Comparative Genomic Hybridization Li-Fraumeni, neurofibromatosis type 2, and von Hip- pel-Lindau. An additional molecular cytogenetic method that has been efficacious in identifying chromosomal and gene Loss of Heterozygosity (LOH) Analysis alterations in solid tumors is comparative genomic hybridization (CGH) (Kallioniemi et al., 1992). This DNA polymorphisms have also been utilized to locate method is based on the competition between two dif- TSGs in sporadic tumors through a process known as ferent DNAs, one from a tumor and one from a nor- deletion mapping. A chromosomal deletion map is mal tissue, for hybridization to normal metaphase obtained through the application of loss of heterozy- chromosomes. Before hybridization, the normal and gosity (LOH) analysis (Lasko et al., 1991) in which tumor DNAs are labeled with different fluorochromes the patterns of restriction enzyme or polymerase (usually red and green, respectively), and as a result chain reaction (PCR) DNA fragments are compared most regions of hybridized metaphase chromosomes in a patient’s normal and tumor DNAs. Loss of a re- show a fluorescence color combination that is equally striction or PCR fragment-length allele in a tumor DNA balanced between the two labeled DNAs. When the tu- sample is indicative of a genetic alteration directed at 3601_e09_p237-251 2/19/02 9:36 AM Page 241 Genetic and Molecular Basis of Primary CNS Tumors 241 the deletion of a TSG. By applying a battery of mapped gane et al., 1997). The protein products of onco- probes (markers) from a chromosome of interest, genes promote cell proliferation, and oncogenes one can limit the chromosomal location of a TSG by can be activated by increasing the synthesis of their determining the smallest common region of deletion corresponding protein, in its normal form, or by al- among a panel of similar tumors. This type of analy- teration of corresponding protein function through sis has been applied extensively to brain tumors and gene mutation. In general, oncogene alterations only has revealed several associations between detectable involve one of the two copies of a specific oncogene alterations and tumor histopathology. within a cell. In contrast, both copies of a specific tumor suppressor gene (TSG) must be inactivated through deletion or mutation for a cell to gain a GENES IMPORTANT TO NERVOUS growth advantage. As might be suspected from their SYSTEM TUMORS name, proteins encoded by TSGs inhibit cell growth. Table 9–1 summarizes the oncogene and TSG al- terations important to the development of various Classification of Cancer Genes types of nervous system tumors, and following are As in all human cancers, two families of genes ap- brief discussions of specific genes that are fre- pear to be involved in the pathogenesis of brain tu- quently altered during the development of specific mors: