ESTRUCTURAS GRUPALES

For m any years analysis of m etaphase chrom osom es has been th e cornerstone of cancer genetics. G-banding analysis has proved to be useful in identifying chrom osom e abnorm alities in leukaem ias a n d lym phom as b u t has been less successful in studying solid tu m o u rs because of the difficulty in obtaining good quality m etap h ases preparations (Gray and Collins, 2000). CGH was first developed by Kallioniemi and colleagues in 1992 and has allow ed the entire g en o m e to be studied in a single experim ent from ju st a few nanogram s of starting DNA, w ithout the need for cell culture (Forozan et al, 1997). It enables the screening of tu m o u rs for DNA copy-num ber changes an d provides a m ap of chrom osom al regions th at are lost or gained, w h ic h can help in locating specific chrom osom al regions th at m ight play a role in the pathogenesis or progression of tum ours. Once results fro m CGH have been obtained, m ore specific m olecular genetic techniques, such as FISH, LOH analysis and sequencing can be used in order to identify oncogenes a n d /o r tu m o u r suppressor genes in these regions (Weiss et al, 1999).

CGH is based on a m odified in situ hybridisation m ethod w h ere differentially labelled test (tum our) and reference (norm al) genom ic D N A are co-hybridised in the presence of h u m an Cot-1 DNA to n o rm a l h u m a n m etaphase chrom osom e spreads. The relative am ounts of test and reference DNA that bind at any given chrom osom e locus are dep en d en t on the abundance of those sequences in the test a n d reference DNA sam ples. Copy num ber differences betw een the tw o sam ples can be seen as greenired fluorescence intensity differences o n m etaphase chrom osom es. Ratios of the signal intensities can be quantitated using a digital image analysis system w hich calculates intensity profiles for both colours and the green:red ratio along each chrom osom e (Kallioniem i et al, 1992). C ains and am plifications in th e test DNA are seen as chrom osom al regions w ith an increased fluorescence ratio (>1) w hereas losses and deletions show a reduced

fluorescence ratio (<1) (Forozan et al, 1997; Weiss et al, 1999). The m a in lim itations of CGH are its resolution (10-20Mb), it does not p ro v id e quantitative inform ation about gene dosage and it is insensitive to structural aberrations that do not result in a DNA sequence copy num ber change (Gray and Collins, 2000). H ow ever it is sen sitiv e enough to detect am plification events (e.g. 5-10 fold am plifications of Im b regions). The technique cannot be used to detect balanced rearrangem ents such as inversions and translocations, and deletions w ithin a chrom osomal band (Biegal, 1999; Forozan et al, 1997).

CGH has helped identify the androgen receptor (AR) gene located in a

region of increased copy num ber at Xql2 in horm one refractory prostate cancers (Koivisto et al, 1997) and the PIK3CA gene located at 3q26 in ovarian cancer (Shayesteh et al, 1999). Several genes have been identified in regions of am plification at 20q, including A l B l a steroid receptor co-activator (Anzick et al, 1997), ZNF217 a p u tativ e Z n-finger transcription factor associated w ith instability and im m o rta lisa tio n

(Collins et al, 1998) and STK15 a centrosom e-associated

serin e/th reo n in e protein kinase (Zhou et al, 1998).

CGH has been used to study brain tum ours in both children and ad u lts and has identified six new regions of am plification at lq32, 4ql2, 7q21.1, 7q21.2-3, 12p and 22ql2, as well as am plification of the EGFR gene at 7pl2 in m alignant gliomas. Gain of chrom osom e 7 and loss of

chrom osom e 10 w ere the m ost com m on events in this group of

tu m o u rs (Schrôck et al, 1994). The w ork of B runner et al. (2000) also show ed gain of 7 and loss of 10 to be the m ost com m on aberrations in adult GBM. They also observed a novel am plification at 20pll-12 in a n oligoastrocytom a. CGH has identified two m ajor sub-groups of h ig h - grade oligodendroglial tum ours, 1 group w ith -lp /- 1 9 q and the o th e r group w ith +7/-10 (Jeuken et al, 1999).

The first group to publish w ork on CGH analysis of ep en d y m o m a identified losses of 6q and 22q as the m ost com m on aberration in prim ary ependym om a in paediatric patients (Reardon et al, 1999). Loss of 22q w as the m ost frequent abnorm ality in a m ixed group of ad u lt an d paediatric ependym om a studied by Zheng et al, (2000). Gains of Iq h a v e been seen to occur exclusively in paediatric ependym om a (Scheil et al,

2001). Loss of 13q was a frequent observation especially in

m yxopapillary ependym om a. Shlom it et al, (2000) show ed gain of Iq and loss of 16q to be the m ost frequent alterations in paediatric low grade ependym om a. Gain of Iq and loss of 9 w ere the m ost frequent alterations in intracranial ependym om a in a study by Hirose et al, (2001), w hilst gain of 7 was seen exclusively in spinal ependym om a. A recent study show ed isochrom osom e Iq to be an early genetic event in a case of prim ary and recurrent ependym om a from a child w ith a n intracranial ependym om a (Vinchon et al, 2001).

O nly a handful of studies have investigated the genetic alterations in paediatric astrocytom a using CGH. Only a sm all n u m b er of ju v e n ile pilocytic astrocytom a have been found to have detectable alterations. The tu m o u rs that did show alterations h ad gains of a single chrom osom e, the m ost com m on being gain of 7 (Sanoudou et al, 2000). These findings are in agreem ent w ith an earlier study that found n o consistent changes in paediatric low grade astrocytom a (Schrôck et al, 1996). A nother study also show ed juvenile pilocytic astrocytom a to have a low num ber of copy num ber aberrations. The m ost c o m m o n alterations w ere gain of 6q and loss of 9q (Shlom it et al, 2000). In paediatric high grade astrocytom a the m ost com m on im balance was loss of 16p and high copy num ber am plifications w ere seen at Ip, 2q22, 7q22-23, 8q21-22,12q and 13qll-14 (Warr et al, 2001). A recent CGH study identified loss of 8p as a sole abnorm ality in a case of paediatric pleom orphic xanthoastrocytom a (Yin et al, 2002).

The application of CGH has been expanded th ro u g h com bination w ith tissue m icrodissection and PGR am plification using a universal p rim e r. This allows test DNA to be obtained from m in u te subregions and e v e n from in dividual cells (Forozan et al, 1997). The com bination of

m icrodissection and CGH allows the detection of cytogenetic

aberrations from clones that may be m issed w hen analysing D N A extracted from large cell populations (Aubele et al, 1999). D egenerate oligonucleotide-prim ed polymerase chain reaction (DOP-PCR) has been reported to be a reliable m ethod to produce a representation of sm all am o u n ts of DNA (Cheung, 1996; H uang et al, 2000; K uukasjarvi et al, 1997; Speicher et al, 1993). DOP-PCR allows the analysis of less than In g of DNA and is useful for amplifying DNA from m icrodissected tissue.