Document YjYbEKM0xBE6bzaO4oKBkk2XN

I- C *' GENES. CHROMOSOMES8 CANCER 31:201-206 (2001) R.. ESEARCH ARTICLES Detection of Unidentified Chromosome UONFIVTEHRESIHTEYAOLFTHPrSnCSIBEuNRCEQSH Abnormalities in Human Neuroblastoma by Spectral Karyotyping (SKY) JUN 6 2001 Ninette Cohen,' David R. Betts: Luba Trakhtenbrot,' Felix K. Niggli? Ninette Amariglio,' Frida Brok-Simoni,' Gideon Rechavi,' and Dafna Meitar'* 'Department of Pediatnc Hemato-Oncology and Institute of Hematology The Chaim Sheba Meaical Center Tel Hashomer Israel 'Department of Oncology Univenity Children's Hospital Zunch Switzerland ; Spectral karyotyping (SKY) is a novel technique based on the simultane@us hybridization of 24 fluorescently labeled chromosome painting probes. It provides a valuable addition to the investigation of many tumors that can be difficult to define by conventional banding techniques. One such tumor is neuroblastoma, which is often characterized by poor chromosome morphology and complex karyotypes. Ten primary neuroblastoma tumor samples initially analyzed by C-banding were analyzed by SKY. In 8/10 tumors, we were able t o obtain additional cytogenetic information. This included the identification of complex rearrangementsand material of previouslyunknown origin. Structurally rearrangedchromosomes can be identified even in highly condensed metaphase chromosomes. Followingthe SKY results, the C-banding findings were reevaluated, and the combination of the two techniques resulted in a more accurate karyotype. This combination allows identification not only of mam$ial gained and lost, but also of breakpoints and chromosomalassociations. The use of SKY is therefore a powerful tool im,&'&etic characterization of neuroblastoma and can contribute to a better understanding of the molecular evenfs with this tumor. 8 2001 Wiley-Liss, Inc rnrcei fI': ge 3:,-ite INTRODUCTION Neuroblastoma is the most common tumor in (Nakagawara et al., 1993) are associated with a- favorable prognosis. infants younger than 1 year and the second most T h e karyotypic analysis of neuroblastoma tu- common solid tumor of childhood (Grovas et al., mors with conventional methods is often a difficult 1997; Gurney et al., 1997). It is an embryonal can- process that may be hindered by limited numbers cer of the postganglionic sympathetic nervous sys- of metaphase cells and poor chromosome morphol- I tem. ogy. often leading to on]! partial characterization of T h e clinical hallmark of neuroblastoma is heterogeneity. Some patients can be cured by surgery alone, but others have a fatal outcome. Further- the chromosomal events. However, prior to the use of fluorescence in Situ hybridization (FISH) and comparative genomic hybridization (CGH), conventional cytogenetic analysis had identified a more, in some patients the tumor undergoes spon- number of nonrandom abnormalities, including ab- taneous regression or differentiation. This clinical errations of l p , 2p, 4p, 5q, l l q , 17q. and 19p diversity correlates with several molecular biologic (Franke et al.. 1986; Kaneko et al., 1987; Hayashi et features of neuroblastoma. al., 1989: Aver-Loiseau et al., 1995; Caron et ai., Established indicators of the aggressiveness of 1996; Lastowska et al., 1997). the tumor and poor outcome include the amplifi- Spectral karyotyping (SKY) is a recently devel- cation of the MYCX gene (Brodeur et al., 1984, oped technique based on the hybridization of 24 1992: Seeger et al., 1985), deletion of the short arm fluorescently labeled chromosome painting probes of chromosome 1 (lp), a near-diploidy or near- (Garmi et al., 1996). It has been shown in a number tetraploidy (Ambros et al.. 19961, angiogenic phe- of studies that highly complex karyotypes with notype (Meitar et al., 1996), the recently identified unidentified marker chromosomes can be defined gain of chromosome arm 17q (Bown et al., 1999). b? this method O'eldman et al.. 1997: Huang et al., and telomerase activity (Poremba et al., 2000). , However, the presence of single copies of MYCA', a normal lp, near-triploidy, the presence of an expanded CD10' cell population in the bone marrow (Mandel et al., 19941, absence of an angiogenic phenotype, and the expression of the TRK gene *Correspondence to: Dr. Dafna Meitar. Department of Pediatric Hematc+Oncolog\.. Chaim Sheha Medicai Center. I'el Hashomer 52621. Israel. E-mail: darneita@nervision.net.il Received 2 Jul) 2000. .4cceptrd Ih Nolember 2000 Published online 26 April 2001 e 200 I Wiky-Lizr, Inc. 202 COHEN ET A L 1998: Pulivarthi et al., 1998: Zattara-Cannoni et al., 1998; Fleischman et al.. 1999). We used SKY for the analysis of 10 primary human neuroblastoma tumors. which had previously been anal?-zed by conventional cytogenetics. MATERIALS AND METHODS Cytogenetics Cases 1-9 were derived from a consecutive series of 17 primary neuroblastomas from which material was sent by various centers in Switzerland for cytogenetic investigation. T h e tumor material was processed as previously described (Betts et al., 1997). Multiple cultures were set up, although the most reliable culture employed Colcemid (final concentration, 0.005 g/ml; Life Technologies, Basel. Switzerland) added overnight on the day of sample receipt. Slides were made from fixed cell suspensions and were trypsin G-banded and Giemsa-stained. All other slides were aged before a full analysis was undertaken. Case 10 was described previously (Trakhtenbrot et al., 1999). Spectral Karyotyping T h e cases investigated by SKY were selected on the basis that fixed suspension material was still availabie following cytogenetic and FISH investigations. Slides for SKY were prepared by use of chromosome preparations stored at -20C. Chromosome labeling was performed with the SKY flu- orescent labeling kit (.4pplied Spectral Imaging, Migdal HaEmek. Israel) according to the manufacturer's protocol. Chromosomes were counterstained with DAPI. Image acquisition was performed by use of an SD2OO Spectracube (Applied Spectral Imaging) mounted on an Olympus BH-2 microscope using a custom-designed optical filter (SKY-1. Chroma Technology, Brattleboro, VT). .Automatic identification of chromosomes was based on the measurement of the spectrum for each chromosome. Fluorescence In Situ Hybridization (FISH) A range of probes (Vysis, Downers Grove, IL) was employed, including an .IfYCLV-specificprobe to test for the presence of .LfYC.V amplification, and whole-chromosome painting probes for chromosomes 6. 12, 18. 21, and 22 were used to verify some of the translocations detected by SKY. LAll probes were hybridized according to the manufacturer's instructions. RESULTS SKY provided additional cytogenetic information in 8 of the 10 cases (Table 1). In cases 1 and 2, SKY supported the previously identified abnormalities determined by G-banding and FISH. By combining the SKY and G-banding/FISH results, an almost full definition of the tumor karyotypes was possible (Table 2). Two examples of SKY-classitied karyotypes (cases 6 and 9) are presented in Figures 1A and 2A. T h e abnormalities that were newly determined or reclassified by SKY are shown in bold in both Tables 1 and 2. T h e majority of the changes identified by SKY were previously referred to as markers or add(v) chromosomes. In cases 4,6, 9, and 10, a number of breakpoints could be defined more accurately by referring back to the Gbanding analysis. In cases 5, 6, 7, and 10, five chromosome segments were reclassified. T h e vast majority of rearrangements (19/27) de- tected were unbalanced and, in some cases, complex. 4 n example of the complexity observed was the der(l1) chromosomes present in case 9 (Fig. ZB). In case 3, a possible jumping translocation (JT) was seen, involving 18qll with recipient chromosomes 21q22 and 22q13. J T is defined as a translocation of the same chromosome segment on two or more recipient chromosomes in different cell clones of the same patient (Lejeune et al., 1979 Bernard et al., 2000). JT is a rare cytogenetic event reported in leukemias. lymphomas, and solid tu- mors (i :., carcinomas, renal tumors). To our knowledge, this could be the first report of such an event in neuroblastoma. Not all rearrangements were defined after iden- tification by SKY.T h a t could be the combination of three reasons: additional material detected by G-banding was too small to be detected by SKY the rearrangement is a complex internal event that SKY will not detect; and different cultures were analyzed by SKY and G-banding, and different subclones were analyzed. An example is add(1)(q32) and add(1l)(p15) in case 4. Both were found only in a clone not detected by G-banding. In case 7, the additional segment on add(l)(q42) and on add(13)(qX) was not detected by SKY because of its small size or due to similarity of spectral characteristics between the chromosome and the additional segment. A further strength of SKY was that a greater number of metaphase cells could be fully or partially analyzed than is typically possible with the average G-banding analysis, principally because SKY was able to provide information on metaphase UNIDENTIFIED CHROMOSOME ABNORMALITIES 203 TABLE I . Reclassification of Abnormalities Detected Originally by G-Banding by SKY* Case number G-banding anaiysis Reclassification by SKY FISH verifications with painting probes -3 1. add(4)(p 14). add(12)(q24), add(7)(p22) 1. der(4)t(4;I9)(p 14;qI3), der( I2)t( I2;2 I)(q24;q2I), 1 2 6mar [IS]/ +der(7)t(Y;7)(q II;p22), der(22)t( I8;22)(q II;q 13) II.clone not detected -t611 II. 65 69. X, -X. - Y, idem but instead of der(22) pI8 and p22 t(18;22)(ql l;q13) there is der(2l)t(l8;21) (9 I 1~q22)t91 p18 and p2I add(4)(q3I), +mart91 der(4)t(4;8)(q26q2 I).der(5)t(5; I8)(p I?;q2 I), der(6)t(6; I2)(!;?)[ I21 p6 and p12 I)mder(I)t(X; l)(q24;p34), add(16)(q22). der(2I)t(! I;2 l)(p34;pI der(l)t( 1;2)(p34;!). der( I6)t(X, 16)(q26;q22), der(2 I ) t( I9;2l)(q I3;pl I)[141 I. add(l)(p36), add(6)(q23-25), add(7) I. der( I)t( I;7)(p36?), der(6)t(6; I4)(q2 I;q II),der(7) (p2I). der( 16)t(l6?;17)(p13;!;q2I), t(6;7)(~21.1;~22).der(16)t(1617)(pl3;q2l)t(9;17) der( I 7)t(X I 7)(q2I ;q IO) [IO] (92 I;q25). der(I7)t(4; 17)(q2&28p I I )[6]/ 11. clone not detected It. 73-91, XXIY, idem t o (I) X2[3]/ 111. clone not detected 111. 91, XXYY, idem to (11). but instead of der(l6) t( 16;17)(p13;q2l)t(9;17)(q2l;q25)X2 there are der( I6)t( 16; I7)(p I3;q2 1)x2,[ 11/92, XXYY. idem, t(4;7)(?;?)x 2[ I] 7 der(l6)t(l617)(ql2q2I), +I-Smar [lo] der( I9)t(17; I9)(q2 I;p I3)[ 131 8 1. +2-3mar [IO] 1. +del(7)(q3 I),der(l I)del( Il)(p 12- l3)[ I9]/ 11. 87-89,idem to (I),+de1(7)(q3I), +de1(7)(q3 I) 9 addfC)@36), add(6)(q16-22), +8- I4mar[IO] [41 der( I)t(1;17)(~36;qll),dcr(6)t(6;12)(q2l;ql?4), der( Il)(7qter-+7q II. 2 IIpI3+1 Iq22.7q II.2-+ 7qter), der(l I)(I 8 q t e r 4 8 q l 1.2::7q3?+7qZI:: II p l 5 - 4 l q 2 2 7 q I 1.2+7qter), der(l2)t(5;12) (?;?),der( I4)t( 14; I 7Xp II;q2 I)PO] IO I. del(l)(!p34)X2 der(16)t(!5;16)(!q31; 1. der(l)t(l;l2)(pl3;ql I-IZ)XZ, der(16)t(1617) 44). der( I9)t( 19;?)(q13;?)[33]/ (q22q II). der(I9)t(8; 19)(?;q I3)[45]/ 11. 47, idem, der(I O)t(I O?)(qW;!)[ 121 It. 47, idem to (I),der( IO)t(4; I O)(q22q26)[ IS] *Abnormalities detected only by SKY are shown in bold. cells that would not be analyzable by G-banding. This provided further clues to the clonal evolution of tumors and made the analysis of polyploid metaphases possible. T h e advantage of the latter was shown in case 6, where one of the polyploid metaphase cells contained a der(16)t(16:17)(p13;qZl) instead of the der(l6)t(16;17)(pl3;q21)t(9;17)(q21; q25) that characterized the diploid and an additional polyploid clone (Fig. IB). This demonstrated the presence of divergent clonal evolution in this tumor. -411 cases were investigated by FISH with a probe for MYOY, and amplification of the MYCAregion was shown to be present in cases 1. 2, 4, 6, 8. and 10. which in all cases was in the form of double minutes (dmin). In cases 4 and 10. only the signal pattern of the MYCAYprobe, in both interphase and metaphase cells. allowed the identification of dmin, as they were apparently too small to be visualized under a light microscope. In case 3, despite the presence of dmin, no MYCh- amplification was demonstrated, thereby implying amplification of another chromosomal region. In three cases (1, 6, and 8). SKY detected the presence of double-minute chromosomes, painted with the color of chromosome 2. In cases 2-4, SKY did not detect the dmin. probably due to either their small size or low copy number. SKY was successful in demonstrating five cases with I p deletion; all were the results of unbalanced translocations (cases 1.5.6,9. and 10).In two cases. this involved chromosome arm 17q. and the other three involved chromosomes 2, 7, and 12. DISCUSSION Neuroblastoma is often characterized b) gains and losses of genetic material and complex chromosomal abnormalities CBiedler et al.. 1973: Bro- 204 COHEN ET A L TABLE 2. Final Tumor Karyotype Combining G-Banding and SKY Results* Case I 2 3 4 5 6 7 8 9 IO Age (Y. M) 392 0.4 6. IO 3, I I 2.5 I. 6 6.9 4.0 2.7 5,s Stage IV I 111 IV IV IV 111 IV IV IV Survival (M) 46+ 1st 13 IO II+ I I days 14-r 34t 31 + 6 Final tumor karyotype 46.XY.der( I)t( I;I7)(p36. I;q I2).der(22)t( I722)(q2I :p 12),50- IOOdmin [8]/46.idern.del( I l)(q23. I q23.3)[121 46,XX,der(3)t(3; I7)(p I3;q23). IO- I00dmin[cp 161 66-69.XXY, -3.der(4)t(24)(p2I-22;q33),der(4)t(4 I9)(p 14q13). +der(7)t(Y,7)(ql l;p22),- loader(12)t(1221)(q24q21),L 13.+ 17. - 19,+20,-21,-21,der(22)t( 18;22)(ql I;ql3),10-20dmin[cp20]/ 65-69.X.-X,-Y,-3,der(4)t(24)(p21 -22q33),der(4)t(4 19)(p14qI3), L6.+der(7)t(Y;7)(ql l;p22),- IO,der(12)t(1221)(q24q21)+ 13,- 19, +20, -2I.der(2 I)t(I8;2 I)(q Il;q22),+der(?)(!; I7)(!;q 12). IO-ZOdmin[cp IO]/ 130- I38,idemX2[cp3] -56-6 I(3n).XX,-X,der(4)t(48)(q26;q2 I),der(S)t(S; I8)(p I?;q2 I), der(6)t(6; I2)(!;?),-8,-9,- IO,- I I, 14,- I6.-2 I[cp IO]/S6-61, idern,add(l)(q32),+del(2)(pl6).+7,add(l I)(plS),- 13.- 15.- 18,- 19. -20[cp IS I 46,-Xder(X)t(X7)(q22;q I I.2).der( I)t(I;2)(p34?),der(4)t(4I7)(p I 2 q2l),der( 16)t(X;I6)(q26;q22),t 17.der(2l)t( 19;21)(q13;pl l)m/46, idern,del( I l)(q23)[3]/47,idcm,+ 18[3] 46,XY,der( l)t( I;7)(p36,?),der(6)t(6; I4)(q2 I :qIl).der(7)t(6;7) (p2I.I:p22),der( I6)t( I 6I7)(p I3;q2 I)t(R I7)(q2 I;q25).der( I7)t (4; I7)(q26-28;p I1),20- I00dmin[cp2 1]/92,1demX2[2] 70-72,XY.-X.add( l)(q42)x2.del( l)(q32q42),-3,+8,- I I,del( I I) (q14q23),+13,-14.-15,-16,+18.+18.+18,+18,-19.der(l9)t (17;19)(q2l;pl3)[cpl3]/70-72.idem.+7.-9.der(9)t(6;9)(pl I;pl I). +lO,-I2,add(13)(q32),+ 17,+ 18.+18,-19.+20,-22[cpIO]/46,XY, del(l l)(q14-q23)c[l] 87-89.XX,-Y.-Y,-3,-5,+deI(7)(q?3 1),-9,- IO,der( II)del( II) (pI2)del( II)(q 13). +20.20-200dmin[cp33]/46,XY[S] 8 3 - 8 9 W . -X,del( I)(p34),der( I)t(I;I7)(p36q2 I).- 3, -4, -S.der(S) t(S;I2)(?;?),der(6)t(6I2)(q2I;q1?4),-i(6)(p10).-9,dcr( I1)(7qter-, 7q21::I IplS-I lq22::7qll-+lqter),der(f I)(l8qter-.l8qI 1.2: 7q31-7q2I::I IplS-I Iq22:7q2I-+7qter),der(l4)t(l4;17) (pl l;q2l),- 19,-21;der(22)t(l7;22)(qI I 2q13)X2[cp20] 47JX,der( 1)t(I;6)(q4;3).de I)t(I;I2)(p I3;q II-I2) x 2,der (I6)t( 16; I7)(qU;q 1 l),dcr( I9)t(8; I9)O;q I3)[45]/47,idem,der (IO)t(+ IO)(q22;q26)[ I51 MYCN verification dmin, MYCN' dmin, MYCN' dmin, MYCN- MYCN 2-1 5 copies MYCNdmin, MYCN' MYCN- dmin, MYCN' ins(!;l)(!;p23-24) MYCN' MYCN' *Abnormalities facilitated by SKY are shown in bold. deur et al., 1977). T h e interpretation of the full karyotypic picture can be complicated further by the presence of divergent clonal evolution and nonclonal rearrangements. We have used SKY combined with classical G-banding to characterize a series of primary neuroblastomas karyotypically. Classical cytogenetic investigation of neuroblastoma is notoriously problematic, and unidentified chromosomal regions will often remain even after analysis by a skilled cytogeneticist. SKY is a novel technique that, when used alone, allows the identification of chromosomes involved in numerical and structural aberrations in the investigated tumor. However, when the SKY results are used in conjunction with G-banding, specific chromosomal segments and breakpoints can be defined, thereby allowing an almost full characterization of the karyotype. We showed that the majority of rearrangements detected were unbalanced (Table 21, particularly so in the case of l p and 17q. Deletions of l p have been associated with a poor prognosis in neuroblastoma (Franke et al., 1986; Fong et al., 1989 Caron et al., 1996; Schleiermacher et al., 1996). T h e five cases with a I p abnormality in this series were all the result of an unbalanced translocation. This observation would suggest that the majority of l p deletions in neuroblastoma result from such an event rather than solely from a deletion event. Two of the del( l p ) cases resulted from translocations involving 17q. Such a rearrangement has previously been suggested to be the most common rearrangement event in neuroblastoma (Franke et al., 1986). Chromosome arm 17q was found to be involved in unbalanced translocations, in addition to lp, with five chromosomal regions: 4q26-28, 9q21, 1 6 ~ 1 3 , UNlDENT/FlED CHROMOSOME ABNORMALITIES 205 A B Figure I Case 6 A SKY spectra-based colors classificauor colors of chromosomes 13 14, 15. and 21 centromeres differ from the rest of the chromosome due t o nonspeclfic sequences in those regions The following chromosomal abnormalitiesare seen clearlr ir the kayogram der( I)t( I.7)(p36.') der(6)t16. i4)(q2I.q 1 I). der(7\t(6.7)('~2I 1 . ~ 2 2 ) der( I 6 k ( 16.I7)(p' 3.02 I )t(F i 7)1qi i.q251 and der( 17)t14.I7)iq26-26 p I I) E Polyptola memphase celi in RGB display colors SKY can detect markers ever in polyplotd cells where full analysis cannot be aone The arrows indicate rearranged chromosomes a dep(7)t(6.7)f!~21 .~221b der( l6)t(I 6 l7)(pi 3.q2 I )t(9 '7)(q2I .q25) c der( 17~14I.7)(o26-28.p 1 1 1 d der(6)t16 14);q2 I.q I ) e dmins-MYCkpositivc ir the coio" LS chro- mosome i 206 COHEN ET A L B i Figure 2. Case 9. A Chromosomalaberrations identified by SKY are seen in chromosomes I . 5, 6, I I, 14. and 2 2 der(l)t(l;17)(p36q2I). der(5)t(5;12)(?;?). der(6)~(6;I2)(q2l;qI?4), der(\ 1)'7qter-7q21:: I Ip I5+1 Iq22:7q2 I+7qter). der( I I)( I % 1::7q3?+7qZI:: I IpI5- I Iq227q2 I +7qrer), der( I4)t( 14; And der(22)t(l7: ii iii 22)tq I I.2:q I3)x2. B: Normal and aberrant chromosomes I I, after G- banding and SKY. i. normal chromosome I I; ii. der(l 1)(7qter+ 7q.11::I IplS-I Iq227q2I-+7qter): iii. der(l I)(IBqter-I8qi 1.2: 7q3?-7q2 I::I Ip 1 5 4 1 Iq22:7q2 I+7qrer). 16q22, and 1 9 ~ 1 3I.nterestingly, cases were found to have multiple 17q rearrangements, with different breakpoints on chromosome Arm 17q. In- deed, further use of SKY in the identification of partner chromosomes in these types of prognosti- cdly important rearrangements may ultimately lead to a refinement of the clinical implications of these events. -IlYCLV~mplificationis a common event in neu- roblastoma and one that has great importance in the clinical outcome (Brodeur et al., 1992: Yoshi- mot0 et al.. 1999). SKY was able to detect only 3/4 cases with dmin-.MYC.L- amplification. previously verified by FISH. This illustrates that, although SKY is a powerful technique, it still has its limita- Lions. Thus. small dmins may not always be de- tected. .\ further current limitation is that intra- chromosomal rearrangements such as inversions will not be detected. showing that SKY alone is insufficient. In addition to the previously described involve- ment of l p and 17q, a number of other chromo- soma1 bands were involved in more than one tumor: lq26, 7p22, 7q11.2, 18q11.2, and 19q13. It should also be noted chat there was frequent partial gain through structural rearrangements of chromosome 7 . T h e involvement of Iq. 7/7q, and 18q could be significant because gains of these segments have also been reported frequently in cytogenetic and/or CGH studies in neuroblastoma (AItura et al., 1997; Van Roy et al., 1997). T h e contribution of genes in these regions to neuroblastoma tumorigenesis should be the target of further investigation. h coordinate association between the loss of 3p 3nd 1l q has recently been described by Breen et al. (3000).In this present study, only one case (case 2 ) had a deletion of 3p, with a further three having loss of the whole chromosome (cases 7, 8, 9). Five cases also had deletions of 1l q or loss of the whole chromosome (cases 1, 5, 7, 8. 9). Therefore, our results can be said neither to support nor disprove the association. T h e regions 3p and l l q probably have an important role to play in the tumorigenesis UNIDENTIFIED CHROMaSOME ABNORMAUTIES 207 of neuroblastoma, but whether their combined loss is nonrandom requires further proof. Our work shows the capability of the SKY tech- nique to identify novel marker chromosomes, to clarify complex chromosome aberrations, and to describe clonal evolution in neuroblastoma. T h e combination of conventional karyotyping and the SKY technique allows an improved understanding of solid tumors. T h e contribution of SKY to the field of neuroblastoma cytogenetics is potentiall) of great value, because more precise definition of chromosomal rearrangements may lead to new in- formation c0ncernir.g genetic events in neuroblas- toma oncogenesis and to the identification of prog- nostic markers. In summar)., the full strength of SKY is observed when it is used in combination with conventional banding techniques, resulting in the following gains and thus leading to further clarification of neuroblastomas: reconfirmation of the G-banding results; identification of partially characterized re- arrangements; definition of markers of unknown 1 ,L* origin; reclassification of G-ba her refine- ments of breakpoints original ned by G- banding; and analysis of both and poor- morpholog?, metaphase cell typically cannot be fully analyzed by classic Cytogenetics. ACKNOWLEDGMENTS We thank Mrs. Bella Weisman for excellent technical assistance. This work was performed in partial fulfillment of N.C.'srequirements for a PhD at the Sackler School of Medicine, Tel Aviv University. REFERENCES Altura RA. Maris JM, Li H, Bovett JM. Brodeur GM, Look .4T. 1997. Novel regions of chromosomal loss in familial neurohlastoma by comparative genomic hybridization. Genes Chromosomes Cancer 19:176-184. 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