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Chromosome analysis of human neuroblastoma
Abstracts
275
lines with the cosmid clone DIS98 and VNTR-probes (e.g. DIS32, DIZ2), all p r o b e s are s p e c i f i c for c h r o m o s o m e r e g i o n ip36. Using ISH c o n d i t i o n s that suppress u n s p e c i f i c signals we could d e t e r m i n e the exact spacial assignment of t h e p r o b e s to e a c h o t h e r a n d visualize the a b s e n c e or p r e s e n c e of t h e s e s e q u e n c e s in n e u r o blastoma c e l l lines. ISH on i n t e r p h a s e nuclei with chromosome s p e c i f i c probes offers the p o s s i b i l i t y to d e t e c t n u m e r i c a l chromosome aberrations and d e l e t i o n s w i t h o u t the n e c e s s i t y to c u l t u r e t u m o r c e l l s or to p r e p a r e c h r o m o s o m e spreads. In n e u r o b l a s t o m a a d e l e t i o n on c h r o m o s o m e ip is c o n s i s t e n t l y c o r r e l a t e d w i t h a poor p r o g n o s i s w h e r e a s only numerical c h r o m o s o m e changes (±3x) indicate a b e t t e r outcome. We p e r f o r m e d d o u b l e - t a r g e t non i s o t o p i c ISH on tumor cells from p a t i e n t s of d i f f e r e n t c l i n i c a l s t a g e s as well as on n e u r o b l a s t o m a cell lines u s i n g V N T R - p r o b e s (e.g. DIS32, DIZ2) s p e c i f i c for c h r o m o s o m e r e g i o n ip36 and t h e lqh s p e c i f i c p r o b e DIZI. We could clearly evaluate the number of intact c h r o m o s o m e s 1 and the ratio of c h r o m o s o m e s 1 with a d e l e t i o n at ip36.
109
D E M O N S T R A T I O N OF N M Y C A M P L I F I C A T I O N IN N E U R O B L A S T O M A . C. Rudduck, T. M c R o b e r t , R. Lukeis, U. K e e s * & O.M. Garson. Depts. of Medicine and C y t o g e n e t i c s , St. Vincent's Hospital Melbourne and * C l i n i c a l Immunology Research Unit, Princess M a r g a r e t Hospital, Perth, A u s t r a l i a . A n o n - r a d i o a c t i v e c h r o m o s o m a l in situ h y b r i d i z a t i o n technique utilizing a biotin-strepavidin-polyalkaline-phosphatase complex, was a p p l i e d to three n e u r o b l a s t o m a cell lines and to b o n e marrow c h r o m o s o m e p r e p a r a t i o n s f r o m two p a t i e n t s w i t h n e u r o b l a s t o m a , for detection of N-myc a m p l i f i c a t i o n . The c e l l lines w e r e derived from consecutJive b o n e m a r r o w samples from a p a t i e n t w i t h stage IV neuroblastoma. The c e l l line d e r i v e d at d i a g n o s i s had N-myc a m p l i f i c a t i o n in the form of d o u b l e m i n u t e c h r o m o s o m e s , fragments and rings of v a r y i n g n u m b e r and size. The cell lines derived from subsequent samples contained amplified N-myc as two homogeneously s t a i n i n g r e g i o n s (HSR). N-myc amplification was analyzed in c h r o m o s o m e p r e p a r a t i o n s from a p r i m a r y tumor in a patient with stage III n e u r o b l a s t o m a and f r o m the infiltrated bone n a r r o w of a p a t i e n t w i t h stage IV, b o t h at d i a g n o s i s . The p r i m a r y tumor had two H S R s of amlmlified N - m y c d e t e c t e d by in situ hybridization. O n l y one of these had b e e n i d e n t i f i e d as an HSR by standard cytogenetic techniques, the other having been d e s i g n a t e d a m a r k e r c h r o m o s o m e . P r e p a r a t i o n s f r o m the i n f i l t r a t e d bone marrow had N-myc a m p l i f i c a t i o n in the fo~m of double minutes, the h e t e r o g e n e i t y of these also being noticeable in interphase cells. Therefore this t~chnique is a useful i n d i c a t o r of gene a m p l i f i c a t i o n in both m e t a p h a s e a n d Interphase cells.
275
lines with the cosmid clone DIS98 and VNTR-probes (e.g. DIS32, DIZ2), all p r o b e s are s p e c i f i c for c h r o m o s o m e r e g i o n ip36. Using ISH c o n d i t i o n s that suppress u n s p e c i f i c signals we could d e t e r m i n e the exact spacial assignment of t h e p r o b e s to e a c h o t h e r a n d visualize the a b s e n c e or p r e s e n c e of t h e s e s e q u e n c e s in n e u r o blastoma c e l l lines. ISH on i n t e r p h a s e nuclei with chromosome s p e c i f i c probes offers the p o s s i b i l i t y to d e t e c t n u m e r i c a l chromosome aberrations and d e l e t i o n s w i t h o u t the n e c e s s i t y to c u l t u r e t u m o r c e l l s or to p r e p a r e c h r o m o s o m e spreads. In n e u r o b l a s t o m a a d e l e t i o n on c h r o m o s o m e ip is c o n s i s t e n t l y c o r r e l a t e d w i t h a poor p r o g n o s i s w h e r e a s only numerical c h r o m o s o m e changes (±3x) indicate a b e t t e r outcome. We p e r f o r m e d d o u b l e - t a r g e t non i s o t o p i c ISH on tumor cells from p a t i e n t s of d i f f e r e n t c l i n i c a l s t a g e s as well as on n e u r o b l a s t o m a cell lines u s i n g V N T R - p r o b e s (e.g. DIS32, DIZ2) s p e c i f i c for c h r o m o s o m e r e g i o n ip36 and t h e lqh s p e c i f i c p r o b e DIZI. We could clearly evaluate the number of intact c h r o m o s o m e s 1 and the ratio of c h r o m o s o m e s 1 with a d e l e t i o n at ip36.
109
D E M O N S T R A T I O N OF N M Y C A M P L I F I C A T I O N IN N E U R O B L A S T O M A . C. Rudduck, T. M c R o b e r t , R. Lukeis, U. K e e s * & O.M. Garson. Depts. of Medicine and C y t o g e n e t i c s , St. Vincent's Hospital Melbourne and * C l i n i c a l Immunology Research Unit, Princess M a r g a r e t Hospital, Perth, A u s t r a l i a . A n o n - r a d i o a c t i v e c h r o m o s o m a l in situ h y b r i d i z a t i o n technique utilizing a biotin-strepavidin-polyalkaline-phosphatase complex, was a p p l i e d to three n e u r o b l a s t o m a cell lines and to b o n e marrow c h r o m o s o m e p r e p a r a t i o n s f r o m two p a t i e n t s w i t h n e u r o b l a s t o m a , for detection of N-myc a m p l i f i c a t i o n . The c e l l lines w e r e derived from consecutJive b o n e m a r r o w samples from a p a t i e n t w i t h stage IV neuroblastoma. The c e l l line d e r i v e d at d i a g n o s i s had N-myc a m p l i f i c a t i o n in the form of d o u b l e m i n u t e c h r o m o s o m e s , fragments and rings of v a r y i n g n u m b e r and size. The cell lines derived from subsequent samples contained amplified N-myc as two homogeneously s t a i n i n g r e g i o n s (HSR). N-myc amplification was analyzed in c h r o m o s o m e p r e p a r a t i o n s from a p r i m a r y tumor in a patient with stage III n e u r o b l a s t o m a and f r o m the infiltrated bone n a r r o w of a p a t i e n t w i t h stage IV, b o t h at d i a g n o s i s . The p r i m a r y tumor had two H S R s of amlmlified N - m y c d e t e c t e d by in situ hybridization. O n l y one of these had b e e n i d e n t i f i e d as an HSR by standard cytogenetic techniques, the other having been d e s i g n a t e d a m a r k e r c h r o m o s o m e . P r e p a r a t i o n s f r o m the i n f i l t r a t e d bone marrow had N-myc a m p l i f i c a t i o n in the fo~m of double minutes, the h e t e r o g e n e i t y of these also being noticeable in interphase cells. Therefore this t~chnique is a useful i n d i c a t o r of gene a m p l i f i c a t i o n in both m e t a p h a s e a n d Interphase cells.