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CHOP
200
COMPARATIVE GENOMIC HYBRIDIZATION AS A TOOL TO DEFINE TWO DISTINCT CHROMOSOME 12-DERIVED AMPLIFICATION UNITS IN WELL-DIFFERENTIATED LIPOSARCOMAS Ron F. SUIJKERBUIJK ~ Daniel E.M. OLDE WEGHU1S~, Margot VAN DEN BERG~, Florence PEDEUTOUR 2, Anne FORUS3, Ola MYKLEBOST 3, Cliff GLIER4, Claude TURC-CAREL:, Ad GEURTS VAN KESSEL~ JDepartment of Human Genetics, University Hospital, Nijmegen, The Netherlands 2Laboratoire de G4naique Mol&ulaire des Cancers Humains, URA CNRS 1462, Nice, France 3Department of Tumor Biology, the Norwegian Radium Hospital, Oslo, Norway 4Biological Detection Systems, Inc., Rockville (MD), USA The characteristic presence of supernumerary ring and/or giant rod-shaped marker chromosomes as sole detectable cytogenetic abnormalities in human welldifferentiated liposarcomas (WDLPS) suggests an important role for these markers in the development of this type of soft tissue tumors. Recently, we and others have demonstrated the consistent involvement of chromosome 12-derived sequences in the formation of these rings and giant marker chromosomes [1]. In addition, our studies indicated the participation of one (or more) other chromosomes (i.e., chromosome 1, 4, 8 and 16) in most of these extraordinary rearrangements. No 12p-derived sequences were found to be present in the markers of 3 out of 3 WDLPS examined, suggesting overrepresentation of only 12q-derived material. The observation of a consistent amplification of selected 12q-derived genes (i.e., SAS and MDM2) in WDLPS, underlines this latter suggestion. In order to ascertain that (some of) these amplification events represent non-coincidental features in WDLPS, we have initiated the examination of a series of WDLPS tumor samples for the presence of common regions of DNA amplification by means of comparative genomic hybridization, CGH [2,3]. Eight tumor samples, i.e., six primary WDLPS cases and two primary myxoid liposarcomas, were investigated. Interestingly, two distinct chromosome 12derived amplification units could be identified in all WDLPS tumors examined, one being located in the q14-q15 region as expected, and a second one in the q21.3-q22 region, which was unexpected. No amplifications were observed in the myxoid liposarcomas. These results indicate that the concerted amplification of two distinct but specific regions on the long arm of chromosome 12 may be a consistent characteristic of WDLPS. These amplifications are most likely directly related to the presence of the supernumerary ring and/or giant marker chromosomes in this group of soft tissue tumors. [ 1] Pedeutour F. et al., Cancer Genet Cytogenet 66:133-134 (1993). [2] Kallioniemi A. et al., Science 258:818-821 (1992). [3] Du Manoir S. et al., Hum Genet 90:590-610 (1993).
12q 13-14 AMPLICA IN HUMAN S ARCOMAS WITHOUT MDM2 INCLUDE CDK4 , SAS AND GADD 153~CHOP. Anne Forus. Vivi A. Flcrenes, Gunhild M. Maelandsmo, Paul S. Meltzer, ~ystein Fodstad and Ola Myklebost. Dept. of Tumor Biology, The Norwegian Radium Hospital, 0310 OSLO, Norway. In human sarcomas, amplification of several genes located to the q13-14 region of chromosome 12 has been reported. A selective growth advantage has been assigned to increased copy number and expression of MDM2, because the mdm2 protein may inactivate the tumor suppressor protein p53. However, the CDK4 gene, coding for a cyclin-dependent kinase, has recently been located between SAS and MDM2 (Meltzer etal, unpublished results), and could be another candidate gene. We have previously analyzed the amplification of SAS, MDM2, GADD153/CHOP, GLI and A2MR in a panel of 98 human sarcomas of different subtypes, and in two additional cell lines with GLI amplifications (Forus etal 1993, Cell Growth & Diff 4: 1065-1070). SAS was amplified in 12 samples, MDM2 in 10, CHOP in 6, GL/in 4 andA2MR in 3. In most cases, amplification was associated with increased expression of the corresponding gene. MDM2 and SAS were co-amplified in 9 samples. One liposarcoma showed amplification of MDM2 alone whereas two osteosarcomas an a rhabdomyosarcoma celt line showed amplification of SAS and CHOP but not MDM2. Interestingly, 2/2 fibrosarcomas had co-amplification of MDM2 and SAS, but not the other genes. We have now analyzed amplification of CDK4 in samples with amplifications of SAS or MDM2, and found that CDK4 was always co-amplified with SAS. Neither CDK4 nor any of the other genes studied fulfil the criteria of a common selective gene, but a possible common target could be located between CDK4 and MDM2. However, it is not unlikely that more than one mechanism is responsible for selection of 12q 13-14 amplica in different sarcoma types.
Abstracts
B R E A K P O I N T A T B A N D q13 ON BOTH C H R O M O S O M E S 12 IN A
PARAOSTEAL CHONDROMA. A.VERHEST,G.MfllIer,JM.Dangou,M.Petein. Dept.of Cyto genetics,Institut Jules Bordet, Brussels - Belgium. Our knowledge of the role of chromomosomal changes in benign tumors is still sparse however chromosome 12 already appears as non randomly involved in various types of e p i t h e l i a l and m e s e n c h y m a l neoplams. The few reported benign cartilaginous tumors are chromosomally different in each single case. It is therefore relevant to present a second case of paraosteal chondroma with both chromosomes 12 rearranged in q13. (N.Mandhal et al., Genes,Chrom & Cancer 6,121-123,1993) A 23-year-old man underwent a partial excision of the proximal part of the humerus for an osteolytic lesion of 1.5 cm size with outward extension. The histologic feature was characteristic of a para osteal chondroma with production of m y x o i d cartilage circumscribed by a shell of endosteal cortex. A sample of the tumor was cultured for cytogenetic analysis. G banding performed on 24 mitoses showed a pseudodiploid karyotype 46 XY, t(7;12) (pll.2; q13.1), t(12;16) (q13;p13). Paraesteal chondroma is a rare cartilaginous tumor, its behavior is benign if completely removed but already hallmarked by an abnormal karyotype from its clinical onset. In spite of the few data, the very similar breakpoints strongly suggest the presence of a gene near 12q13 keynoting this type of benign tumor.
MOLECULAR CYTOGENETIC STUDY OF SOFT TISSUE S A R C O M A . F a b i o l a Minoletti. G a b r i e l l a Sozzi, M o n i c a Miozzo, L a u r a Sard, A l b e r t o Azzarelli, Silvana Pilotti, M a r c o A. Pierotti Istituto N a z i o n a l e T u m o r i , Milan, Italy. Soft tissue s a r c o m a s a r e a h e t e r o g e n e o u s g r o u p o f m a l i g n a n t t u m o r s with a w i d e r a n g e o f clinical p r e s e n t a t i o n , m o r p h o l o g i c features and biologic behaviour. These features and the recent d e v e l o p m e n t o f d i f f e r e n t i a t e d t r e a t m e n t r e g i m e n s f o r d i f f e r e n t soft tissue s a r c o m a s stress the n e e d f o r a r e f i n e d histologic classification u s i n g o t h e r c o m p l e m e n t a r y a p p r o a c h e s s u c h as cytogenetic a n d m o l e c u l a r g e n e t i c analyses. In t h e p r e s e n t s t u d y w e c o u p l e d classical c y t o g e n e t i c s a n d f l u o r e s c e n t in situ h y b r i d i z a t i o n ( F I S H ) b o t h o n m e t a p h a s e s a n d i n t e r p h a s e nuclei to d e m o n s t r a t e t h e feasibility o f this a p p r o a c h for studying t u m o r - s p e c i f i c c h r o m o s o m e r e a r r a n g e m e n t s in soft tissue s a r c o m a s of d i f f e r e n t histotype. T h e t u m o r tissue w e r e disaggre[gated with c o l l a g e n a s e II a n d the c h r o m o s o m e p r e p a r a t i o n s w e r e o b t a i n e d a f t e r s h o r t - t e r m i n c u b a t i o n following s t a n d a r d p r o c e d u r e s . In 31 cases o f d i f f e r e n t histotype successfully analyzed, 24 s h o w e d the p r e s e n c e of specific c h r o m o s o m e r e a r r a n g e m e n t s s u c h as t(X;18) in synovial s a r c o m a , t(12;16) in myxoid l i p o s a r c o m a , t(11,22) in primitive n e u r o e c t o d e r m a l t u m o r s , t(2;13) in r h a b d o m y o s a r c o m a a n d r i n g c h r o m o s o m e s in d e r m a t o f i b r o s a r c o m a p r o t u b e r a n s . In o t h e r cases the a b s e n c e o f specific c h r o m o s o m e c h a n g e s o r the c o m p l e x i t y o f the k a r y o t y p e w e r e of help for a differential diagnosis. T h e F I S H analysis using c e n t r o m e r i c a n d / o r p a i n t i n g p r o b e s n o t only c o n f i r m e d t h e c y t o g e n e t i c results b u t also a l l o w e d the identification of t u m o r specific c h r o m o s o m e c h a n g e s in t h o s e cases p r e s e n t i n g low m i t o t i c i n d e x o r with p o o r q u a l i t y c h r o m o s o m e s . In a b s e n c e of a n a l y z a b l e m e t a p h a s e s , F I S H w a s successfully p e r f o r m e d o n i n t e r p h a s e nuclei. N o t e w o r t h y w e w e r e a b l e to a n a l y s e by F I S H also G - b a n d e d c h r o m o s o m e s p r e a d s a g e d u p to 1 y e a r to d e t e c t t h e p r e s e n c e o f t u m o r - s p e c i f i c c h r o m o s o m e c h a n g e s . T h e results sug~gest a clinical r e l e v a n c e o f m o l e c u l a r c y t o g e n e t i c analysis in soft Ussue s a r c o m a s . This w o r k is p a r t i a l l y s u p p o r t e d b y A I R C a n d C N R Special P r o j e c t "ACRO".
COMPARATIVE GENOMIC HYBRIDIZATION AS A TOOL TO DEFINE TWO DISTINCT CHROMOSOME 12-DERIVED AMPLIFICATION UNITS IN WELL-DIFFERENTIATED LIPOSARCOMAS Ron F. SUIJKERBUIJK ~ Daniel E.M. OLDE WEGHU1S~, Margot VAN DEN BERG~, Florence PEDEUTOUR 2, Anne FORUS3, Ola MYKLEBOST 3, Cliff GLIER4, Claude TURC-CAREL:, Ad GEURTS VAN KESSEL~ JDepartment of Human Genetics, University Hospital, Nijmegen, The Netherlands 2Laboratoire de G4naique Mol&ulaire des Cancers Humains, URA CNRS 1462, Nice, France 3Department of Tumor Biology, the Norwegian Radium Hospital, Oslo, Norway 4Biological Detection Systems, Inc., Rockville (MD), USA The characteristic presence of supernumerary ring and/or giant rod-shaped marker chromosomes as sole detectable cytogenetic abnormalities in human welldifferentiated liposarcomas (WDLPS) suggests an important role for these markers in the development of this type of soft tissue tumors. Recently, we and others have demonstrated the consistent involvement of chromosome 12-derived sequences in the formation of these rings and giant marker chromosomes [1]. In addition, our studies indicated the participation of one (or more) other chromosomes (i.e., chromosome 1, 4, 8 and 16) in most of these extraordinary rearrangements. No 12p-derived sequences were found to be present in the markers of 3 out of 3 WDLPS examined, suggesting overrepresentation of only 12q-derived material. The observation of a consistent amplification of selected 12q-derived genes (i.e., SAS and MDM2) in WDLPS, underlines this latter suggestion. In order to ascertain that (some of) these amplification events represent non-coincidental features in WDLPS, we have initiated the examination of a series of WDLPS tumor samples for the presence of common regions of DNA amplification by means of comparative genomic hybridization, CGH [2,3]. Eight tumor samples, i.e., six primary WDLPS cases and two primary myxoid liposarcomas, were investigated. Interestingly, two distinct chromosome 12derived amplification units could be identified in all WDLPS tumors examined, one being located in the q14-q15 region as expected, and a second one in the q21.3-q22 region, which was unexpected. No amplifications were observed in the myxoid liposarcomas. These results indicate that the concerted amplification of two distinct but specific regions on the long arm of chromosome 12 may be a consistent characteristic of WDLPS. These amplifications are most likely directly related to the presence of the supernumerary ring and/or giant marker chromosomes in this group of soft tissue tumors. [ 1] Pedeutour F. et al., Cancer Genet Cytogenet 66:133-134 (1993). [2] Kallioniemi A. et al., Science 258:818-821 (1992). [3] Du Manoir S. et al., Hum Genet 90:590-610 (1993).
12q 13-14 AMPLICA IN HUMAN S ARCOMAS WITHOUT MDM2 INCLUDE CDK4 , SAS AND GADD 153~CHOP. Anne Forus. Vivi A. Flcrenes, Gunhild M. Maelandsmo, Paul S. Meltzer, ~ystein Fodstad and Ola Myklebost. Dept. of Tumor Biology, The Norwegian Radium Hospital, 0310 OSLO, Norway. In human sarcomas, amplification of several genes located to the q13-14 region of chromosome 12 has been reported. A selective growth advantage has been assigned to increased copy number and expression of MDM2, because the mdm2 protein may inactivate the tumor suppressor protein p53. However, the CDK4 gene, coding for a cyclin-dependent kinase, has recently been located between SAS and MDM2 (Meltzer etal, unpublished results), and could be another candidate gene. We have previously analyzed the amplification of SAS, MDM2, GADD153/CHOP, GLI and A2MR in a panel of 98 human sarcomas of different subtypes, and in two additional cell lines with GLI amplifications (Forus etal 1993, Cell Growth & Diff 4: 1065-1070). SAS was amplified in 12 samples, MDM2 in 10, CHOP in 6, GL/in 4 andA2MR in 3. In most cases, amplification was associated with increased expression of the corresponding gene. MDM2 and SAS were co-amplified in 9 samples. One liposarcoma showed amplification of MDM2 alone whereas two osteosarcomas an a rhabdomyosarcoma celt line showed amplification of SAS and CHOP but not MDM2. Interestingly, 2/2 fibrosarcomas had co-amplification of MDM2 and SAS, but not the other genes. We have now analyzed amplification of CDK4 in samples with amplifications of SAS or MDM2, and found that CDK4 was always co-amplified with SAS. Neither CDK4 nor any of the other genes studied fulfil the criteria of a common selective gene, but a possible common target could be located between CDK4 and MDM2. However, it is not unlikely that more than one mechanism is responsible for selection of 12q 13-14 amplica in different sarcoma types.
Abstracts
B R E A K P O I N T A T B A N D q13 ON BOTH C H R O M O S O M E S 12 IN A
PARAOSTEAL CHONDROMA. A.VERHEST,G.MfllIer,JM.Dangou,M.Petein. Dept.of Cyto genetics,Institut Jules Bordet, Brussels - Belgium. Our knowledge of the role of chromomosomal changes in benign tumors is still sparse however chromosome 12 already appears as non randomly involved in various types of e p i t h e l i a l and m e s e n c h y m a l neoplams. The few reported benign cartilaginous tumors are chromosomally different in each single case. It is therefore relevant to present a second case of paraosteal chondroma with both chromosomes 12 rearranged in q13. (N.Mandhal et al., Genes,Chrom & Cancer 6,121-123,1993) A 23-year-old man underwent a partial excision of the proximal part of the humerus for an osteolytic lesion of 1.5 cm size with outward extension. The histologic feature was characteristic of a para osteal chondroma with production of m y x o i d cartilage circumscribed by a shell of endosteal cortex. A sample of the tumor was cultured for cytogenetic analysis. G banding performed on 24 mitoses showed a pseudodiploid karyotype 46 XY, t(7;12) (pll.2; q13.1), t(12;16) (q13;p13). Paraesteal chondroma is a rare cartilaginous tumor, its behavior is benign if completely removed but already hallmarked by an abnormal karyotype from its clinical onset. In spite of the few data, the very similar breakpoints strongly suggest the presence of a gene near 12q13 keynoting this type of benign tumor.
MOLECULAR CYTOGENETIC STUDY OF SOFT TISSUE S A R C O M A . F a b i o l a Minoletti. G a b r i e l l a Sozzi, M o n i c a Miozzo, L a u r a Sard, A l b e r t o Azzarelli, Silvana Pilotti, M a r c o A. Pierotti Istituto N a z i o n a l e T u m o r i , Milan, Italy. Soft tissue s a r c o m a s a r e a h e t e r o g e n e o u s g r o u p o f m a l i g n a n t t u m o r s with a w i d e r a n g e o f clinical p r e s e n t a t i o n , m o r p h o l o g i c features and biologic behaviour. These features and the recent d e v e l o p m e n t o f d i f f e r e n t i a t e d t r e a t m e n t r e g i m e n s f o r d i f f e r e n t soft tissue s a r c o m a s stress the n e e d f o r a r e f i n e d histologic classification u s i n g o t h e r c o m p l e m e n t a r y a p p r o a c h e s s u c h as cytogenetic a n d m o l e c u l a r g e n e t i c analyses. In t h e p r e s e n t s t u d y w e c o u p l e d classical c y t o g e n e t i c s a n d f l u o r e s c e n t in situ h y b r i d i z a t i o n ( F I S H ) b o t h o n m e t a p h a s e s a n d i n t e r p h a s e nuclei to d e m o n s t r a t e t h e feasibility o f this a p p r o a c h for studying t u m o r - s p e c i f i c c h r o m o s o m e r e a r r a n g e m e n t s in soft tissue s a r c o m a s of d i f f e r e n t histotype. T h e t u m o r tissue w e r e disaggre[gated with c o l l a g e n a s e II a n d the c h r o m o s o m e p r e p a r a t i o n s w e r e o b t a i n e d a f t e r s h o r t - t e r m i n c u b a t i o n following s t a n d a r d p r o c e d u r e s . In 31 cases o f d i f f e r e n t histotype successfully analyzed, 24 s h o w e d the p r e s e n c e of specific c h r o m o s o m e r e a r r a n g e m e n t s s u c h as t(X;18) in synovial s a r c o m a , t(12;16) in myxoid l i p o s a r c o m a , t(11,22) in primitive n e u r o e c t o d e r m a l t u m o r s , t(2;13) in r h a b d o m y o s a r c o m a a n d r i n g c h r o m o s o m e s in d e r m a t o f i b r o s a r c o m a p r o t u b e r a n s . In o t h e r cases the a b s e n c e o f specific c h r o m o s o m e c h a n g e s o r the c o m p l e x i t y o f the k a r y o t y p e w e r e of help for a differential diagnosis. T h e F I S H analysis using c e n t r o m e r i c a n d / o r p a i n t i n g p r o b e s n o t only c o n f i r m e d t h e c y t o g e n e t i c results b u t also a l l o w e d the identification of t u m o r specific c h r o m o s o m e c h a n g e s in t h o s e cases p r e s e n t i n g low m i t o t i c i n d e x o r with p o o r q u a l i t y c h r o m o s o m e s . In a b s e n c e of a n a l y z a b l e m e t a p h a s e s , F I S H w a s successfully p e r f o r m e d o n i n t e r p h a s e nuclei. N o t e w o r t h y w e w e r e a b l e to a n a l y s e by F I S H also G - b a n d e d c h r o m o s o m e s p r e a d s a g e d u p to 1 y e a r to d e t e c t t h e p r e s e n c e o f t u m o r - s p e c i f i c c h r o m o s o m e c h a n g e s . T h e results sug~gest a clinical r e l e v a n c e o f m o l e c u l a r c y t o g e n e t i c analysis in soft Ussue s a r c o m a s . This w o r k is p a r t i a l l y s u p p o r t e d b y A I R C a n d C N R Special P r o j e c t "ACRO".