- Email: [email protected]
Chromosomal translocations in human malignant diseases
ABSTRACTS 1
Abstract Chromesomal Translocatlons in Human Malignant Diseases Janet 0. Rowley, H.D.
Chromosome translocations have been most closely associated with human leukemias and lymphomas. Recently, they have been shown to occur in sarcomas as well. In each type of tumor, the translocattons are r e l a t i v e l y specifically associated with particular subtypes of these tumors. Cloning the translocatton junctions has identified the genes affected by the breakpoint in these diseases. Although the genes that are affected participate in a number of steps in the complex pathway of transmitting growth regulatory signals from the cell surface to the nucleus, most of those i d e n t i f i e d in the leukmlas and lymphooas act as transcriptional activators. That ts they are ONA binding proteins that d i r e c t l y regulate the level of transcription of the target genes. Some of the genes recently cloned freo breakpoint junctions in sarcomas, also act to regulate transcription. Part of the challenge for the future is to understand the mechanisms leadtng to recurring translocattons and also to understand the tumors p e c i f i c i t y of the translocattons. I t ts hoped that this increasing understanding of the btology of these malignant diseases w i l l lead to more accurate diagnosis, to improved therapy, and possibly to more effective prevention.
2
C H a R A ~ A T I O N OF~
USINGSI'B3~C~
GLOBAL DNA SEQUENCE COPY NUMBER ANALYSIS OF H U M A N TUMORS BY COMPARATIVE GENOMIC HYBRIDIZATION D. Pinkel 1, A. KaUioniemi 1, O. Kallioniemi 1, M. Sakamoto 1, F. Waldman 1, D. Sudar 1, J. Gray l, D. Rutovitz 2, J. Piper 2. 1University of California, San Francisco, 2MRC Edinburgh. F l u o r e s c e n c e in situ h y b r i d i z a t i o n p e r m i t s simultaneous analysis of DNA sequence copy number and structural rearrangements at several specific genetic loci in individual cells. However surveying the entire genome is not practical using specific probes. We have recently d e v e l o p e d a new technique, C o m p a r a t i v e Genomic Hybridization (CGH), which uses the kinetics of in situ h y b r i d i z a t i o n to efficiently o b t a i n g e n o m e - w i d e comparisons of DNA sequence copy number. In a typical application genomic DNAs from tumor and normal cells are differentially labeled and simultaneously hybridized to n o r m a l metaphase chromosomes, and detected with different fluorochromes. Properly registered images of each fluorochrome are obtained using a microscope equipped with multi-band filters and a CCD camera. Digital image analysis permits measurement of intensity ratio profiles along each of the target chromosomes. Studies of cells with known aberrations indicate that the intensity ratio at each position is proportional to the copy n u m b e r of the sequences in the tumor that bind there. Knowledge of the actual copy number at one position permits calibration of the profiles so that copy n u m b e r is known everywhere. Deletions, duplications and higher level amplifications can be detected. Thus comparisons of tumor and normal DNA permit development of a "copy number karyotype" for the t u m o r genome. Alternatively, abnormal genomes, for example a primary t u m o r and its metastases can be compared to detect differences between them.
TUMORS
I.W.Cmq,
A. Kallkw;,~; O. ~,n;a-i-~i, M. Stkamete,EL Nakamm'a,I(. ~ M. hllavl~t~F.Wtldman, D. PtnkeL ~ d ~ o f Z ~ ~,MC~230, Box
0s0s ~ ~
,
of Labon,e=y Medidat. U=~-may of San F ~ ,
CA 94143-080S
~Pluoa~et~e la alttt hyln:kll=atimt w l d t ~ probes is i n u r i n g paz'doAmiy uaoful fo~ d-*-,,v,,~ amt characterization of deletion ~ t m p l i t k a t i o n ev-m= ar,aociated with ere genesis or p m ~ of mlid h m m n t u m o n . This presentation will ~ memt appUeatiom of 1~;-I/=r ~ ar,d ~ o f tivme evertts. S p e e i ~ t o p k = t o b e e o v e m d ~,,,4,,A,.- 1) Detection of eye, rut ustn8 FJ,SH e,.at meeelat~ with lore of l~rz78~ty. S~_,~_' ~ on 17p a n d 16q~bt'm~t ~ and
wglbee=mm=~4~ 2) ~ a n a l y ~ a f f p m ==~e~_eo~ ~ soua e==e=mw(th qwwdtt ~ S ~
mhow~g m u b ~
on
ke1~- madt n ~
he=~p=m~y ~ c u ~ c .ad eeee= ==eeecm~= ~ ov=h~ md bmm ea~mC~esl~iv~y, wel be immmtm~ bapozlance of mu~t ~
~ eaam¢ tt~mmemtm~l
propmt~eonwinbedt~me~ 3) ~ h ~ anatym of e~e extmt o~ ~ m a phy~ d~t md amp.e~uon Va=cm=dby W ~ mdy=~ = by = = e m e v . genomic~ , ~ _ H,n~ exp~____-ncewith daeaonaappi~ o~ l ~ t In b:e~t eane~wln t~ di~me~d. ~ = ~ m el
pzebeiy maeeed,q=e~i= k~,= pmbu wm be kk=em,d. 4) D e t e c t i o n o ~ r m ' e , ~ c c e l l L Recm~mq~kmce with ~ of metasta~: c~ll= In the bone maxzow of patte=ts wittt breast and pzm~W ~ ___-¢eemdfm~ __ to
geno~pe ,rid ~
3
~
v~n b, d ~ m ~ .
FLUORESCENCE IN SITU HYBRIDIZATION TO 4 METAPHASE AND INTERPHASE CHROMOSOMES Barbara Trask 1, Anne Fetlitta2, Mari Christensen2, Susan Allen2. Marge Segravcs 2, Anne Bergmann2, Hillary Massa1, Rainer Sachs 3, and Ger van den Engh z, ]-Dept. Molecular Biotechnoiogy, Univ. Washington, Seattle, WA; 2Lawrence Livermom National Laboratory, Livermom CA; 3Dept. Mathematics, Univ. of California, Berkeley, CA Fluorescence in situ hybridization is rapidly replacing radioactive in situ hybridization and somatic cell hybrid analysis as the method of choice to determine the chromosomal location of DNA sequences. DNA sequences ranging in size from several kbp (plasmids) to several 100 kbps (YACs) can be mapped to chromosome bands by FISH. The development of multicolor FISH allows probes to be mapped relative to each other and, thus. at higher resolution than is possible by single probe mapping relative to chromosome length or to bands. Two probes can be accurately mapped relative to each other in metaphnse chromatin if the distance between them exceeds -1 Mop. Higher map resolution can he achieved by mapping probes relative to each other in interphase chromatin, lnterphnse chromatin behaves as a random polymer in the 50 kbp to 1-2 Mbp range and follows a random walk model (mean square interphase distance C( genomio distance). Therefore, the relative spacing of DNA sequences can be deduced from pair-wise interphase distance measurements. Based on these results, a multilevel strategy has been developed for producing a 100-kbp average density probe map using FISH. Examples of the application of this strategy to build maps of regions of chromosomes 4 and 19 will be shown. Together, an understanding of the organization of inter-phase chromatin after hybridization and the identification of probes mapped along each chromosome can be used to diagnose chromosome abnormalities below the detection limits of conventional metaphase cytogenetic analysis. Work funded by U.S.D.O.E. and NIH (HG00256).
Abstract Chromesomal Translocatlons in Human Malignant Diseases Janet 0. Rowley, H.D.
Chromosome translocations have been most closely associated with human leukemias and lymphomas. Recently, they have been shown to occur in sarcomas as well. In each type of tumor, the translocattons are r e l a t i v e l y specifically associated with particular subtypes of these tumors. Cloning the translocatton junctions has identified the genes affected by the breakpoint in these diseases. Although the genes that are affected participate in a number of steps in the complex pathway of transmitting growth regulatory signals from the cell surface to the nucleus, most of those i d e n t i f i e d in the leukmlas and lymphooas act as transcriptional activators. That ts they are ONA binding proteins that d i r e c t l y regulate the level of transcription of the target genes. Some of the genes recently cloned freo breakpoint junctions in sarcomas, also act to regulate transcription. Part of the challenge for the future is to understand the mechanisms leadtng to recurring translocattons and also to understand the tumors p e c i f i c i t y of the translocattons. I t ts hoped that this increasing understanding of the btology of these malignant diseases w i l l lead to more accurate diagnosis, to improved therapy, and possibly to more effective prevention.
2
C H a R A ~ A T I O N OF~
USINGSI'B3~C~
GLOBAL DNA SEQUENCE COPY NUMBER ANALYSIS OF H U M A N TUMORS BY COMPARATIVE GENOMIC HYBRIDIZATION D. Pinkel 1, A. KaUioniemi 1, O. Kallioniemi 1, M. Sakamoto 1, F. Waldman 1, D. Sudar 1, J. Gray l, D. Rutovitz 2, J. Piper 2. 1University of California, San Francisco, 2MRC Edinburgh. F l u o r e s c e n c e in situ h y b r i d i z a t i o n p e r m i t s simultaneous analysis of DNA sequence copy number and structural rearrangements at several specific genetic loci in individual cells. However surveying the entire genome is not practical using specific probes. We have recently d e v e l o p e d a new technique, C o m p a r a t i v e Genomic Hybridization (CGH), which uses the kinetics of in situ h y b r i d i z a t i o n to efficiently o b t a i n g e n o m e - w i d e comparisons of DNA sequence copy number. In a typical application genomic DNAs from tumor and normal cells are differentially labeled and simultaneously hybridized to n o r m a l metaphase chromosomes, and detected with different fluorochromes. Properly registered images of each fluorochrome are obtained using a microscope equipped with multi-band filters and a CCD camera. Digital image analysis permits measurement of intensity ratio profiles along each of the target chromosomes. Studies of cells with known aberrations indicate that the intensity ratio at each position is proportional to the copy n u m b e r of the sequences in the tumor that bind there. Knowledge of the actual copy number at one position permits calibration of the profiles so that copy n u m b e r is known everywhere. Deletions, duplications and higher level amplifications can be detected. Thus comparisons of tumor and normal DNA permit development of a "copy number karyotype" for the t u m o r genome. Alternatively, abnormal genomes, for example a primary t u m o r and its metastases can be compared to detect differences between them.
TUMORS
I.W.Cmq,
A. Kallkw;,~; O. ~,n;a-i-~i, M. Stkamete,EL Nakamm'a,I(. ~ M. hllavl~t~F.Wtldman, D. PtnkeL ~ d ~ o f Z ~ ~,MC~230, Box
0s0s ~ ~
,
of Labon,e=y Medidat. U=~-may of San F ~ ,
CA 94143-080S
~Pluoa~et~e la alttt hyln:kll=atimt w l d t ~ probes is i n u r i n g paz'doAmiy uaoful fo~ d-*-,,v,,~ amt characterization of deletion ~ t m p l i t k a t i o n ev-m= ar,aociated with ere genesis or p m ~ of mlid h m m n t u m o n . This presentation will ~ memt appUeatiom of 1~;-I/=r ~ ar,d ~ o f tivme evertts. S p e e i ~ t o p k = t o b e e o v e m d ~,,,4,,A,.- 1) Detection of eye, rut ustn8 FJ,SH e,.at meeelat~ with lore of l~rz78~ty. S~_,~_' ~ on 17p a n d 16q~bt'm~t ~ and
wglbee=mm=~4~ 2) ~ a n a l y ~ a f f p m ==~e~_eo~ ~ soua e==e=mw(th qwwdtt ~ S ~
mhow~g m u b ~
on
ke1~- madt n ~
he=~p=m~y ~ c u ~ c .ad eeee= ==eeecm~= ~ ov=h~ md bmm ea~mC~esl~iv~y, wel be immmtm~ bapozlance of mu~t ~
~ eaam¢ tt~mmemtm~l
propmt~eonwinbedt~me~ 3) ~ h ~ anatym of e~e extmt o~ ~ m a phy~ d~t md amp.e~uon Va=cm=dby W ~ mdy=~ = by = = e m e v . genomic~ , ~ _ H,n~ exp~____-ncewith daeaonaappi~ o~ l ~ t In b:e~t eane~wln t~ di~me~d. ~ = ~ m el
pzebeiy maeeed,q=e~i= k~,= pmbu wm be kk=em,d. 4) D e t e c t i o n o ~ r m ' e , ~ c c e l l L Recm~mq~kmce with ~ of metasta~: c~ll= In the bone maxzow of patte=ts wittt breast and pzm~W ~ ___-¢eemdfm~ __ to
geno~pe ,rid ~
3
~
v~n b, d ~ m ~ .
FLUORESCENCE IN SITU HYBRIDIZATION TO 4 METAPHASE AND INTERPHASE CHROMOSOMES Barbara Trask 1, Anne Fetlitta2, Mari Christensen2, Susan Allen2. Marge Segravcs 2, Anne Bergmann2, Hillary Massa1, Rainer Sachs 3, and Ger van den Engh z, ]-Dept. Molecular Biotechnoiogy, Univ. Washington, Seattle, WA; 2Lawrence Livermom National Laboratory, Livermom CA; 3Dept. Mathematics, Univ. of California, Berkeley, CA Fluorescence in situ hybridization is rapidly replacing radioactive in situ hybridization and somatic cell hybrid analysis as the method of choice to determine the chromosomal location of DNA sequences. DNA sequences ranging in size from several kbp (plasmids) to several 100 kbps (YACs) can be mapped to chromosome bands by FISH. The development of multicolor FISH allows probes to be mapped relative to each other and, thus. at higher resolution than is possible by single probe mapping relative to chromosome length or to bands. Two probes can be accurately mapped relative to each other in metaphnse chromatin if the distance between them exceeds -1 Mop. Higher map resolution can he achieved by mapping probes relative to each other in interphase chromatin, lnterphnse chromatin behaves as a random polymer in the 50 kbp to 1-2 Mbp range and follows a random walk model (mean square interphase distance C( genomio distance). Therefore, the relative spacing of DNA sequences can be deduced from pair-wise interphase distance measurements. Based on these results, a multilevel strategy has been developed for producing a 100-kbp average density probe map using FISH. Examples of the application of this strategy to build maps of regions of chromosomes 4 and 19 will be shown. Together, an understanding of the organization of inter-phase chromatin after hybridization and the identification of probes mapped along each chromosome can be used to diagnose chromosome abnormalities below the detection limits of conventional metaphase cytogenetic analysis. Work funded by U.S.D.O.E. and NIH (HG00256).