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The use of flow cytometry in the classification of leukemias and the detection of residual disease
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Scientific Program and Abstracts
T H E USE OF FLOW C Y T O M E T R Y IN THE C L A S S I F I C A T I O N OF L E U K E M I A S A N D T H E D E T E C T I O N OF R E S I D U A L D I S E A S E Ger J. van den Engh BiomedicaI Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA, U.S.A. Flow cytometry and cell sorting are ideally suited for the study of hematological disorders. With these techniques it is possible to inspect larger numbers of cells individually and to determine their characteristics. This can be useful in several areas in the study of leukemias. I) The classification of tumors. 2) Measuring the response of tumor cells to chemotherapy. 3) Detecting low numbers of tumor cells during remission. The surface antigens of leukemic cells are widely studied. Classifications of tumor ceils based on the expression of normal differentiation antigens or unique tumor antigens have been proposed. Recently, techniques have become available for the analysis of chromosomes in flow cytometry. If culture methods for leukemic cells can be improved, it will be possible to analyze chromosomal abnormalities by flow cytometry. Cell surface and chromosomal markers can be used to uniquely define the tumor population. Once their unique characteristics are known, the hemopoietic tissues can be screened for tumor cells during remission. A unique surface marker will allow the detection of as few as l:105 cells. Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48.
SPECIFIC BREAKPOINT ANALYSIS: POTENTIALS FOR DETECTION OF MINIMAL RESIDUAL DISEASE G. G r o s v e l d , D. Bootsma, A. de K l e i n , N. Heisterkamp a, K. Stama and J. Groffen a Dept. Ceil B i o l o g y and G e n e t i c s , Erasmus U n i v e r s i t y , Rotterdam, The Netherlands and (a) Oncogene Science I n c . , M i n o ] a , N . Y . , U.S.A. Chronic mye]oid leukemia (CML) is c h a r a c t e r i z e d by the presence of ~he P h i l a d e l p h i a (Ph I ) chromosome in the leukemic c e i l s of 96% o f a ] ] CML p a t i e n t s . The Ph" chromosome (22q-) i s the r e s u l t of a r e c i p r o c a l t r a n s I o c a t i o n between chromosome 22 and chromosome 9, t ( g q 3 4 , 2 2 q 1 1 ) . P r e v i o u s l y we d e s c r i b e d the l o c a l i z a t i o n of the human c-ab] oncogene on chromosome 9 and demonstrated i t s t r a n s ] o c a t i o n t o the Ph chromosome in CML p a t i e n t s . The c l o n i n g and a n a l y s i s o f b r e a k p o i n t fragments r e v e a l e d t h a t the b r e a k p o i n t s on chromosome 22 a l l c l u s t e r in a v e r y l i m i t e d area, the b r e a k p o i n t c l u s t e r r e g i o n , bcr. B r e a k p o i n t s on chromosome 9, however, are s c a t t e r e d o v e r a l a r g e area which may v a r y from 14 kb up t o more as 100 kb upstream of the v - a b ] homologous sequences) o f the c-abl gene. The d e t e c t i o n o f a c h i m e r i c mRNA (5' bcr and 3 " abl sequences) in the leukemic c e i l s o f CML p a t i e n t s and the c l o n i n g of c h i m e r i c cDNAs from a CML d e r i v e d c e i l l i n e K562 s t r o n g l y i n d i c a t e t h a t bcr and c-ab] coding sequences are l i n k e d by RNA s p l i c i n g , independent from the d i s t a n c e between the two genes on the Ph I chromosome. Sequence a n a l y s i s o f the f u s i o n r e g i o n o f the c h i m e r i c RNA shows t h a t i t codes f o r a b c r / c - a b ] f u s i o n p r o t e i n . A n t i b o d i e s r a i s e d a g a i n s t s y n t h e t i c o ] i g o p e p t i d e s from the f u s i o n r e g i o n w i l l be h i g h l y s p e c i f i c f o r Ph- CML c e l l s and could be used f o r d i a g n o s i s and d e t e c t i o n of r e l a p s e a f t e r bone marrow t r a n s p l a n t a t i o n . THE MAMMALIAN ETS GENES: TWO UNIQUE CHROMOSOMAL LOCATIONS IN CAT, MICE, AND MAN AND NOVEL TRANSLOCATED POSITION IN HUMAN LEUKEMIAS. T. S. Papas, D. K. Watson, N. Sacchi, M.J. McWilliams-Smith, L a b o r a t o r y of M o l e c u l a r Oncology, N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h , F r e d e r i c k , MD 21701 The mammalian homologues of the e t s - r e g i o n from t h e t r a n s f o r m i n g gene of t h e E26 v i r u s , c o n s i s t s of two d i s t i n c t domains l o c a t e d on d i f f e r e n t chromosomes. Using somatic c e l l h y b r i d p a n e l s , t h e mammalian homolog of the 5' v - e t s - d o m a i n ( e t s - 1 ) was mapped t o c h r o mosome 11 in man, t o chromosome 9 in mouse, and t ~ h r o m o s o m e ~ in c a t . The mammalian homolog of t h e 3' v - e t s domain ( e t s - 2 ) was s i m i l a r l y mapped t o human chromosome 21, t o mouse chromosome 16, and t o f e l i n e chromosome C2. To d e f i n e t h e human domains, DNA sequences were m o l e c u l a r l y cloned and a n a l y z e d . The e t s - r e l a t e d sequences of human DNA on chromosomes 11 and 21 are d i s c o n t i g u o u s , except f o r a small o v e r l a p r e g i o n . We conclude t h a t t h e ets sequence shared by t h e v i r u s , the chicken and man is l i k e l y t o cont a i n at l e a s t two d i s s o c i a b l e f u n c t i o n a l domains, e t s - i and e t s - 2 . The human e t s - i locus encodes a s i n g l e mRNA of 6.8 kb, w h i l e the second l o c u s , e t s - 2 encodes t h r e e mRNAs of 4 . 7 , 3.2 and 2.7 kb. Because chromosome t r a n s l o c a t i o n s have been a s s o c i a t e d w i t h d i f f e r e n t human disorders, we have used our human probes and two panels of rodent-human cell hybrids to study specific translocations occurring in acute n~eloid leukemias (AML). The human ets-1 gene was found to translocate from chromosome 11 to 4 in t(4;11)(q21;q23) and the human ets-2 gene was found to translocate from chromosome 8 to 21 in t(8;21) (q22;q22). B o t h translocations were found associated with altered expression of ets.
Scientific Program and Abstracts
T H E USE OF FLOW C Y T O M E T R Y IN THE C L A S S I F I C A T I O N OF L E U K E M I A S A N D T H E D E T E C T I O N OF R E S I D U A L D I S E A S E Ger J. van den Engh BiomedicaI Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA, U.S.A. Flow cytometry and cell sorting are ideally suited for the study of hematological disorders. With these techniques it is possible to inspect larger numbers of cells individually and to determine their characteristics. This can be useful in several areas in the study of leukemias. I) The classification of tumors. 2) Measuring the response of tumor cells to chemotherapy. 3) Detecting low numbers of tumor cells during remission. The surface antigens of leukemic cells are widely studied. Classifications of tumor ceils based on the expression of normal differentiation antigens or unique tumor antigens have been proposed. Recently, techniques have become available for the analysis of chromosomes in flow cytometry. If culture methods for leukemic cells can be improved, it will be possible to analyze chromosomal abnormalities by flow cytometry. Cell surface and chromosomal markers can be used to uniquely define the tumor population. Once their unique characteristics are known, the hemopoietic tissues can be screened for tumor cells during remission. A unique surface marker will allow the detection of as few as l:105 cells. Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48.
SPECIFIC BREAKPOINT ANALYSIS: POTENTIALS FOR DETECTION OF MINIMAL RESIDUAL DISEASE G. G r o s v e l d , D. Bootsma, A. de K l e i n , N. Heisterkamp a, K. Stama and J. Groffen a Dept. Ceil B i o l o g y and G e n e t i c s , Erasmus U n i v e r s i t y , Rotterdam, The Netherlands and (a) Oncogene Science I n c . , M i n o ] a , N . Y . , U.S.A. Chronic mye]oid leukemia (CML) is c h a r a c t e r i z e d by the presence of ~he P h i l a d e l p h i a (Ph I ) chromosome in the leukemic c e i l s of 96% o f a ] ] CML p a t i e n t s . The Ph" chromosome (22q-) i s the r e s u l t of a r e c i p r o c a l t r a n s I o c a t i o n between chromosome 22 and chromosome 9, t ( g q 3 4 , 2 2 q 1 1 ) . P r e v i o u s l y we d e s c r i b e d the l o c a l i z a t i o n of the human c-ab] oncogene on chromosome 9 and demonstrated i t s t r a n s ] o c a t i o n t o the Ph chromosome in CML p a t i e n t s . The c l o n i n g and a n a l y s i s o f b r e a k p o i n t fragments r e v e a l e d t h a t the b r e a k p o i n t s on chromosome 22 a l l c l u s t e r in a v e r y l i m i t e d area, the b r e a k p o i n t c l u s t e r r e g i o n , bcr. B r e a k p o i n t s on chromosome 9, however, are s c a t t e r e d o v e r a l a r g e area which may v a r y from 14 kb up t o more as 100 kb upstream of the v - a b ] homologous sequences) o f the c-abl gene. The d e t e c t i o n o f a c h i m e r i c mRNA (5' bcr and 3 " abl sequences) in the leukemic c e i l s o f CML p a t i e n t s and the c l o n i n g of c h i m e r i c cDNAs from a CML d e r i v e d c e i l l i n e K562 s t r o n g l y i n d i c a t e t h a t bcr and c-ab] coding sequences are l i n k e d by RNA s p l i c i n g , independent from the d i s t a n c e between the two genes on the Ph I chromosome. Sequence a n a l y s i s o f the f u s i o n r e g i o n o f the c h i m e r i c RNA shows t h a t i t codes f o r a b c r / c - a b ] f u s i o n p r o t e i n . A n t i b o d i e s r a i s e d a g a i n s t s y n t h e t i c o ] i g o p e p t i d e s from the f u s i o n r e g i o n w i l l be h i g h l y s p e c i f i c f o r Ph- CML c e l l s and could be used f o r d i a g n o s i s and d e t e c t i o n of r e l a p s e a f t e r bone marrow t r a n s p l a n t a t i o n . THE MAMMALIAN ETS GENES: TWO UNIQUE CHROMOSOMAL LOCATIONS IN CAT, MICE, AND MAN AND NOVEL TRANSLOCATED POSITION IN HUMAN LEUKEMIAS. T. S. Papas, D. K. Watson, N. Sacchi, M.J. McWilliams-Smith, L a b o r a t o r y of M o l e c u l a r Oncology, N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h , F r e d e r i c k , MD 21701 The mammalian homologues of the e t s - r e g i o n from t h e t r a n s f o r m i n g gene of t h e E26 v i r u s , c o n s i s t s of two d i s t i n c t domains l o c a t e d on d i f f e r e n t chromosomes. Using somatic c e l l h y b r i d p a n e l s , t h e mammalian homolog of the 5' v - e t s - d o m a i n ( e t s - 1 ) was mapped t o c h r o mosome 11 in man, t o chromosome 9 in mouse, and t ~ h r o m o s o m e ~ in c a t . The mammalian homolog of t h e 3' v - e t s domain ( e t s - 2 ) was s i m i l a r l y mapped t o human chromosome 21, t o mouse chromosome 16, and t o f e l i n e chromosome C2. To d e f i n e t h e human domains, DNA sequences were m o l e c u l a r l y cloned and a n a l y z e d . The e t s - r e l a t e d sequences of human DNA on chromosomes 11 and 21 are d i s c o n t i g u o u s , except f o r a small o v e r l a p r e g i o n . We conclude t h a t t h e ets sequence shared by t h e v i r u s , the chicken and man is l i k e l y t o cont a i n at l e a s t two d i s s o c i a b l e f u n c t i o n a l domains, e t s - i and e t s - 2 . The human e t s - i locus encodes a s i n g l e mRNA of 6.8 kb, w h i l e the second l o c u s , e t s - 2 encodes t h r e e mRNAs of 4 . 7 , 3.2 and 2.7 kb. Because chromosome t r a n s l o c a t i o n s have been a s s o c i a t e d w i t h d i f f e r e n t human disorders, we have used our human probes and two panels of rodent-human cell hybrids to study specific translocations occurring in acute n~eloid leukemias (AML). The human ets-1 gene was found to translocate from chromosome 11 to 4 in t(4;11)(q21;q23) and the human ets-2 gene was found to translocate from chromosome 8 to 21 in t(8;21) (q22;q22). B o t h translocations were found associated with altered expression of ets.