A Rare Translocation (4;11)(q21;p14-15) in an Acute Lymphoblastic Leukemia Expressing T-Cell and Myeloid Markers Jennifer E. Hardingham, Gregory B. Peters, Alexander Dobrovic, Barry M. Dale, Dusan Kotasek, Helen E. Ford, Colin J. Story, and Robert E. Sage
ABSTRACT: A 21-year-old male presented with a large mediastinal mass and a white cell count of 420 × 109/L. A diagnosis of acute lymphoblastic leukemia (ALL) was made, with 90% of cells in the bone marrow (BM) and 99% in the peripheral blood (PB) being lymphoblasts (FAB L1 ). Cytogenetic analysis of these cells revealed a rare variant of the t14;11) translocation involving chromosome arm 1 lp rather than 11 q, namely t(4;I 1)(q21 ;p 14-15). The standard form of the (4; 11 ) translocation has been associated with leukemias with mixed-lineage phenotypes. Three cases of ALL with t(4q;l lp) have previously been reported. One of these cases showed phenotypic heterogeneity involving myeloid and lymphoid lineages. The leukemia reported here also exhibits lymphoid/myeloid features, lmmunophenotyping of the blasts showed that most of the cells were positive for CD2, CD5, CD7, CDI O ICALLA ), CD34, and HLA-DR. A significant proportion of the cells expressed CD33. These results suggest a biphenotypic rather than a biclonol disease. Molecular analysis showed rearrangement of both immunoglobulin heavy-chain genes (JH) and of a single allele of the T-cell receptor (TCR) 71 gene, while retaining germline TCR i~ genes.
INTRODUCTION C h r o m o s o m a l t r a n s l o c a t i o n s h a v e b e e n useful in d e t e r m i n i n g the p a t h o g e n e s i s of m a l i g n a n c i e s b e c a u s e of the i d e n t i f i c a t i o n of o n c o g e n e s located at or n e a r the breakpoints. T h e 4;11 t r a n s l o c a t i o n is a m o n g the t r a n s l o c a t i o n s in w h i c h the critical s e q u e n c e s h a v e yet to be d e t e r m i n e d . T w o " v a r i a n t s " of t(4;11) h a v e b e e n recognized; the " s t a n d a r d " form, t ( 4 ; l l ) ( q 2 1 ; q 2 3 ) and a s e c o n d f o r m (or forms), w h e r e the b r e a k p o i n t on c h r o m o s o m e 11 i n v o l v e s the p arm [1]. T h r e e cases of this translocation h a v e b e e n r e p o r t e d [2, 3]. O n e o t h e r case was r e p o r t e d w h e r e the t(4q;11p) arose as a s e c o n d a r y c h a n g e in an infant w i t h the s t a n d a r d t(4;11) [4]. T h e s t a n d a r d transloc a t i o n is a s s o c i a t e d w i t h a g r o u p of a c u t e l e u k a e m i a s c h a r a c t e r i z e d by m a r k e d leucocytosis, s p l e n o m e g a l y , and p o o r prognosis, and i n c l u d e s a n u m b e r of cases w h e r e the blasts s h o w lineage h e t e r o g e n e i t y [5-7]. From the Departments of Hematology-Oncology (J. E. H., A. D., B. M. D., D. K., H. E. F., R. E. S.) and Genetics (G. B. P.J, The Queen Elizabeth Hospital, Woodville; and Hematology Department (C. J. S.), Flinders Medical Centre, Bedford Park, South Australia. Address reprint requests to: Jennifer E. Hardingham, Department of Hematology-Oncology, The Queen Elizabeth Hospital, Woodville S.A. 5011, Australia. Received January 2, 1991; accepted April 26, 1991. 255 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 56:255 262 (1991) 0165-4608/91/$03.50
256
I.E. Hardingham et al. Rearrangements of genes encoding the i m m u n o g l o b u l i n (Ig) chains and T-cell receptors are useful markers of clonality and lineage in lymphoid malignancies [8]. It is apparent, however, that these rearrangements are not entirely restricted to a given lineage and there have been several reports of cross-lineage rearrangements occurring in B- and T-cell malignancies and in acute myeloid leukemia [9 11]. We describe a new case of ALL with the 4q;11p translocation. We have monitored the progress of this case using cytogenetics, i m n m n o p h e n o t y p i n g , and molecular genetics.
CASE HISTORY A 21-year-old male presented in November 1986 with a 6-month history of progressive lethargy, sweats, and recurrent respiratory infections. On physical examination the only abnormal finding was moderate hepato-splenomegaly. A chest X-ray revealed gross mediastinal enlargement. Full blood examination showed a leucocyte count of 420 x 109/L with 99% blasts, a hemoglobin of 7.8 g/dL, and slight thrombocytopenia. The bone marrow was hypercellular with greater than 90% of all cells being lymphoblasts (FAB L1 type). Cytochemical staining showed the cells to be PAS positive and S u d a n Black negative. The patient was treated with combination chemotherapy and entered comp|ete remission in December 1986. In February 1987, he u n d e r w e n t allogeneic bone marrow transplantation using his fully HLA-matched sister as donor. Complete engraftment was established as was a normal female karyotype. In May 1988, cytogenetic analysis detected the re-emergence of the original clonal abnormality (Table 1). h n n m n o p h e n o t y p i n g and DNA studies confirmed this leukemic relapse in the bone marrow and a second matched allogeneic transplant was carried out in July 1988, .using the same donor. Full engraftment was again established and the patient remained in remission until January 1990. At this time he presented with a paravertebral mass that was morphologically identical to the original clone. Cytogenetic analysis revealed new abnormalities in addition to those seen at presentation. Bone marrow biopsy showed one cell with the new karyotype in a total of 95 metaphases analyzed, the remainder of which were donor derived. The patient received systemic and intrathecal chemotherapy with 11o change in the size ot the mass. Local radiotherapy was given in May 1990, but 1 month later, the patient presented with a frank leukemic relapse. Intensive chemotherapy was reinstituted but the patient died of septicemia in July 1990.
Table 1
Karyotype and DNA studies Specimen karyotype
1. 48,XY,t(4;ll)(q21;p14-151 '~ 2. 46,XY 3. 46,XX/46,XY 4. 46,XX/48,XY,t(4;11)(q21;p14-15)"/46,XY
5. 46,XX 6. 49,XY,+ 8,3q-,t(4;11/(q21 ;p14-15), 5p+,11p ,19q+,+mar1,+mar2
No. metaphases
lgH
TCRB
TCR~/
15 30 38/2 22/7/1 23 7
R G G R G R
G G G G G G
R G G R G R
Specimens 1: presentationPB 5.11.86;2: remissionBM 17.12.86; 3: BM post 1st. transplantd+ 366;4: relapse BM d+452; 5: BM post 2nd transplant d+21; 6: relapse paravertebral mass d+553. " With additional changes.
257
t(4;11)(q21;p14-15) in A c u t e L y m p h o b l a s t i c L e u k e m i a
MATERIALS AND METHODS P e r i p h e r a l b l o o d and b o n e m a r r o w s a m p l e s w e r e o b t a i n e d from the patient at presentation and f o l l o w up for c y t o g e n e t i c s , surface markers, and D N A studies.
Cytogenetics P e r i p h e r a l b l o o d s p e c i m e n s w e r e c u l t u r e d for 72 h o u r s at 37°C in RPMI 1640 med i u m w i t h 20% fetal calf serum. M e t h o t r e x a t e (10 7 M final) was a d d e d 18 h o u r s prior to harvest. It was w a s h e d out w i t h two c h a n g e s of m e d i a 6 h o u r s prior to harvest. Cells w e r e r e s u s p e n d e d in fresh m e d i a w i t h 10 6 M b r o m o d e o x y u r i d i n e and i n c u b a t e d for 5 hours. C o l c h i c i n e (1 /xg/mL final) was a d d e d 30 m i n u t e s prior to harvest. B o n e m a r r o w aspirates w e r e i n c u b a t e d o v e r n i g h t at 37°C in RPMI 1640 c o n t a i n i n g 5% fetal calf serum. As w i t h b l o o d culture, c o l c h i c i n e was a d d e d 30 m i n u t e s prior to harvest. T h e cells w e r e fixed in 3 : 1 m e t h a n o l : acetic acid. C h r o m o s o m e s w e r e b a n d e d by a s t a n d a r d GTG p r o c e d u r e . Blood and b o n e m a r r o w specim e n s w e r e also c u l t u r e d (separately) in the p r e s e n c e of the m i t o g e n s PHA and 12-0t e t r a d e c a n y l p h o r b o l - 1 3 - a c e t a t e (TPA).
Cell Surface Marker Studies I m m u n o p h e n o t y p i n g of m o n o n u c l e a r cells was d o n e by i n d i r e c t i m m u n o f l u o r e s cence, u s i n g m o n o c l o n a l a n t i b o d i e s (Table 2) and F I T C - c o n j u g a t e d s h e e p a n t i - m o u s e F(ab')2 fraction as s e c o n d a n t i b o d y . Results w e r e e x p r e s s e d as a p e r c e n t a g e of fluorescent cells in a s a m p l e of 200. Viable cells f r o m c r y o p r e s e r v e d s a m p l e s of the p r e s e n t a t i o n and final relapse s p e c i m e n s w e r e also a n a l y z e d on the Becton Dickinson F A C S c a n for CD2, CD3, CD4, CD5, CD7, CD8, CD10, C D l l b , CD14, CD19, CD20, CD33, CD34, CD71, and HLA DR.
DNA Analysis D N A was extracted from b l o o d or b o n e m a r r o w m o n o n u c l e a r cells using s t a n d a r d t e c h n i q u e s . T e n m i c r o g r a m a l i q u o t s w e r e digested w i t h a p p r o p r i a t e restriction enzymes, size f r a c t i o n a t e d in a 0.8% agarose gel, and transferred to n y l o n m e m b r a n e s (Gene S c r e e n Plus, NEN R e s e a r c h Products). D N A s a m p l e s w e r e a n a l y z e d by hybridization w i t h the f o l l o w i n g gene probes : JH, a 5.6-kb f r a g m e n t of the i m m u n o g l o b u l i n
Table 2
Surface m a r k e r analysis (% p o s i t i v e cells) Specimen
Marker
1
2
3
4
5
6
HLA DR CD2 CD4 CD8 CD7 CD10 CD71 FMC1 CD14
77 95
ABSTRACT: A 21-year-old male presented with a large mediastinal mass and a white cell count of 420 × 109/L. A diagnosis of acute lymphoblastic leukemia (ALL) was made, with 90% of cells in the bone marrow (BM) and 99% in the peripheral blood (PB) being lymphoblasts (FAB L1 ). Cytogenetic analysis of these cells revealed a rare variant of the t14;11) translocation involving chromosome arm 1 lp rather than 11 q, namely t(4;I 1)(q21 ;p 14-15). The standard form of the (4; 11 ) translocation has been associated with leukemias with mixed-lineage phenotypes. Three cases of ALL with t(4q;l lp) have previously been reported. One of these cases showed phenotypic heterogeneity involving myeloid and lymphoid lineages. The leukemia reported here also exhibits lymphoid/myeloid features, lmmunophenotyping of the blasts showed that most of the cells were positive for CD2, CD5, CD7, CDI O ICALLA ), CD34, and HLA-DR. A significant proportion of the cells expressed CD33. These results suggest a biphenotypic rather than a biclonol disease. Molecular analysis showed rearrangement of both immunoglobulin heavy-chain genes (JH) and of a single allele of the T-cell receptor (TCR) 71 gene, while retaining germline TCR i~ genes.
INTRODUCTION C h r o m o s o m a l t r a n s l o c a t i o n s h a v e b e e n useful in d e t e r m i n i n g the p a t h o g e n e s i s of m a l i g n a n c i e s b e c a u s e of the i d e n t i f i c a t i o n of o n c o g e n e s located at or n e a r the breakpoints. T h e 4;11 t r a n s l o c a t i o n is a m o n g the t r a n s l o c a t i o n s in w h i c h the critical s e q u e n c e s h a v e yet to be d e t e r m i n e d . T w o " v a r i a n t s " of t(4;11) h a v e b e e n recognized; the " s t a n d a r d " form, t ( 4 ; l l ) ( q 2 1 ; q 2 3 ) and a s e c o n d f o r m (or forms), w h e r e the b r e a k p o i n t on c h r o m o s o m e 11 i n v o l v e s the p arm [1]. T h r e e cases of this translocation h a v e b e e n r e p o r t e d [2, 3]. O n e o t h e r case was r e p o r t e d w h e r e the t(4q;11p) arose as a s e c o n d a r y c h a n g e in an infant w i t h the s t a n d a r d t(4;11) [4]. T h e s t a n d a r d transloc a t i o n is a s s o c i a t e d w i t h a g r o u p of a c u t e l e u k a e m i a s c h a r a c t e r i z e d by m a r k e d leucocytosis, s p l e n o m e g a l y , and p o o r prognosis, and i n c l u d e s a n u m b e r of cases w h e r e the blasts s h o w lineage h e t e r o g e n e i t y [5-7]. From the Departments of Hematology-Oncology (J. E. H., A. D., B. M. D., D. K., H. E. F., R. E. S.) and Genetics (G. B. P.J, The Queen Elizabeth Hospital, Woodville; and Hematology Department (C. J. S.), Flinders Medical Centre, Bedford Park, South Australia. Address reprint requests to: Jennifer E. Hardingham, Department of Hematology-Oncology, The Queen Elizabeth Hospital, Woodville S.A. 5011, Australia. Received January 2, 1991; accepted April 26, 1991. 255 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 56:255 262 (1991) 0165-4608/91/$03.50
256
I.E. Hardingham et al. Rearrangements of genes encoding the i m m u n o g l o b u l i n (Ig) chains and T-cell receptors are useful markers of clonality and lineage in lymphoid malignancies [8]. It is apparent, however, that these rearrangements are not entirely restricted to a given lineage and there have been several reports of cross-lineage rearrangements occurring in B- and T-cell malignancies and in acute myeloid leukemia [9 11]. We describe a new case of ALL with the 4q;11p translocation. We have monitored the progress of this case using cytogenetics, i m n m n o p h e n o t y p i n g , and molecular genetics.
CASE HISTORY A 21-year-old male presented in November 1986 with a 6-month history of progressive lethargy, sweats, and recurrent respiratory infections. On physical examination the only abnormal finding was moderate hepato-splenomegaly. A chest X-ray revealed gross mediastinal enlargement. Full blood examination showed a leucocyte count of 420 x 109/L with 99% blasts, a hemoglobin of 7.8 g/dL, and slight thrombocytopenia. The bone marrow was hypercellular with greater than 90% of all cells being lymphoblasts (FAB L1 type). Cytochemical staining showed the cells to be PAS positive and S u d a n Black negative. The patient was treated with combination chemotherapy and entered comp|ete remission in December 1986. In February 1987, he u n d e r w e n t allogeneic bone marrow transplantation using his fully HLA-matched sister as donor. Complete engraftment was established as was a normal female karyotype. In May 1988, cytogenetic analysis detected the re-emergence of the original clonal abnormality (Table 1). h n n m n o p h e n o t y p i n g and DNA studies confirmed this leukemic relapse in the bone marrow and a second matched allogeneic transplant was carried out in July 1988, .using the same donor. Full engraftment was again established and the patient remained in remission until January 1990. At this time he presented with a paravertebral mass that was morphologically identical to the original clone. Cytogenetic analysis revealed new abnormalities in addition to those seen at presentation. Bone marrow biopsy showed one cell with the new karyotype in a total of 95 metaphases analyzed, the remainder of which were donor derived. The patient received systemic and intrathecal chemotherapy with 11o change in the size ot the mass. Local radiotherapy was given in May 1990, but 1 month later, the patient presented with a frank leukemic relapse. Intensive chemotherapy was reinstituted but the patient died of septicemia in July 1990.
Table 1
Karyotype and DNA studies Specimen karyotype
1. 48,XY,t(4;ll)(q21;p14-151 '~ 2. 46,XY 3. 46,XX/46,XY 4. 46,XX/48,XY,t(4;11)(q21;p14-15)"/46,XY
5. 46,XX 6. 49,XY,+ 8,3q-,t(4;11/(q21 ;p14-15), 5p+,11p ,19q+,+mar1,+mar2
No. metaphases
lgH
TCRB
TCR~/
15 30 38/2 22/7/1 23 7
R G G R G R
G G G G G G
R G G R G R
Specimens 1: presentationPB 5.11.86;2: remissionBM 17.12.86; 3: BM post 1st. transplantd+ 366;4: relapse BM d+452; 5: BM post 2nd transplant d+21; 6: relapse paravertebral mass d+553. " With additional changes.
257
t(4;11)(q21;p14-15) in A c u t e L y m p h o b l a s t i c L e u k e m i a
MATERIALS AND METHODS P e r i p h e r a l b l o o d and b o n e m a r r o w s a m p l e s w e r e o b t a i n e d from the patient at presentation and f o l l o w up for c y t o g e n e t i c s , surface markers, and D N A studies.
Cytogenetics P e r i p h e r a l b l o o d s p e c i m e n s w e r e c u l t u r e d for 72 h o u r s at 37°C in RPMI 1640 med i u m w i t h 20% fetal calf serum. M e t h o t r e x a t e (10 7 M final) was a d d e d 18 h o u r s prior to harvest. It was w a s h e d out w i t h two c h a n g e s of m e d i a 6 h o u r s prior to harvest. Cells w e r e r e s u s p e n d e d in fresh m e d i a w i t h 10 6 M b r o m o d e o x y u r i d i n e and i n c u b a t e d for 5 hours. C o l c h i c i n e (1 /xg/mL final) was a d d e d 30 m i n u t e s prior to harvest. B o n e m a r r o w aspirates w e r e i n c u b a t e d o v e r n i g h t at 37°C in RPMI 1640 c o n t a i n i n g 5% fetal calf serum. As w i t h b l o o d culture, c o l c h i c i n e was a d d e d 30 m i n u t e s prior to harvest. T h e cells w e r e fixed in 3 : 1 m e t h a n o l : acetic acid. C h r o m o s o m e s w e r e b a n d e d by a s t a n d a r d GTG p r o c e d u r e . Blood and b o n e m a r r o w specim e n s w e r e also c u l t u r e d (separately) in the p r e s e n c e of the m i t o g e n s PHA and 12-0t e t r a d e c a n y l p h o r b o l - 1 3 - a c e t a t e (TPA).
Cell Surface Marker Studies I m m u n o p h e n o t y p i n g of m o n o n u c l e a r cells was d o n e by i n d i r e c t i m m u n o f l u o r e s cence, u s i n g m o n o c l o n a l a n t i b o d i e s (Table 2) and F I T C - c o n j u g a t e d s h e e p a n t i - m o u s e F(ab')2 fraction as s e c o n d a n t i b o d y . Results w e r e e x p r e s s e d as a p e r c e n t a g e of fluorescent cells in a s a m p l e of 200. Viable cells f r o m c r y o p r e s e r v e d s a m p l e s of the p r e s e n t a t i o n and final relapse s p e c i m e n s w e r e also a n a l y z e d on the Becton Dickinson F A C S c a n for CD2, CD3, CD4, CD5, CD7, CD8, CD10, C D l l b , CD14, CD19, CD20, CD33, CD34, CD71, and HLA DR.
DNA Analysis D N A was extracted from b l o o d or b o n e m a r r o w m o n o n u c l e a r cells using s t a n d a r d t e c h n i q u e s . T e n m i c r o g r a m a l i q u o t s w e r e digested w i t h a p p r o p r i a t e restriction enzymes, size f r a c t i o n a t e d in a 0.8% agarose gel, and transferred to n y l o n m e m b r a n e s (Gene S c r e e n Plus, NEN R e s e a r c h Products). D N A s a m p l e s w e r e a n a l y z e d by hybridization w i t h the f o l l o w i n g gene probes : JH, a 5.6-kb f r a g m e n t of the i m m u n o g l o b u l i n
Table 2
Surface m a r k e r analysis (% p o s i t i v e cells) Specimen
Marker
1
2
3
4
5
6
HLA DR CD2 CD4 CD8 CD7 CD10 CD71 FMC1 CD14
77 95
