LEAD ARTICLE 8;20 Chromosomal Translocation in a Case of Acute Leukemia Cytogenetic, Immunophenotypic, Ultrastructural, and Molecular Characteristics Keith F. Jorgenson, Gamil R. Antoun, Craig C. Childs, Edward A. Felix, Ann Cork, Garland Yee, Jose M. Trujillo, Donald P. Pinkel, and Theodore F. Zipf
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INTRODIICTION Kiiryot~,,t)i(: ~d)norm~dilies hi~v~ I)een ~ ; s u n ~ , d Io 1)I~,' an~ iml)ortluH roh~ i[~ Ih(', i)~ltho(ltn~;ntly il re(:il)ro(:~d triH~.sl()(:~lti()n theft h~s I)~eH sh(~wn to h~l~ (; iml)()rliHH t)ro~nosti(: i~x~(I th~',HI)~,uti(: siy, nifi(:~u~(:(~ l)oth in (:hildhoo(I ~(:ut~ ~, l),'mt)hoi(I h,~nk~mii~ (,,\1,1,) (2) iu~(I ~:ut~ ~, I~on~ lyml)hoi(t l~,'uk~,mill (,,\NI,I~) (3 5). \,%'~; h~v~ [)een tr~;iHi[k~ ~ (:hild w i t h ~(:ut~; l~,uk~;mii~ whos~; h~lnk~mi~l ~:~lls d ~ m o ~ ~lr~lt(,'(I ~u~ ~g:20 ¢:hromosonn~d t['iu~lo~:~Hion ~t both di~Lq~osi~ ~nd r~d~q)~;, l~ili~dl',', th~ ~,i)~dielH's ]~;~3kemi~l (:~dl,'-; d~;l~()l~str~d~(I m\'~,qoid fe,~dui~,~s t)x' both inorl)holo~i~: ~ 1 ¢:vlo(:hemi(:~d (:rile[ii~. t)ut th(~,~,' ~li~(l~,~rwe~[H t)]o~,r(;ssi:'e (:ll~u~g(~s to l).,rll~l)h()i(] (:hi|l'~l(:l,~,l"
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CASE REPORT A 3-year-old black female was admitted to The University of Texas M.D. Anderson Cancer Center for evaluation. At the time of admission a physical examination revealed generalized l y m p h a d e n o p a t h y and hepatosplenomegaly. Bh)od counts revealed hemogh)bin of 98 gin/L, WBC of 57 x 10"/1, and platelets 166 × 10~/1. A bone marrow aspirate deinonstrated 84% leukemia cells and decreased normal hematopoiesis. Morphologic and cytochemical features were felt to be consistent with the diagnosis of ANL1, When the disease proved to be only partially responsive to a regimen consisting of vincristine, daunorubicin, etoposide, prednisone, and mercaptopurine, the therapy was changed to cytarabine achninistered by contimmus infusion for 5 clays with d a u n o r u b i c i n injections on days 1 and 2. Subsequently, complete remission cteveloped two months after diagnosis. Courses of daunorubicin-cytarabine therapy were repeated at monthly intervals for four months before chemotherapy was discontinued 6 months after diagnosis. Bone marrow aspirates performed monthly for the next two months c o n t i n u e d to demonstrate complete remission. However, a marrow aspirate at month nine revealed a predolninance of undifferentiated leukemia cells. Treatment with viucristine, prednisone, c y c l o p h o s p h a m i d e , I~-Asparaginase, methotrexate, and m e r c a p t o p u r i n e failed to clear the leukemia cells and the disease was rapidly progressive 12 months after diagnosis. The patient was then started on weekly cytarabine and thioguanine with clinical and hematologic improvemeu! but with the persistent appearance of leukemia cells in her peripheral blood. Further bone marrow examination had not been done at the time of this writing.
MATERIALS AND METHODS Cells After informed consent, patient cells were obtained from bone marrow aspirate specimens taken for diagnostic purposes. The mononuclear cell fraction was isolated on a Ficoll-Hypaque gradient of density 1.077 gm/cm :~ (Pharmacia, Piscataway, NJ). The HL-60 cell line was obtained from the American Type Culture Collection, Bethesda. MD.
Cytogenetic Studies Karyotypic analysis of the leukemia cells was performed on bone marrow aspirates using well established cytogenetic techniques (6). At least 25 metaphases were analyzed from each short term culture. Karyotypes were prepared froin two well spread metaphases of each cell type and reported according to the International System for Human Cytogenetic Nomenclature (7).
Morphology, Cytochemistries, Terminal Deoxynucleotidyl Transferase, and Ultrastructure Bone marrow aspirate smears were stained with Wright-Giemsa stains and with stains specific for m y e l o p e r o x i d a s e (MPO), alpha naphthyl butyrate, periodic ac:id-Schiff, and chloracetate esterase. The leukemia cells were stained for terminal deoxynucleotidyl transferase (TdT) with an indirect staining technique and examined under fluorescence microscopy as previously described (8). Electron microscopy for the determination of the presence of MPO and platelet peroxidase was performed according to standard methods {9, "10).
8:20 Chromosomal Translocation
3
Immunophenotyping The m o n o c l o n a l antibody panel used to establish the i m m u n o p h e n o t y p e of the patient's leukemia cells by single color immunofluorescence included Leu2/CD8, Leu3/ CD4, Leu4/CD3, Leul/CD5, Leu9/CD7, CALLA/CD10, Leu12/CD19, Leu16/CD20. and anti-HLA-DR (Becton-Dickinson, Mountain View, CA), MY4/CD14 and MY9/CD33 (Coulter, Hialeah, FL). All of these monoclonal antibodies were conjugated with fluorescein isothiocyanate (FITC). Multiparameter measurements utilized R-phycoerythrin (RPE) and a l l o p h y c o c y a n i n {APC) conjugated to streptavidan (Caltag, South San Francisco, CA), APC conjugated goat-anti-mouse (Caltag), purified MY9/CD33 (Coulter) and biotin conjugated Leul/CD5 {Becton Dickinson). Three color staining was performed as described (11, 12). Quantitative fluorescence analysis utilized a modified System 50H Cytofluorograf (Ortho Diagnostic Systems, Westwood, MA) (131.
Analysis of Ig and T-cell Receptor Genes Configuration High m o l e c u l a r weight DNA was extracted from both the leukemia and HL-60 cells, digested with the restriction enzymes and subjected to electrophoretic fractionation (14). Following electrophoresis, the DNA was transferred to NYTRAN (Scbleicher and Schuell, Keene, NH) filters (15). Oligolabelled probes (16) for the/3-(17) and 7{18) chain genes of the TCR and the Ig heavy chain gene {19) were then hvbridized with the transferred DNA under stringent conditions and the filters exposed with an intensifying screen to Kodak XR fihn at - 70°C. Samples of DNA extracted from HL60 cells in w h i c h these three genes are in the germline configuration were run in parallel for comparison.
RESULTS Cytogenetics Chromosome preparations of leukemia cells in the bone marrow aspirate taken at diagnosis y i e l d e d a p s e u d o d i p l o i d clone with the t(8:20)(q13:q13) present in every metaphase examined (Fig. 1A and B). No abnormality was observed in the cells from the remission marrow obtained at the end of the 2nd month: a 46,XX karyotype was seen in every metaphase. The t(8;20) was observed in 22/25 metaphases recovered from the month 9 aspirate when recurrent leukemia was observed. In the same preparation both a second translocation t(3;17)(q25:q25) and a chromosome 12 short arm deletion at band p13 appeared in 2/22 metaphases with the t(8;20). This same pattern was present in the specimen taken at month 12. Cytogenetic studies were performed a total of five times during the 12 months following diagnosis (Table 1).
Morphology, Cytochemistry and Ultrastructure At diagnosis the leukemia cells had the morphologic and cytochemical characteristics of acute m y e l o i d leukemia (AML), FAB M1 subtype. Ten percent of the cells stained for MPO, 15% for TdT and none for nonspecific esterase. Electron microscopy demonstrated MPO activity confined to small developmental granules. At relapse, in m o n t h 9, the leukemia cell morphology by Wright-Giemsa stain was u n c h a n g e d from that at diagnosis. However, less than 3% of the leukemia cells were MPO positive by light microscopy and 10% of the cells were positive for TdT. Ultrastructural examination demonstrated MPO positive cytoplasmic granules in less
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INTRODIICTION Kiiryot~,,t)i(: ~d)norm~dilies hi~v~ I)een ~ ; s u n ~ , d Io 1)I~,' an~ iml)ortluH roh~ i[~ Ih(', i)~ltho(ltn~;ntly il re(:il)ro(:~d triH~.sl()(:~lti()n theft h~s I)~eH sh(~wn to h~l~ (; iml)()rliHH t)ro~nosti(: i~x~(I th~',HI)~,uti(: siy, nifi(:~u~(:(~ l)oth in (:hildhoo(I ~(:ut~ ~, l),'mt)hoi(I h,~nk~mii~ (,,\1,1,) (2) iu~(I ~:ut~ ~, I~on~ lyml)hoi(t l~,'uk~,mill (,,\NI,I~) (3 5). \,%'~; h~v~ [)een tr~;iHi[k~ ~ (:hild w i t h ~(:ut~; l~,uk~;mii~ whos~; h~lnk~mi~l ~:~lls d ~ m o ~ ~lr~lt(,'(I ~u~ ~g:20 ¢:hromosonn~d t['iu~lo~:~Hion ~t both di~Lq~osi~ ~nd r~d~q)~;, l~ili~dl',', th~ ~,i)~dielH's ]~;~3kemi~l (:~dl,'-; d~;l~()l~str~d~(I m\'~,qoid fe,~dui~,~s t)x' both inorl)holo~i~: ~ 1 ¢:vlo(:hemi(:~d (:rile[ii~. t)ut th(~,~,' ~li~(l~,~rwe~[H t)]o~,r(;ssi:'e (:ll~u~g(~s to l).,rll~l)h()i(] (:hi|l'~l(:l,~,l"
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CASE REPORT A 3-year-old black female was admitted to The University of Texas M.D. Anderson Cancer Center for evaluation. At the time of admission a physical examination revealed generalized l y m p h a d e n o p a t h y and hepatosplenomegaly. Bh)od counts revealed hemogh)bin of 98 gin/L, WBC of 57 x 10"/1, and platelets 166 × 10~/1. A bone marrow aspirate deinonstrated 84% leukemia cells and decreased normal hematopoiesis. Morphologic and cytochemical features were felt to be consistent with the diagnosis of ANL1, When the disease proved to be only partially responsive to a regimen consisting of vincristine, daunorubicin, etoposide, prednisone, and mercaptopurine, the therapy was changed to cytarabine achninistered by contimmus infusion for 5 clays with d a u n o r u b i c i n injections on days 1 and 2. Subsequently, complete remission cteveloped two months after diagnosis. Courses of daunorubicin-cytarabine therapy were repeated at monthly intervals for four months before chemotherapy was discontinued 6 months after diagnosis. Bone marrow aspirates performed monthly for the next two months c o n t i n u e d to demonstrate complete remission. However, a marrow aspirate at month nine revealed a predolninance of undifferentiated leukemia cells. Treatment with viucristine, prednisone, c y c l o p h o s p h a m i d e , I~-Asparaginase, methotrexate, and m e r c a p t o p u r i n e failed to clear the leukemia cells and the disease was rapidly progressive 12 months after diagnosis. The patient was then started on weekly cytarabine and thioguanine with clinical and hematologic improvemeu! but with the persistent appearance of leukemia cells in her peripheral blood. Further bone marrow examination had not been done at the time of this writing.
MATERIALS AND METHODS Cells After informed consent, patient cells were obtained from bone marrow aspirate specimens taken for diagnostic purposes. The mononuclear cell fraction was isolated on a Ficoll-Hypaque gradient of density 1.077 gm/cm :~ (Pharmacia, Piscataway, NJ). The HL-60 cell line was obtained from the American Type Culture Collection, Bethesda. MD.
Cytogenetic Studies Karyotypic analysis of the leukemia cells was performed on bone marrow aspirates using well established cytogenetic techniques (6). At least 25 metaphases were analyzed from each short term culture. Karyotypes were prepared froin two well spread metaphases of each cell type and reported according to the International System for Human Cytogenetic Nomenclature (7).
Morphology, Cytochemistries, Terminal Deoxynucleotidyl Transferase, and Ultrastructure Bone marrow aspirate smears were stained with Wright-Giemsa stains and with stains specific for m y e l o p e r o x i d a s e (MPO), alpha naphthyl butyrate, periodic ac:id-Schiff, and chloracetate esterase. The leukemia cells were stained for terminal deoxynucleotidyl transferase (TdT) with an indirect staining technique and examined under fluorescence microscopy as previously described (8). Electron microscopy for the determination of the presence of MPO and platelet peroxidase was performed according to standard methods {9, "10).
8:20 Chromosomal Translocation
3
Immunophenotyping The m o n o c l o n a l antibody panel used to establish the i m m u n o p h e n o t y p e of the patient's leukemia cells by single color immunofluorescence included Leu2/CD8, Leu3/ CD4, Leu4/CD3, Leul/CD5, Leu9/CD7, CALLA/CD10, Leu12/CD19, Leu16/CD20. and anti-HLA-DR (Becton-Dickinson, Mountain View, CA), MY4/CD14 and MY9/CD33 (Coulter, Hialeah, FL). All of these monoclonal antibodies were conjugated with fluorescein isothiocyanate (FITC). Multiparameter measurements utilized R-phycoerythrin (RPE) and a l l o p h y c o c y a n i n {APC) conjugated to streptavidan (Caltag, South San Francisco, CA), APC conjugated goat-anti-mouse (Caltag), purified MY9/CD33 (Coulter) and biotin conjugated Leul/CD5 {Becton Dickinson). Three color staining was performed as described (11, 12). Quantitative fluorescence analysis utilized a modified System 50H Cytofluorograf (Ortho Diagnostic Systems, Westwood, MA) (131.
Analysis of Ig and T-cell Receptor Genes Configuration High m o l e c u l a r weight DNA was extracted from both the leukemia and HL-60 cells, digested with the restriction enzymes and subjected to electrophoretic fractionation (14). Following electrophoresis, the DNA was transferred to NYTRAN (Scbleicher and Schuell, Keene, NH) filters (15). Oligolabelled probes (16) for the/3-(17) and 7{18) chain genes of the TCR and the Ig heavy chain gene {19) were then hvbridized with the transferred DNA under stringent conditions and the filters exposed with an intensifying screen to Kodak XR fihn at - 70°C. Samples of DNA extracted from HL60 cells in w h i c h these three genes are in the germline configuration were run in parallel for comparison.
RESULTS Cytogenetics Chromosome preparations of leukemia cells in the bone marrow aspirate taken at diagnosis y i e l d e d a p s e u d o d i p l o i d clone with the t(8:20)(q13:q13) present in every metaphase examined (Fig. 1A and B). No abnormality was observed in the cells from the remission marrow obtained at the end of the 2nd month: a 46,XX karyotype was seen in every metaphase. The t(8;20) was observed in 22/25 metaphases recovered from the month 9 aspirate when recurrent leukemia was observed. In the same preparation both a second translocation t(3;17)(q25:q25) and a chromosome 12 short arm deletion at band p13 appeared in 2/22 metaphases with the t(8;20). This same pattern was present in the specimen taken at month 12. Cytogenetic studies were performed a total of five times during the 12 months following diagnosis (Table 1).
Morphology, Cytochemistry and Ultrastructure At diagnosis the leukemia cells had the morphologic and cytochemical characteristics of acute m y e l o i d leukemia (AML), FAB M1 subtype. Ten percent of the cells stained for MPO, 15% for TdT and none for nonspecific esterase. Electron microscopy demonstrated MPO activity confined to small developmental granules. At relapse, in m o n t h 9, the leukemia cell morphology by Wright-Giemsa stain was u n c h a n g e d from that at diagnosis. However, less than 3% of the leukemia cells were MPO positive by light microscopy and 10% of the cells were positive for TdT. Ultrastructural examination demonstrated MPO positive cytoplasmic granules in less
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