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A variant t(X;15)(p11;q22) translocation in acute promyelocytic leukemia
A Variant t(X;15)(p11;q22) Translocation in Acute Promyelocytic Leukemia Arun Srivastava, Nyla Heerema, Richard C. Lauer, Piruz Nahreini, H. Scott Boswell, Ronald Hoffman, and Asok C. Antony
ABSTRACT: Nonrandom reciprocal translocations involving chromosomes #15 and #17 are characteristic anomalies in a great majority of cases of acute prumyelocytic leukemia (APL). Other complex translocations in APL that invariably involve chromosome #17 also have been described. We describe a patient with clinical and morphologic characteristics of APL but with a previously undescribed acquired karyotype, t(X;15)(p11;q22). This is the first translocation in APL described in which chromosome #17 is not involved. Although a comparative structure/function analysis of potentially relevant genes to the translocation breakpoints in bdth t(X;15) and t(15;17) APL showed no major alterations, the enhanced expression of the c-Kiras oncogene observed in t(X;15) APL supports the concept of heterogeneity in APL at the cytogenetic and molecular levels. INTRODUCTION A chromosomal translocation involving h u m a n chromosomes #15 and #17, t(15;17)(q22;q12 or 21), appears to be a characteristic aberration in acute promyelocytic leukemia (APL). This translocation is specific for APL, and is found in 6 4 % 86% of all cases as determined by chromosome b a n d i n g techniques [1-4]. Recently, complex rearrangements involving chromosomes #15, #17, and a third chromosome also have been reported [5-7], as have rearrangements involving chromosome #17, but not chromosome #15 [8-10]. In all cases reported, however, the translocation invariably has involved a segment of chromosome #17. A n updated account on t(15;17) APL confirming these observations was recently published in this journal [11]. Taken together, these observations suggest that the genes located in this segment of 17q12 or 21 may play a role in the genesis of APL. We report a u n i q u e case of morphologically typical APL that had an acquired translocation between one part of the X chromosome and chromosome #15, t(X;15)(p11;q22) without the apparent i n v o l v e m e n t of chromosome #17, as analyzed by cytogenetic techniques.
From the Divisionof Hematology/Oncology,Departments of Medicine, Medical Genetics,Microbiology and Immunology,and Indiana Elks CancerCenter, Indiana UniversitySchool of Medicine,Indianapolis,IN 46223. Address requests for reprints to Arun Srivastava, Ph.D., Department of Microbiology and Immunology, 635 Barnhill Dr., Rm 255, Indiana University School of Medicine, Indianapolis, IN 46223. Received February 13, 1987; accepted April 9, 1987.
65 © 1987 Elsevier SciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet29:65-74(1987) 0165-4608/87/$03.50
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Although the pathogenesis of API, has been suggested Io inwflve tile lransposition of genetic material from 17(t21 to 15(t22 and/or vice w',rsa and a possible rearrangement of a putative protooncogene [11], tile molecular details remain elusive. The neu oncogene, an erb B-homologous gene distinct from, and unlinked to, the gene encoding the epidermal growth factor receptor has been described [12, 13], and recently has been i m p l i c a t e d to play a role in the pathogenesis of h u m a n breast cancer [14]. The h u m a n neu oncogene has been localized by in situ hybridization and somatic cell techniques to 17q21 (13). Because this band is involved in the majority of cases of t(15;17)(q22;q21) APL, the possibility of a causal relationship between the neu oncogene and APL was raised [13]. Although our patient with t(X;15) APL did not have abnormalities involving c h r o m o s o m e #17, it was of significant interest to ascertain whether or not the neu oncogene was involved in this variant APL. We present a comparative study on the structure/function relationship of the neu oncogene in t(X;15) APL, t(15;17) APL, and normal cells.
CASE REPORT
An 18-year-old white female presented to Indiana University Hospital in July 1985, with a 1-week history of headache, blurred vision, nausea, generalized bone pain and splenomegaly, associated with a white blood cell count of 220,000/mm 3 with 95% promyelocytes, h e m o g l o b i n 14.2 g/dl, and platelet count 50,000/mm 3. Bone marrow aspiration and b i o p s y revealed 90% replacement of normal bone marrow with promyelocytes. The leukemic promyelocytes in both p e r i p h e r a l blood and bone marrow aspirate had high nuclear/cytoplasmic ratios with large nucleoli within i n d e n t e d or folded nuclei. There were n u m e r o u s coarse azurophilic granules and Auer rods in the cytoplasm. These findings were confirmed (by five hematologists and two pathologists) to be consistent with acute p r o m y e l o c y t i c leukemia (M3 by FAB classification). Coagulation studies revealed findings consistent with a low grade d i s s e m i n a t e d intravascular coagulation with m i l d prolongation of the prothrombin time, partial t h r o m b o p l a s t i c time and t h r o m b i n time with decreased fibrinogen and increased fibrinogen/fibrin split products. The patient was treated with leukophoresis on the first 2 days after admission, w h i c h r e d u c e d the p e r i p h e r a l leukocyte count to 100,000/mm 3 and 50,000/mm 3, respectively. Specific therapy with daunorubicin, cytosine arabiuoside, and 6-thioguanine was instituted on the second day, together with continuous heparin infusion and platelet transfusions. A bone marrow aspirate on day 14 of chemotherapy revealed severe hypocellularity. By day 21 and again on day 28, the patient had a progressive r e p o p u l a t i o n of the bone marrow with normal hematopoietic elements with no reemergence of p r o m y e l o c y t i c leukemia cells. Following one cycle of consolidation therapy with the same drugs used for i n d u c t i o n therapy in attenuated doses while the patient was in complete remission, she subsequently had an allogeneic bone marrow transplantation from an HLA-identical sister. She remains in continuous complete remission with m i n i m a l grade I g r a f t - v e r s u s - h o s t disease. EXPERIMENTAL PROCEDURES
Leukophoresed blood containing greater than 99% promyelocytes, obtained from the patient prior to c h e m o t h e r a p y , was frozen at -70°C. Cytogenetic analysis was performed on bone marrow aspirates at admission, and on phytohemagglutininstimulated peripheral blood cells 1 m o n t h after achieving complete remission. Total genomic DNA was isolated as described [15] from the patient's leukophoresed blood cells, from bone marrow cells of her sibling at the time of bone marrow trans-
Variant t(X;15) in APL
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plantation, and from marrow cells of another patient with t(15;17) APL at initial presentation. Total RNA from these cells was prepared by the m e t h o d of Glisin et al. and Ullrich et al. [16, 17]. The cloned neu oncogene (obtained from Dr. Robert A. Weinberg, W h i t e h e a d Institute, Massachusetts Institute of Technology, Cambridge, MA) was propagated in E. coli HB101 [18], and the 420 bp neu-specific insert was excised from the p l a s m i d by digestion with Barn H1 restriction endonuclease and electrophoresis on p o l y a c r y l a m i d e gels, as described [19]. The neu insert was radioactively labeled with 32p-dCTP by nick-translation to a specific activity of 5 × 108 cpm/~g DNA, as described [20]. All other cloned DNA probes (Oncor, Inc., Gaithersburg, MD) were radioactively labeled to specific activities ranging from 2 to 8 × 108 cpm/~g DNA, as described above. Southern blots of genomic DNA [21], quantitative RNA dot blots [22], and hybridizations were carried out as described [23]. The studies involving h u m a n tissues were performed in accordance with guidelines specified by the Indiana University Committee on Protection of H u m a n Subjects. All experiments dealing with recombinant DNA molecules were carried out u n d e r BL-1 c o n t a i n m e n t level, as specified by the current National Institutes of Health guidelines on recombinant DNA molecules. RESULTS A N D DISCUSSION
The characteristic m o r p h o l o g y of the bone marrow cells from the patient with t(X;15) APL is shown in Figure 1A. Cytogenetic examination of the bone marrow cells was carried out by analysis of 30 metaphases and karyotyping of five cells. As shown in Figure 1B, all cells had a reciprocal translocation between one of the X c h r o m o s o m e s and c h r o m o s o m e #15, such that part of the p arm of the X chromosome was translocated to the q arm of #15, and vice versa. This abnormality was seen in each of the 30 cells analysed, leading to the cytogenetic diagnosis of 46,X,t(X; 15) (p11;q22). Although the cytogenetic examination at the 400-band level with GTGb a n d i n g revealed that c h r o m o s o m e #17 was normal in all cells examined, it is possible that the observed karyotype was a " h i d d e n " t(15;17), involving a threeway translocation including a small part of c h r o m o s o m e #17. Qninicrine fluorescence staining of these metaphases, however, also failed to reveal any abnormalities of c h r o m o s o m e #17. In situ h y b r i d i z a t i o n studies utilizing specific gene probes relevant to the translocation breakpoint or further somatic cell genetic studies to establish the karyotype were h a m p e r e d due to unavailability of sufficient n u m b e r of viable cells because, following treatment, the patient went into complete remission. The r e m a i n d e r of the studies were limited to the analysis of the structure/ function relationship of the putative relevant protooncogenes in APL, both t(X;15) and t(15;17). Because no normal cells were seen in the initial bone marrow of the patient with t(X;15) APL, it was not possible to determine w h e t h e r the chromosomal a b n o r m a l i t y was constitutional or acquired due to, or associated with, the leukemic process. Subsequent analysis of the patient's stimulated p e r i p h e r a l blood (when she was in complete remission) revealed a normal 46,XX constitutional karyotype after analyzing 25 metaphases and karyotyping four cells. These findings confirmed that the observed translocation in the patient's bone marrow cells was associated with acute p r o m y e l o c y t i c leukemia. Total genomic DNA samples isolated from the patient with t(X;15) APL, her normal sibling, another patient with classic t(15;17) APL, as well as normal h u m a n placenta were digested to c o m p l e t i o n with EcoRI, electrophoresed on agarose gels, and blotted to nitrocellulose filters as described by Southern [21]. The Southern blots were probed with nick-translated 32p-labeled neu insert and autoradiographed. The results of these studies are s h o w n in Figure 2. The probe detected a 7.2-kb EcoRI fragment homologous to the neu oncogene in all cases [13]. Under nonstrin-
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Figure 1 (A) A representative view of Wright's-Giemsa stained bone marrow aspirate from the patient with t(X;15) APL (magnification 1000 x ).
gent h y b r i d i z a t i o n and washing conditions, and u p o n deliberate overexposure, the probe also detected a 1.6-kb EcoRI fragment in all DNA samples examined. The significance of the 1.6-kb fragment is not currently known. These results ruled out major structural rearrangements in the neu oncogene on c h r o m o s o m e #17 in the patient with t(X;15) APL. Next, we e x a m i n e d whether or not altered expression of the neu oncogene was associated w i t h this variant t(X;15) APL. Total cellular RNA from the patient, her HLA-identical sibling's bone marrow cells, and another patient with classic t(15;17) APL were isolated, and probed for the expression of a variety of cellular genes on quantitative RNA dot blots. Representative autoradiograms are depicted in Figure 3. It is evident that the relatively low-level expression of the neu oncogene in t(X;15) APL (Panel a, bottom row) and in t(15;17) APL (not shown) was not significantly different from a normal donor (Panal a, top row). Panel b shows the comparable levels of expression of the ~-actin gene in these experiments that was used as a specific positive control, w h i c h also ensured the equivalency of RNA loads.
69
Variant t(X;15) in APL
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Figure I{B) a. Complete karyotype of patient with 46,X,t(X;15)(p11;q22) APL. b. Partial karyotype showing chromosomes #15, X, and #17 from this patient. Arrows indicate the translocation. Because c h r o m o s o m e #15 was specifically involved in the translocation in this patient, we also e x a m i n e d whether or not altered expression of the c-fes oncogene (which is located on c h r o m o s o m e #15, and is involved in cellular growth and differentiation), was associated with t(X;15) APL. The results showed no significant differences in the levels of expression of the c-fes oncogene (panel c). In addition, we also probed identical RNA dot blots for the expression of the cofms oncogene, w h i c h is located on c h r o m o s o m e #5, because 5q deletion (and, thereby, involving a loss of the c-fms oncogene) is frequently associated with neoplastic m y e l o i d disorders [24]. The levels of expression of the c-fins oncogene also r e m a i n e d unchanged (panel d). It r e m a i n e d possible, however, that the bone marrow cells from the t(X;15) APL patient and her normal sibling were a s y n c h r o n o u s with respect to cell-cycle rates at the time of this analysis. To examine this possibility, identical RNA dot blots were also probed for the expression of the comyc oncogene, w h i c h is early G1 stage-specific [25]. The results (panel e) revealed no apparent difference in the expression of the c-myc oncogene, suggesting that the cell-cycle rates of the two samples were not significantly different. Finally, we probed an identical RNA dot blot with a probe for the c-Ki-ras oncogene, w h i c h has been s h o w n to be overexpressed in a variety of leukemic and other malignant disorders [26, 27, 29]. It is interesting to note that there was a p p r o x i m a t e l y a sixfold enhanced expression of
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Southern b|et analysis of total genomic DNA (10 p~g each) from normal placenta (lane 1), normal peripheral blood lymphocytes (lane 2), normal bone marrow cells (lane 3), and t(X;t 5) APL bone marrow cells (lane 4} digested with EcoRI restriction endonuclease and probed with :~2P-labeled neu-specific probe. Hybridization was carried out under low-stringency conditions (30% formamide, 42°C). Filters were washed in 2 x SSC at 50°C and autoradiographed at --70°C for 10 (lays using Kodak XAR-5 autoradiographic film and Crone× Lightning Plus intensifying screens. HiudIII-digested PM2 DNA was electrophoresed to serve as molecular weight markers.
the c-Ki-ras o n c o g e n e (panel f) in cells f r o m the p a t i e n t w i t h t(X;15) APL c o m p a r e d w i t h her n o r m a l sibling and w i t h classic t(15;17) APL. T h e s e results raise the possibility that the c-Ki-ras m a y be e i t h e r d i r e c t l y or i n d i r e c t l y (perhaps as a collaborating o n c o g e n e ) i n v o l v e d in the genesis of t(X;15) APL c o m p a r e d w i t h classic t(15;17) APL.
71
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Figure 3 RNA dot blot analysis of normal bone marrow cells (top row of each panel) and t(X;15) APL cells (bottom row of each panel) using 32p-labeled cloned DNA probes. Twofold serial dilutions of RNA samples, beginning with 50 p.g for each sample, were probed with neu oncogene (panel a); [3-actin, (panel b); c-fes, (panel c); v-fms, (panel d); c-myc, (panel e); and c-Ki-ras (panel f). All filters were hybridized under moderately-stringent conditions (40% formamide, 42°C) and washed in 0.5 × SSC at 55°C, except for the filter probed with neu, which was hybridized under low-stringency conditions (30% formamide, 42°C) and washed in 2 × SSC at 50°C. All filters were autoradiographed for 16 hours at -70°C, except those probed with neu, which were for 96 hours at - 70°C.
Next, we e x a m i n e d w h e t h e r or not the e n h a n c e d expression of the c-Ki-ras oncogene in t(X;15) APL was the c o n s e q u e n c e of oncogene amplification, as has been observed in several neoplastic malignancies [26-28]. Total genomic DNA samples isolated from the patient with t(X;15) APL, as well as various normal h u m a n tissues were cleaved with EcoRI restriction endonuclease and subjected to Southern blot analysis, as described above. The results are sh o w n in Figure 4. The probe detected a 2.9-kb EcoRI fragment homologous to the c-Ki-ras locus in all cases. The equivalency of the hybridization intensity with DNA loads ruled out any significant amplification in the c-Ki-ras oncogene copy n u m b e r per haploid genome. Several lines of evidence, therefore, suggest that in this patient with t(X;15) APL, amplification, overexpression, and gross structural alteration of the neu oncogene on c h r o m o s o m e # 1 7 are not involved. It remains possible, however, that activation of the neu oncogene by a point mutation [30] could still result in the formation of an abnormal protein product (p185); this remains to be determined. The possibility of genetic alterations in the structure/function of the neu oncogene in other patients with classic t(15;17) APL warrants further study of the role
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F i g u r e 4 Southern blot analysis of total genomic DNA (10 ~g each) cleaved with EcoRI restriction endonuclease and probed with 32P-labeled v-Ki-ras cloned DNA probe. Normal human placenta (lane 1), normal peripheral lymphocytes (lane 2), normal human fibroblasts (lane 3), normal human bone marrow cells (lane 4), and t(X;15) APL bone marrow cells (lane 5). Hybridization was carried out in 40% formamide at 42°C, and the filter was washed in 0.5 × SSC at 55°C and autoradiographed at - 70°C for 10 days.
of n e u o n c o g e n e in the genesis of APL. It is possible, n e v e r t h e l e s s , that the findings in our patient w i t h t(X;15) APL are u n i q u e . If so, the o b s e r v e d c h r o m o s o m a l transl o c a t i o n w o u l d s u p p o r t the c o n c e p t of h e t e r o g e n e i t y in APL at the c y t o g e n e t i c and m o l e c u l a r level. E v a l u a t i o n of the m e c h a n i s m of a c t i v a t i o n of the c-Ki-ras o n c o g e n e in this patient w i t h t(X;15) APL m a y add to further u n d e r s t a n d i n g of the m u l t i s t e p process of l e u k e m o g e n e s i s . Supported in part by Grants 1-RO1-HD-20889 (A.C.A.), 1-RO1-CA-34841 (R.H.) from the National Institutes of Health, and an American Cancer Society Institutional Grant IN-161A (A.S.). R.C.L is an American Cancer Society Clinical Fellow.
V a r i a n t t(X;15) i n A P L
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The authors thank Dr. Robert A. Weinberg for generously providing the plasmid containing the cloned n e u onc0gene. The excellent secretarial assistance of Shirley Duke and Stephanie Moore during the preparation of this manuscript is gratefully acknowledged.
REFERENCES 1. Rowley JD, Golomb HM, Dougherty C (1977): 15, 17 translocation, a consistent chromosomal change in acute promyelocytic leukemia. Lancet i;549. 2. Van Den Berghe H, Louwagie A, Brockaert-Van Orshoven A, David G, Verwilghen R, Michaux JL, Sokal G (1979): Chromosome abnormalities in acute promyelocytic leukemia. Cancer 43:558. 3. Larson RA, Kondo K, Vardiman JW, Butler AE, Golomb HM, Rowley JD (1984): Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia. Am J Med 76:827. 4. Misawa S, Lee E, Schiffer CA, Liu Z, Testa JR (1986): Association of the translocation (15;17) with malignant proliferation of promyelocytes in acute and chronic myelogenous leukemia at blastic crisis. Blood. 67:270. 5. Bernstein R, Mendelow B, Pinto MR, Morcom G, Bezwoda W (1980): Complex translocations involving chromosomes 15 and 17 in acute promyelocytic leukemia. Br J Haematol 46:311. 6. Sonoda Y, Misawa S, Maekawa T, Taniwaki M, Abe T, Takino T (1985): A variant 15;17 translocation in a patient with acute promyelocytic leukemia. Cancer Genet Cytogenet 17:61. 7. Ohyashiki K, Oshimura M, Uchida H, Nomoto S, Sakai N, Tonomura A, Ito H (1985): Cytogenetic and ultrastructural studies on ten patients with acute promyelocytic leukemia including one case with a complex translocation. Cancer Genet Cytogenet 14:247. 8. Kondo K, Sasaki M (1982): Further cytogenetic studies on acute promyelocytic leukemia. Cancer Genet Cytogenet 6:39. 9. Garson OM, Hagemeijer A, Kondo K, Rowley JD (1984): Chromosomes in acute promyelocytic leukemia. Fourth International Workshop on Chromosomes in Leukemia, 1982. Cancer Genet Cytogenet 11:288. 10. Mitelman F (1985). Catalogue of chromosome aberrations in cancer. Cytogenet Cell Genet 36:1. 11. De Braekeleer M (1986): The occurrence of translocation (15;17) in acute promyelocytic leukemia: An update. Cancer Genet Cytogenet 23:275. 12. King CR, Kraus MH, Aaronson SA (1985): Amplification of a novel v - e r b B-related gene in a h u m a n mammary carcinoma. Science 229:974. 13. Schechter AL, Hung M-C, Weinberg RA, Yang-Feng TL, Francke U, Ullrich A, Coussens L (1985): The neu gene: An erb B-homologous gene distinct form and unlinked to the gene encoding the EGF receptor. Science 229:976. 14. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL (1987): Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177. 15. Srivastava A, Norris JS, Reis RJS, Goldstein S (1985): c-Haoras-1 proto-oncogene amplification and over-expression during the limited replicative lifespan of normal h u m a n fibroblasts. J Biol Chem 260:6404. 16. Glisin V, Crkvenjakov R, Buys C (1974): Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633. 17. Ullrich A, Shine J, Chirgwin J, Pictet R, Tisher E, Rutter WJ, Goodman HM (1977): Rat insulin genes: Construction of plasmids containing the coding sequences. Science 196:1313. 18. Mandel M, Higa N (1970): Calcium dependent bacteriophage DNA infection. J Molec Biol 53:154. 19. Maxam A, Gilbert W (1980): Sequencing end-labeled DNA with base-specific chemical cleavages. Meth Enzymol 65:499.
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20. Maniatis T, [effrey A, Kleid DC (1975): Nucleotide sequence of the rightward operator ot phage k. Proc Natl Acad Sci (USA) 72:1184. 21. Southern EM (1975): Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Molec Biol 98:503. 22. Thomas PS (1980): Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci {USA) 77:5201. 23. Wahl GM, Stern M, Stark GR (1979): Efficient transfer of large DNA fragments from agarose to diazobenzloxymethal-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci (USA) 76:3683. 24. Le Beau MM, Westbrook CA, Diaz MO, Larson RA, Rowley JD, Gasson JC, Golde DW, Sherr CJ {1986): Evidence for the involvement of GM-CSF and fins in the deletion {5q) in myeloid disorders. Science 231:984. 25. Kelly K, Cochran HB, Stiles CD, Leder P (1983): Cell-specific regulation of the c - m y c gene by lymphocyte mitogens and platelet derived growth factor. Cell 35:603. 26. Schwab M, Alitalo K, Varmus HE, Bishop JM, George D (1983): A cellular oncogene {c-Kiras) is amplified, over-expressed and located within karyotypic abnormalities in mouse adrenocortical tumor cells. Nature 303:497. 27. George D, Powers V (1982): Amplified DNA sequences in Y1 mouse adrenal tumor ceils: Association with double minutes and localization to a homogeneously staining chromosomal region. Proc Natl Acad Sci (USA) 79:1597. 28. Capon DJ, Seeburg PH, McGrath JP, Hayflick JS, Edman V, Levinson AD, Goeddel DV (1983): Activation of Ki-ras-2 gene in h u m a n colon and lung carcinomas by two different point mutations. Nature 304:507. 29. Slamon DJ, deKernion JB, Verma IM, Cline MJ (1984): Expression of cellular oncogenes in h u m a n malignancies. Science 224:256. 30. Bargmann CI, Hung M-C, Weinberg RA (1986): Multiple independent activations of the hen oncogene by a point mutation altering the transmembrane domain of p185. Cell 45:649.
ABSTRACT: Nonrandom reciprocal translocations involving chromosomes #15 and #17 are characteristic anomalies in a great majority of cases of acute prumyelocytic leukemia (APL). Other complex translocations in APL that invariably involve chromosome #17 also have been described. We describe a patient with clinical and morphologic characteristics of APL but with a previously undescribed acquired karyotype, t(X;15)(p11;q22). This is the first translocation in APL described in which chromosome #17 is not involved. Although a comparative structure/function analysis of potentially relevant genes to the translocation breakpoints in bdth t(X;15) and t(15;17) APL showed no major alterations, the enhanced expression of the c-Kiras oncogene observed in t(X;15) APL supports the concept of heterogeneity in APL at the cytogenetic and molecular levels. INTRODUCTION A chromosomal translocation involving h u m a n chromosomes #15 and #17, t(15;17)(q22;q12 or 21), appears to be a characteristic aberration in acute promyelocytic leukemia (APL). This translocation is specific for APL, and is found in 6 4 % 86% of all cases as determined by chromosome b a n d i n g techniques [1-4]. Recently, complex rearrangements involving chromosomes #15, #17, and a third chromosome also have been reported [5-7], as have rearrangements involving chromosome #17, but not chromosome #15 [8-10]. In all cases reported, however, the translocation invariably has involved a segment of chromosome #17. A n updated account on t(15;17) APL confirming these observations was recently published in this journal [11]. Taken together, these observations suggest that the genes located in this segment of 17q12 or 21 may play a role in the genesis of APL. We report a u n i q u e case of morphologically typical APL that had an acquired translocation between one part of the X chromosome and chromosome #15, t(X;15)(p11;q22) without the apparent i n v o l v e m e n t of chromosome #17, as analyzed by cytogenetic techniques.
From the Divisionof Hematology/Oncology,Departments of Medicine, Medical Genetics,Microbiology and Immunology,and Indiana Elks CancerCenter, Indiana UniversitySchool of Medicine,Indianapolis,IN 46223. Address requests for reprints to Arun Srivastava, Ph.D., Department of Microbiology and Immunology, 635 Barnhill Dr., Rm 255, Indiana University School of Medicine, Indianapolis, IN 46223. Received February 13, 1987; accepted April 9, 1987.
65 © 1987 Elsevier SciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet29:65-74(1987) 0165-4608/87/$03.50
66
:~. N r i v a s t a v a el al.
Although the pathogenesis of API, has been suggested Io inwflve tile lransposition of genetic material from 17(t21 to 15(t22 and/or vice w',rsa and a possible rearrangement of a putative protooncogene [11], tile molecular details remain elusive. The neu oncogene, an erb B-homologous gene distinct from, and unlinked to, the gene encoding the epidermal growth factor receptor has been described [12, 13], and recently has been i m p l i c a t e d to play a role in the pathogenesis of h u m a n breast cancer [14]. The h u m a n neu oncogene has been localized by in situ hybridization and somatic cell techniques to 17q21 (13). Because this band is involved in the majority of cases of t(15;17)(q22;q21) APL, the possibility of a causal relationship between the neu oncogene and APL was raised [13]. Although our patient with t(X;15) APL did not have abnormalities involving c h r o m o s o m e #17, it was of significant interest to ascertain whether or not the neu oncogene was involved in this variant APL. We present a comparative study on the structure/function relationship of the neu oncogene in t(X;15) APL, t(15;17) APL, and normal cells.
CASE REPORT
An 18-year-old white female presented to Indiana University Hospital in July 1985, with a 1-week history of headache, blurred vision, nausea, generalized bone pain and splenomegaly, associated with a white blood cell count of 220,000/mm 3 with 95% promyelocytes, h e m o g l o b i n 14.2 g/dl, and platelet count 50,000/mm 3. Bone marrow aspiration and b i o p s y revealed 90% replacement of normal bone marrow with promyelocytes. The leukemic promyelocytes in both p e r i p h e r a l blood and bone marrow aspirate had high nuclear/cytoplasmic ratios with large nucleoli within i n d e n t e d or folded nuclei. There were n u m e r o u s coarse azurophilic granules and Auer rods in the cytoplasm. These findings were confirmed (by five hematologists and two pathologists) to be consistent with acute p r o m y e l o c y t i c leukemia (M3 by FAB classification). Coagulation studies revealed findings consistent with a low grade d i s s e m i n a t e d intravascular coagulation with m i l d prolongation of the prothrombin time, partial t h r o m b o p l a s t i c time and t h r o m b i n time with decreased fibrinogen and increased fibrinogen/fibrin split products. The patient was treated with leukophoresis on the first 2 days after admission, w h i c h r e d u c e d the p e r i p h e r a l leukocyte count to 100,000/mm 3 and 50,000/mm 3, respectively. Specific therapy with daunorubicin, cytosine arabiuoside, and 6-thioguanine was instituted on the second day, together with continuous heparin infusion and platelet transfusions. A bone marrow aspirate on day 14 of chemotherapy revealed severe hypocellularity. By day 21 and again on day 28, the patient had a progressive r e p o p u l a t i o n of the bone marrow with normal hematopoietic elements with no reemergence of p r o m y e l o c y t i c leukemia cells. Following one cycle of consolidation therapy with the same drugs used for i n d u c t i o n therapy in attenuated doses while the patient was in complete remission, she subsequently had an allogeneic bone marrow transplantation from an HLA-identical sister. She remains in continuous complete remission with m i n i m a l grade I g r a f t - v e r s u s - h o s t disease. EXPERIMENTAL PROCEDURES
Leukophoresed blood containing greater than 99% promyelocytes, obtained from the patient prior to c h e m o t h e r a p y , was frozen at -70°C. Cytogenetic analysis was performed on bone marrow aspirates at admission, and on phytohemagglutininstimulated peripheral blood cells 1 m o n t h after achieving complete remission. Total genomic DNA was isolated as described [15] from the patient's leukophoresed blood cells, from bone marrow cells of her sibling at the time of bone marrow trans-
Variant t(X;15) in APL
67
plantation, and from marrow cells of another patient with t(15;17) APL at initial presentation. Total RNA from these cells was prepared by the m e t h o d of Glisin et al. and Ullrich et al. [16, 17]. The cloned neu oncogene (obtained from Dr. Robert A. Weinberg, W h i t e h e a d Institute, Massachusetts Institute of Technology, Cambridge, MA) was propagated in E. coli HB101 [18], and the 420 bp neu-specific insert was excised from the p l a s m i d by digestion with Barn H1 restriction endonuclease and electrophoresis on p o l y a c r y l a m i d e gels, as described [19]. The neu insert was radioactively labeled with 32p-dCTP by nick-translation to a specific activity of 5 × 108 cpm/~g DNA, as described [20]. All other cloned DNA probes (Oncor, Inc., Gaithersburg, MD) were radioactively labeled to specific activities ranging from 2 to 8 × 108 cpm/~g DNA, as described above. Southern blots of genomic DNA [21], quantitative RNA dot blots [22], and hybridizations were carried out as described [23]. The studies involving h u m a n tissues were performed in accordance with guidelines specified by the Indiana University Committee on Protection of H u m a n Subjects. All experiments dealing with recombinant DNA molecules were carried out u n d e r BL-1 c o n t a i n m e n t level, as specified by the current National Institutes of Health guidelines on recombinant DNA molecules. RESULTS A N D DISCUSSION
The characteristic m o r p h o l o g y of the bone marrow cells from the patient with t(X;15) APL is shown in Figure 1A. Cytogenetic examination of the bone marrow cells was carried out by analysis of 30 metaphases and karyotyping of five cells. As shown in Figure 1B, all cells had a reciprocal translocation between one of the X c h r o m o s o m e s and c h r o m o s o m e #15, such that part of the p arm of the X chromosome was translocated to the q arm of #15, and vice versa. This abnormality was seen in each of the 30 cells analysed, leading to the cytogenetic diagnosis of 46,X,t(X; 15) (p11;q22). Although the cytogenetic examination at the 400-band level with GTGb a n d i n g revealed that c h r o m o s o m e #17 was normal in all cells examined, it is possible that the observed karyotype was a " h i d d e n " t(15;17), involving a threeway translocation including a small part of c h r o m o s o m e #17. Qninicrine fluorescence staining of these metaphases, however, also failed to reveal any abnormalities of c h r o m o s o m e #17. In situ h y b r i d i z a t i o n studies utilizing specific gene probes relevant to the translocation breakpoint or further somatic cell genetic studies to establish the karyotype were h a m p e r e d due to unavailability of sufficient n u m b e r of viable cells because, following treatment, the patient went into complete remission. The r e m a i n d e r of the studies were limited to the analysis of the structure/ function relationship of the putative relevant protooncogenes in APL, both t(X;15) and t(15;17). Because no normal cells were seen in the initial bone marrow of the patient with t(X;15) APL, it was not possible to determine w h e t h e r the chromosomal a b n o r m a l i t y was constitutional or acquired due to, or associated with, the leukemic process. Subsequent analysis of the patient's stimulated p e r i p h e r a l blood (when she was in complete remission) revealed a normal 46,XX constitutional karyotype after analyzing 25 metaphases and karyotyping four cells. These findings confirmed that the observed translocation in the patient's bone marrow cells was associated with acute p r o m y e l o c y t i c leukemia. Total genomic DNA samples isolated from the patient with t(X;15) APL, her normal sibling, another patient with classic t(15;17) APL, as well as normal h u m a n placenta were digested to c o m p l e t i o n with EcoRI, electrophoresed on agarose gels, and blotted to nitrocellulose filters as described by Southern [21]. The Southern blots were probed with nick-translated 32p-labeled neu insert and autoradiographed. The results of these studies are s h o w n in Figure 2. The probe detected a 7.2-kb EcoRI fragment homologous to the neu oncogene in all cases [13]. Under nonstrin-
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Figure 1 (A) A representative view of Wright's-Giemsa stained bone marrow aspirate from the patient with t(X;15) APL (magnification 1000 x ).
gent h y b r i d i z a t i o n and washing conditions, and u p o n deliberate overexposure, the probe also detected a 1.6-kb EcoRI fragment in all DNA samples examined. The significance of the 1.6-kb fragment is not currently known. These results ruled out major structural rearrangements in the neu oncogene on c h r o m o s o m e #17 in the patient with t(X;15) APL. Next, we e x a m i n e d whether or not altered expression of the neu oncogene was associated w i t h this variant t(X;15) APL. Total cellular RNA from the patient, her HLA-identical sibling's bone marrow cells, and another patient with classic t(15;17) APL were isolated, and probed for the expression of a variety of cellular genes on quantitative RNA dot blots. Representative autoradiograms are depicted in Figure 3. It is evident that the relatively low-level expression of the neu oncogene in t(X;15) APL (Panel a, bottom row) and in t(15;17) APL (not shown) was not significantly different from a normal donor (Panal a, top row). Panel b shows the comparable levels of expression of the ~-actin gene in these experiments that was used as a specific positive control, w h i c h also ensured the equivalency of RNA loads.
69
Variant t(X;15) in APL
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Figure I{B) a. Complete karyotype of patient with 46,X,t(X;15)(p11;q22) APL. b. Partial karyotype showing chromosomes #15, X, and #17 from this patient. Arrows indicate the translocation. Because c h r o m o s o m e #15 was specifically involved in the translocation in this patient, we also e x a m i n e d whether or not altered expression of the c-fes oncogene (which is located on c h r o m o s o m e #15, and is involved in cellular growth and differentiation), was associated with t(X;15) APL. The results showed no significant differences in the levels of expression of the c-fes oncogene (panel c). In addition, we also probed identical RNA dot blots for the expression of the cofms oncogene, w h i c h is located on c h r o m o s o m e #5, because 5q deletion (and, thereby, involving a loss of the c-fms oncogene) is frequently associated with neoplastic m y e l o i d disorders [24]. The levels of expression of the c-fins oncogene also r e m a i n e d unchanged (panel d). It r e m a i n e d possible, however, that the bone marrow cells from the t(X;15) APL patient and her normal sibling were a s y n c h r o n o u s with respect to cell-cycle rates at the time of this analysis. To examine this possibility, identical RNA dot blots were also probed for the expression of the comyc oncogene, w h i c h is early G1 stage-specific [25]. The results (panel e) revealed no apparent difference in the expression of the c-myc oncogene, suggesting that the cell-cycle rates of the two samples were not significantly different. Finally, we probed an identical RNA dot blot with a probe for the c-Ki-ras oncogene, w h i c h has been s h o w n to be overexpressed in a variety of leukemic and other malignant disorders [26, 27, 29]. It is interesting to note that there was a p p r o x i m a t e l y a sixfold enhanced expression of
70
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Figure 2
Southern b|et analysis of total genomic DNA (10 p~g each) from normal placenta (lane 1), normal peripheral blood lymphocytes (lane 2), normal bone marrow cells (lane 3), and t(X;t 5) APL bone marrow cells (lane 4} digested with EcoRI restriction endonuclease and probed with :~2P-labeled neu-specific probe. Hybridization was carried out under low-stringency conditions (30% formamide, 42°C). Filters were washed in 2 x SSC at 50°C and autoradiographed at --70°C for 10 (lays using Kodak XAR-5 autoradiographic film and Crone× Lightning Plus intensifying screens. HiudIII-digested PM2 DNA was electrophoresed to serve as molecular weight markers.
the c-Ki-ras o n c o g e n e (panel f) in cells f r o m the p a t i e n t w i t h t(X;15) APL c o m p a r e d w i t h her n o r m a l sibling and w i t h classic t(15;17) APL. T h e s e results raise the possibility that the c-Ki-ras m a y be e i t h e r d i r e c t l y or i n d i r e c t l y (perhaps as a collaborating o n c o g e n e ) i n v o l v e d in the genesis of t(X;15) APL c o m p a r e d w i t h classic t(15;17) APL.
71
Variant t(X;15) in APL
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Figure 3 RNA dot blot analysis of normal bone marrow cells (top row of each panel) and t(X;15) APL cells (bottom row of each panel) using 32p-labeled cloned DNA probes. Twofold serial dilutions of RNA samples, beginning with 50 p.g for each sample, were probed with neu oncogene (panel a); [3-actin, (panel b); c-fes, (panel c); v-fms, (panel d); c-myc, (panel e); and c-Ki-ras (panel f). All filters were hybridized under moderately-stringent conditions (40% formamide, 42°C) and washed in 0.5 × SSC at 55°C, except for the filter probed with neu, which was hybridized under low-stringency conditions (30% formamide, 42°C) and washed in 2 × SSC at 50°C. All filters were autoradiographed for 16 hours at -70°C, except those probed with neu, which were for 96 hours at - 70°C.
Next, we e x a m i n e d w h e t h e r or not the e n h a n c e d expression of the c-Ki-ras oncogene in t(X;15) APL was the c o n s e q u e n c e of oncogene amplification, as has been observed in several neoplastic malignancies [26-28]. Total genomic DNA samples isolated from the patient with t(X;15) APL, as well as various normal h u m a n tissues were cleaved with EcoRI restriction endonuclease and subjected to Southern blot analysis, as described above. The results are sh o w n in Figure 4. The probe detected a 2.9-kb EcoRI fragment homologous to the c-Ki-ras locus in all cases. The equivalency of the hybridization intensity with DNA loads ruled out any significant amplification in the c-Ki-ras oncogene copy n u m b e r per haploid genome. Several lines of evidence, therefore, suggest that in this patient with t(X;15) APL, amplification, overexpression, and gross structural alteration of the neu oncogene on c h r o m o s o m e # 1 7 are not involved. It remains possible, however, that activation of the neu oncogene by a point mutation [30] could still result in the formation of an abnormal protein product (p185); this remains to be determined. The possibility of genetic alterations in the structure/function of the neu oncogene in other patients with classic t(15;17) APL warrants further study of the role
72
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1 2 3
4
5
kbp ,7.2 ,5.6
3.0 2.1
'0.9
F i g u r e 4 Southern blot analysis of total genomic DNA (10 ~g each) cleaved with EcoRI restriction endonuclease and probed with 32P-labeled v-Ki-ras cloned DNA probe. Normal human placenta (lane 1), normal peripheral lymphocytes (lane 2), normal human fibroblasts (lane 3), normal human bone marrow cells (lane 4), and t(X;15) APL bone marrow cells (lane 5). Hybridization was carried out in 40% formamide at 42°C, and the filter was washed in 0.5 × SSC at 55°C and autoradiographed at - 70°C for 10 days.
of n e u o n c o g e n e in the genesis of APL. It is possible, n e v e r t h e l e s s , that the findings in our patient w i t h t(X;15) APL are u n i q u e . If so, the o b s e r v e d c h r o m o s o m a l transl o c a t i o n w o u l d s u p p o r t the c o n c e p t of h e t e r o g e n e i t y in APL at the c y t o g e n e t i c and m o l e c u l a r level. E v a l u a t i o n of the m e c h a n i s m of a c t i v a t i o n of the c-Ki-ras o n c o g e n e in this patient w i t h t(X;15) APL m a y add to further u n d e r s t a n d i n g of the m u l t i s t e p process of l e u k e m o g e n e s i s . Supported in part by Grants 1-RO1-HD-20889 (A.C.A.), 1-RO1-CA-34841 (R.H.) from the National Institutes of Health, and an American Cancer Society Institutional Grant IN-161A (A.S.). R.C.L is an American Cancer Society Clinical Fellow.
V a r i a n t t(X;15) i n A P L
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The authors thank Dr. Robert A. Weinberg for generously providing the plasmid containing the cloned n e u onc0gene. The excellent secretarial assistance of Shirley Duke and Stephanie Moore during the preparation of this manuscript is gratefully acknowledged.
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