(CANCER RESEARCH 53. 2954-2956. July I. 19931 Advances in Brief Common Region of ALL-1 Gene Disrupted in Epipodophyllotoxin-related Secondary Acute Myeloid Leukemia Carolyn A. Felix,1 Naomi J. Winick, Massimo Negrini, W. Paul Bowman, Carlo M. Croce, and Beverly J. Lange Division of Oncology. Department of Pediatrics. Children's Hospital of Philadelphia. Philadelphia. Pennsylvania 19104 ¡C.A. F.. B. J. L¡: Center for Cancer and Blood Disorders, Children 's Medical Center of Dallas. University of Texas Southwestern Medical Center. Dallas. Texas 75235 ¡N.J. W.]: Thomas Jefferson University Cancer Research Center. Philadelphia. Pennsylvania 19107 IM. N.. C. M. C./: and Department of Pediatrics. Cook-Fort Worth Children's Medical Center, Fort Worth, Texas 76104 /W. P. B.¡ Abstract Translocations at chromosomal band Hq23 characterize most de novo acute lymphoblastic leukemia* (ALL) of infants, acute myeloid leukemias (AMD of infants and young children, and secondary AMLs following epipodophyllotoxin exposure. The chromosomal breakpoints at Ilq23 have been cloned from isolated cases off/c novo ALL and AMI . Using an 859-base pair /¡«millfragment of human ALL-1 complementary DNA that recognizes the genomic breakpoint region for de novo ALL and AML, we investigated two cases of secondary AML that followed etoposide- treated primary B-lineage ALL. In the first case, the translocation oc curred between chromosomes 9 and 11 and the breakpoint at Hq23 lo calized to the same 9-kilobase region of the ALL-1 gene that is disrupted in most of the de novo leukemias. In the second case the translocation was between chromosomes 11 and 19. The breakpoint occurred outside of the ALL-1 breakpoint cluster region. Introduction Two distinct forms of secondary AML- occur as late effects of cancer treatment. One form is a complication of therapy with alkyl- ating agents and is characterized by myelodysplasia, a mean latency period of 5 to 7 years, and complete or partial monosomy of chro mosomes 5 or 7 ( 1). The other form of secondary AML is associated with the epipodophyllotoxins. teniposide and etoposide (2-5). It is characterized by a relatively shorter latency period. French-American- British classification M4 or M5 morphology, and translocations in volving chromosomal band 1Iq23. The most common I Iq23 translo cation in epipodophyllotoxin-related secondary AML is the t(9;l 1)(p2l:q23) (6) but variant reciprocal partner chromosomes in cluding 1,2, 16, 17, and 19 have been observed (7). Chromosomal translocations at I Iq23 also are involved in de novo pediatrie ALL and AML. The most common of the de novo translo cations at Ilq23 is the t(4;l I)(q21;q23), found in a majority of the ALLs in infants less than 1 year old (8-10). Variant reciprocal trans- locations nonrandomly fuse chromosomal band Ilq23 with partner chromosomes including 1, 6, 9, 10, or 19 and occur in lymphoblastic. myelomonocytic, or monoblastic (French-American-British classifi cation M4 and M5) leukemia in infants and young children (6, 11-15). The chromosomal breakpoints at I Iq23 recently have been cloned from isolated cases of de novo ALL and AML (15-19). The gene on I Iq23 encompassing the breakpoints has been called ALL-1. MLL, or Hirxl ( 15-19). The majority of the breakpoints identified by Southern blot are located within a 9-kilobase Bam\\\ fragment of genomic DNA that spans exons 5-11 (17, 18, 20-22). Occasional breakpoints at chromosomal band 1Iq23 located outside of this region may involve Received 4/12/93: accepted 5/12/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 V.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Division of Oncology. De partment of Pediatrics. Room 9()93. Children's Hospital of Philadelphia. 34th St. and Civic Center Blvd.. Philadelphia. PA 19104. - The abbreviations used are: AML, acute myeloid leukemia; ALL. acute lymphoblas tic leukemia. either another site in ALL-! or the recently identified, more telomeric rck gene (13, 23). During 1986-1992. 235 children with ALL were treated on a Dallas-Fort Worth protocol that included etoposide during consolida tion and maintenance. Ten cases of secondary AML have been re ported at intervals of 23-68 months from diagnosis of the ALL (7). The secondary AML marrow specimens from two of these patients were available for molecular analysis. In the present study we map the translocation breakpoint at I Iq23 in one secondary AML to the same 9-kilobase BciinHl fragment of the ALL-l gene commonly disrupted in de novo acute leukemia. Materials and Methods The diagnoses of ALL and AML were made by morphological and immu- nohistochemical examination of the marrow and by fluorescence-activated cell sorter analysis with standard monoclonal antibodies (7). Mononuclear cells frozen in liquid nitrogen were obtained from the Pediatrie Oncology Group Leukemia Bank. Informed consent was obtained for the performance of mo lecular analyses. Genomic DNA and total cellular RNA were isolated using 4 Mguanidine isothiocyanate-5.7 MCsCl gradients (24, 25). Five /xg of genomic DNA were digested to completion with 15 units of Bum\\\ (Bethesda Research Laboratories. Bethesda. MD). size fractionated on 0.8% agarose gels, and transferred to nitrocellulose using standard methodology. The filters were hybridized with an 859-base pair BtimHl fragment of human ALL-1 comple mentary DNA that was radiolabeled with |'2P)dCTP by the nick translation method. The 859-base pair BiimHl fragment of ALL-1 complementary DNA spans exons 5-11. the region of the ALL-1 gene breakpoint cluster tor de novo ALL and AML (l8). Results The first case of secondary AML occurred 31 months after the diagnosis of the primary B-lineage ALL in a 3.6-year-old male, was of M5a morphology, and showed a t(9;l I)(p22;q23) karyotype (7). Southern analysis of ßwmHI-digestedgenomic DNA with the B859 probe showed both the normal alÃ-eleand two additional bands con sistent with both derivative chromosomes that had resulted from the translocation (Fig. 1). The second case of etoposide-related secondary AML. also morphologically M5a, occurred 16 months after ALL and showed a t(l I;19)(q23;pl3). The primary B-lineage leukemia was diagnosed at age 2.4 years. In this case the translocation breakpoint at Ilq23 was not located within the ALL-l breakpoint cluster region. Discussion We have demonstrated by Southern blot analysis that the location of a breakpoint on 1Iq23 in an epipodophyllotoxin-associated secondary t(9:ll) AML falls within the same breakpoint cluster region of the ALL-l gene as the breakpoints in de novo AML and ALL. De novo pediatrie AMLs of the M4 and M5 morphological subtypes that occur during infancy have been associated with maternal marijuana and ethanol use and the exposure of either parent to pesticides (26-28). Many infant AMLs have translocations at Ilq23, suggesting that 2954 Research. on January 3, 2016. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from