Detection of leukemia-associated MLL-GAS7 translocation early during chemotherapy with DNA topoisomerase II inhibitors

Abstract

Leukemias with MLL gene translocations are a complication of primary cancer treatment with DNA topoisomerase II inhibitors. How early translocations appear during primary cancer treatment has not been investigated. We tracked the leukemic clone with an MLL gene translocation during neuroblastoma therapy in a child who developed acute myeloid leukemia. The karyotype of the leukemic clone showed del(11)(q23). We used panhandle PCR-based methods to isolate the breakpoint junction involving MLL and an unknown partner gene. Marrow DNA from neuroblastoma diagnosis and DNA and RNA from serial preleukemic marrows were examined for the translocation. The karyotypic del(11)(q23) was a cryptic t(11;17). GAS7, a growth arrest-specific gene at chromosome band 17p13, was the partner gene of MLL. Two different MLL-GAS7 fusion transcripts were expressed. The translocation was already detectable by 1.5 months after the start of neuroblastoma treatment. The translocation was not detectable in the marrow at neuroblastoma diagnosis or in peripheral blood lymphocyte DNAs of six normal subjects. GAS7 is a new partner gene of MLL in treatment-related acute myeloid leukemia. MLL gene translocations can be present early during anticancer treatment at low cumulative doses of DNA topoisomerase II inhibitors. Although MLL has many partner genes and most have not been characterized, panhandle PCR strategies afford new means for detecting MLL gene translocations early during therapy when the partner gene is unknown.

Figure 1

(A) Treatment and clinical events in patient diagnosed with neuroblastoma. CPM denotes cyclophosphamide; ADR, doxorubicin; VCR, vincristine; CPPD, cisplatin; VP16, etoposide; XRT, local radiation therapy; MoAb, anti-G_D2 mAbs (3F8); NBL, neuroblastoma; FAB M4 AML, French-American-British AML; DOD, dead of disease. Scale indicates months (mos) from neuroblastoma diagnosis. (B) Identification of MLL rearrangements by Southern blot analysis of cryopreserved marrows obtained during neuroblastoma treatment. Months (mos) from neuroblastoma diagnosis are indicated. BamHI-digested DNAs were hybridized with B859 cDNA fragment of ALL-1 exons 5–11 (34). Control peripheral blood lymphocyte DNA of normal subject shows the germ-line band (dash). Arrows show rearrangements. (C) PCR amplification of der(11) breakpoint junction with clonotypic primers in DNAs prepared from cryopreserved marrows or marrow aspirates on glass slides during neuroblastoma treatment. The translocation was detected at 1.5 mos from neuroblastoma diagnosis and in all specimens thereafter. No specimen was available after the first course of cyclophosphamide, doxorubicin, and vincristine. (D) Absence of der(11) breakpoint junction by PCR analysis of DNA prepared from morphologically normal marrow at neuroblastoma diagnosis (0 mos) with clonotypic primers. Ten reactions were performed on marrow aspirate DNA from a glass slide, five examples of which are shown (Left). Marrow DNA from AML diagnosis at 17 mos was positive control. PCR with p53 exon 8-specific primers shows amplifiable DNA in marrow from neuroblastoma diagnosis (0 mos) (Right).

Figure 2

(A) Panhandle variant PCR products from der(11) chromosome in marrow DNA at 12 mos from neuroblastoma diagnosis. (Right) Negative control reactions, designated (−) ligase and dH₂O. (B) Summary of panhandle variant PCR products containing der(11) breakpoint junction. The 31-base sequence of primer 3 used in final round of PCR and its complement are at 5′ and 3′ ends, respectively. The 4643–4645 bases of bcr sequence, including primer 3, are 5′ of MLL breakpoint at position 4662, 4663, or 4664 in intron 8 (corkscrew arrow). Depending on MLL breakpoint location, 1034–1036 bases of 3′ sequence are GAS7 partner DNA (GenBank accession no. AC005747). MLL breakpoint is 3′ of an AluY. GAS7 breakpoint is 5′ of an AluY. Identical 5′-AT-3′ sequences in MLL and GAS7 (outlined) preclude more precise assignment of breakpoint positions. Short homologies between MLL and GAS7 are underlined. (C) Genomic organization of 167-kb human GAS7 gene derived from alignment of genomic (GenBank accession no. AC005747) and cDNA sequences (GenBank accession nos. AB007854 and AJ224876). Boxes show the 14 exons. GAS7 der(11) breakpoint (corkscrew arrow) corresponds to nucleotide 165462, 165461, or 165460 upstream of exon 1 in GenBank accession no. AC005747; but sequence in GenBank entry is reverse complement relative to transcriptional orientation of the ORF (GenBank accession nos. AB007854 and AJ224876) and breakpoint corresponds to position 1240, 1241, or 1242 if GenBank accession no. AC005747 were in sense orientation. BamHI site indicates 3′ end of GAS7 sequence in panhandle variant PCR products shown in B.

Figure 3

(A) Amplification of der(17) breakpoint junction in marrow DNA at 12 months from neuroblastoma diagnosis using clonotypic primers. A 360-bp product was expected but a 307-bp product was obtained. Peripheral blood lymphocyte DNAs from two normal subjects and dH₂O were negative controls. (B) Sequencing of the 307-bp products from duplicate reactions showed GAS7 breakpoint at position 1203 upstream of exon 1 and MLL breakpoint at position 4680 in intron 8. Homologous sequences are underlined. GAS7 nucleotide position is for sense orientation of genomic sequence (GenBank accession no. AC005747). (C) Expected der(17) breakpoint junction based on der(11) sequence (Fig. 2B). A total of 53 bases, including the indicated 36–38 bases from GAS7 and 15–17 bases from MLL, were lost in the translocation process. It cannot be determined from where AT was lost (outlined).

Figure 4

(A) cDNA panhandle PCR analysis of marrow total RNA from 7 months after neuroblastoma diagnosis. As shown by smear in second lane of gel at left, a population of products of various sizes was obtained by reverse-transcribing first-strand cDNA from total RNA with 5′-MLL-NNNNNN-3′ oligonucleotides, generating second strands by primer 1 extension, forming stem-loop templates, and PCR with MLL-specific primers. As a positive control, a 2-μl aliquot of the same 5′-MLL-NNNNNN-3′-primed first-strand cDNAs was amplified with β-actin primers, which gave the expected 250-bp product (gel at right). (B) Representative recombination PCR-generated subclones of cDNA panhandle PCR products. One-eighth of the cDNA panhandle PCR products, represented by the smear in A, was used in each of three transformations. A total of 413 subclones were obtained, of which 14 were sequenced. (C) Sequences identified by cDNA panhandle PCR analysis. Three subclones contained normal MLL cDNA, three showed an in-frame fusion of MLL exon 7 to GAS7 exon 2, and one contained a fusion of MLL exon 8 to GAS7 exon 2. The other seven subclones contained empty vector sequence. The products from 12 months after neuroblastoma diagnosis were similar (see text).