Bone Marrow Replacement with a Clone Harboring Novel t(4;11)(p12;q23) Involving MLL during Neuroblastoma Treatment Is Not Associated with Leukemia.

Epipodophyllotoxins, anthracyclines and other chemotherapeutic DNA topoisomerase II poisons are associated with MLL-rearranged leukemias and, less often, leukemias with different balanced translocations. Every MLL translocation identified so far in patients has been associated with leukemia and, where tested, murine models have established that the der(11) protein products are leukemogenic. We here describe a highly novel t(4;11)(p12;q23) that emerged in the bone marrow that confers a proliferative advantage but not a leukemia phenotype in a patient with stage 4 neuroblastoma (NB) following N8 therapy, which consisted of 3 cycles of cyclophosphamide/doxorubicin/vincristine, 2 cycles of cisplatin (P)/etoposide (VP), surgery, local radiation, anti-G_D2 monoclonal antibody (3F8), myeloablative therapy (thiotepa, carboplatin, topotecan) plus autologous PBSC transplant (harvested after 1 cycle of PVP), followed by 7 more cycles of 3F8 plus GM-CSF, 4 cycles of oral VP, and 5 cycles of accutane. Cumulative doses of doxorubicin and VP were 225 mg/m² and 5.4 g/m² (4.2 g/m² orally), respectively. Seventeen months after NB diagnosis (11.5 months after transplant) at completion of oral VP, surveillance FISH showed t(4;11)(p12;q23) in 10.5% of marrow cells. The patient is now 32 months from NB diagnosis and 1 year off all therapy. Although both karyotype and FISH showed t(4;11) in 82–100% cells in 7 serial subsequent marrows, all had normal morphology and there is no clinical evidence of leukemia. Molecular analyses of sequential bone marrow samples were undertaken to characterize the translocation. Southern blot analysis of the MLL bcr in BamHI and BglII digested DNAs showed two rearrangements consistent with both derivative chromosomes. A new BglII-based reverse panhandle PCR identified the der(4) breakpoint junction sequence in a 3.2 kb BglII genomic rearrangement and provided the sequence to isolate the der(11) genomic breakpoint junction by clonotypic PCR. The partner DNA was a novel gene localized to chromosome band 4p12. The translocation fused intron 8 of MLL with intron 8 in the new partner gene, and occurred with a loss of 8–13 bases from MLL and 31–36 bases from the partner gene. Both breakpoint junctions contained short homologous sequences suggesting NHEJ. By clonotypic nested PCR (sensitivity 1 cell in 10⁴–10⁵ cells) the translocation was absent at NB diagnosis, and was first detectable at the completion of oral VP (16 months after neuroblastoma diagnosis). RT-PCR revealed in-frame der(11) and der(4) fusion transcripts. Real-time PCR analysis of known MLL target genes and differentially expressed genes in MLL-rearranged leukemias revealed low levels of HOXA4, A5, A7, A9, FLT3, MEIS1, and PBX3 compared to other treatment-related leukemias. Temporal emergence of this translocation after the high total VP dose is of interest because others recently found that cumulative doses of VP >6 g/m² carry an especially high risk of leukemia, raising concern about the role of the oral VP in the genesis of the translocation. This study indicates that the different partner genes of MLL do not confer the same leukemia potential. Because the t(4;11)(p12;q23) confers a proliferative advantage but is insufficient for leukemia, we designated the new partner gene MIFL (MLL Insufficient for Leukemia), however it remains to be determined whether any relevant secondary alteration will result in leukemic transformation.