MYC-containing double minutes in hematologic malignancies: evidence in favor of the episome model and exclusion of MYC as the target gene

Double minutes (dmin)—circular, extra-chromosomal amplifications of specific acentric DNA fragments—are relatively frequent in malignant disorders, particularly in solid tumors. In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), dmin are observed in ∼1% of the cases. Most of them consist of an amplified segment from chromosome band 8q24, always including the MYC gene. Besides this information, little is known about their internal structure. We have characterized in detail the genomic organization of 32 AML and two MDS cases with MYC-containing dmin. The minimally amplified region was shown to be 4.26 Mb in size, harboring five known genes, with the proximal and the distal amplicon breakpoints clustering in two regions of ∼500 and 600 kb, respectively. Interestingly, in 23 (68%) of the studied cases, the amplified region was deleted in one of the chromosome 8 homologs at 8q24, suggesting excision of a DNA segment from the original chromosomal location according to the ‘episome model'. In one case, sequencing of both the dmin and del(8q) junctions was achieved and provided definitive evidence in favor of the episome model for the formation of dmin. Expression status of the TRIB1 and MYC genes, encompassed by the minimally amplified region, was assessed by northern blot analysis. The TRIB1 gene was found over-expressed in only a subset of the AML/MDS cases, whereas MYC, contrary to expectations, was always silent. The present study, therefore, strongly suggests that MYC is not the target gene of the 8q24 amplification

Fusion of ETV6 with an intronic sequence of the BAZ2A gene in a paediatric pre-B acute lymphoblastic leukaemia with a cryptic chromosome 12 rearrangement.

ETV6 at 12p13 is rearranged in a variety of haematological malignancies and solid tumours, with more than 20 different partners having been reported. These fusions result in either chimeric proteins or activation of the partner gene. However, there are a few examples of abnormalities resulting in truncated and, most likely, unproductive ETV6 proteins, suggesting that haploinsufficiency of ETV6 and/or the partner is leukaemogenic. We present a novel ETV6 rearrangement, identified in a paediatric pre-B acute lymphoblastic leukaemia. Fluorescence in situ hybridisation (FISH) and molecular genetic analyses revealed a fusion of ETV6 and BAZ2A (at 12q13), generated through a cryptic rearrangement between 12p13 and 12q13, consisting of exons 1 and 2 of ETV6 and a sequence from intron 1 of BAZ2A. This transcript is not expected to produce any chimeric protein, but may encode a truncated form of ETV6, containing the first 54 amino acids (aa), followed by 16 aa from the 3' fusion sequence, reminiscent of ETV6 fusions with MDS2, LOC115548, PER1, and STL. The rearrangement might also modify the regulation of BAZ2A by either activating a cryptic promoter or by coming under the control of the ETV6 promoter. The present case emphasises that 'unproductive'ETV6 rearrangements may play an important pathogenetic role in leukaemia

Combined binary ratio labeling fluorescence in situ hybridization analysis of chordoma

Chordoma is a rare, low- to intermediate-grade malignant tumor involving the axial spine. Cytogenetic data on these tumors have been limited to 25 cases. The findings of clonal chromosome aberrations in five new cases are presented. One of these and two previously reported cases have been studied with multicolor combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH). The karyotypes were near-diploid, mostly with several numerical and structural aberrations. There were multiple imbalances, with loss of segments from 1p, 3p, 3q, 9p, and chromosome 10 seen in two to four of the seven cases. No clustering of breakpoints was seen and no recurrent recombination between chromosomes was detected. The findings are consistent with previous data and indicate that chordoma tumor development is associated with multiple, nonrandom losses including chromosome segments that are frequently involved in many other solid tumors

Fusion of the NUP98 gene and the homeobox gene HOXC13 in acute myeloid leukemia with t(11;12)(p15;q13).

The NUP98 gene at 11p15 is known to be fused to DDX10, HOXA9, HOXA11, HOXA13, HOXD11, HOXD13, LEDGF, NSD1, NSD3, PMX1, RAP1GDS1, and TOP1 in various hematologic malignancies. The common theme in all NUP98 chimeras is a transcript consisting of the 5' part of NUP98 and the 3' portion of the partner gene; however, apart from the frequent fusion to different homeobox genes, there is no apparent similarity among the other partners. We here report a de novo acute myeloid leukemia with a t(11;12)(p15;q13), resulting in a novel NUP98/HOXC13 fusion. Fluorescence in situ hybridization analyses, by the use of probes covering NUP98 and the HOXC gene cluster at 12q13, revealed a fusion signal at the der(11)t(11;12), indicating a NUP98/HOXC chimera, whereas no fusion was found on the der(12)t(11;12), suggesting that the translocation was accompanied by a deletion of the reciprocal fusion gene. Reverse transcription-PCR and sequence analyses showed that exon 16 (nucleotide 2290) of NUP98 was fused in-frame with exon 2 (nucleotide 852) of HOXC13. Neither the HOXC13/NUP98 transcript nor the normal HOXC13 was expressed. The present results, together with previous studies of NUP98/homeobox gene fusions, strongly indicate that NUP98/HOXC13 is of pathogenetic importance in t(11;12)-positive acute myeloid leukemia

Fusion of NUP98 and the SET binding protein 1 (SETBP1) gene in a paediatric acute T cell lymphoblastic leukaemia with t(11;18)(p15;q12).

Three NUP98 chimaeras have previously been reported in T cell acute lymphoblastic leukaemia (T-ALL): NUP98/ADD3, NUP98/CCDC28A, and NUP98/RAP1GDS1. We report a T-ALL with t(11;18)(p15;q12) resulting in a novel NUP98 fusion. Fluorescent in situ hybridisation showed NUP98 and SET binding protein 1(SETBP1) fusion signals; other analyses showed that exon 12 of NUP98 was fused in-frame with exon 5 of SETBP1. Nested polymerase chain reaction did not amplify the reciprocal SETBP1/NUP98, suggesting that NUP98/SETBP1 transcript is pathogenetically important. SETBP1 has previously not been implicated in leukaemias; however, it encodes a protein that specifically interacts with SET, fused to NUP214 in a case of acute undifferentiated leukaemia

Tiling resolution array CGH of dic(7;9)(p11 approximately 13;p11 approximately 13) in B-cell precursor acute lymphoblastic leukemia reveals clustered breakpoints at 7p11.2 approximately 12.1 and 9p13.1.

The dic( 7; 9)( p11 similar to 13; p11 similar to 13) is a recurrent chromosomal abnormality in acute lymphoblastic leukemia (ALL), mainly of B-lineage. Although more than 20 dic(7; 9)-positive ALLs have been reported to date, the molecular genetic consequences of this aberration are unknown. We performed tiling resolution (32K) genome-wide array-based comparative genomic hybridization ( array CGH) analysis of three cases with dic(7; 9) in order to characterize the breakpoints on 7p and 9p. The analysis showed a clustering of breakpoints within 9p13.1 in all three cases and within 7p11.2 in two cases; the array CGH revealed two different breakpoints - 7p12.1 and 7p14.1 - in the remaining case. Based on these findings the abnormality should hence be designated dic(7; 9)(p11.2 similar to 12.1; p13.1). Locus-specific fluorescence in situ hybridization analysis of one of the cases narrowed down the 7p11.2 breakpoint to a < 500-kb segment in this sub-band, a region containing three known genes. Unfortunately, lack of material precluded further molecular genetic studies, and it thus remains unknown whether the pathogenetically important outcome of the dic(7; 9) is formation of a chimeric gene or loss of 7p and/or 9p material. Copyright (c) 2007 S. Karger AG, Basel

FISH mapping of i(7q) in acute leukemias and myxoid liposarcoma reveals clustered breakpoints in 7p11.2 : implications for formation and pathogenetic outcome of the idic(7)(p11.2)

Isochromosome 7q - i(7q) - is seen in a wide variety of hematologic malignancies and solid tumors, often as a secondary change to a characteristic primary translocation. Despite its high frequency, nothing is known about the formation and the pathogenetic outcome of this abnormality. To address these issues, we performed a detailed fluorescence in situ hybridization (FISH) investigation of four acute lymphoblastic leukemias, one acute myeloid leukemia, and two myxoid liposarcomas with i(7q). Using FISH with bacterial artificial chromosomes (BACs) mapping between 7p12.2 and 7q11.2, the breakpoints (BPs) in all seven cases were shown to cluster to an approximately 340 kb segment at 7p11.2, covered by the overlapping BAC probes RP11-760D2 and RP11-10F11. Thus, the i(7q) should formally be designated idic(7) (p11.2). In one of the cases, FISH with fosmids could narrow down the BP further to an 80-kb sequence delineated by G248P81983A10 and G248P8793H7. No known genes are located in the 340-kb BP cluster region, indicating that the idic(7)(p11.2) does not result in a fusion or deregulation of genes in this segment. The pathogenetically important outcome is thus likely to be an altered gene expression because of copy number changes. The clustering of breakpoints might be due to frequent intrachromosomal duplicons in the BP region