Sabrina Tosi

Delineation of multiple deleted regions in 7q in myeloid disorders

Loss of chromosome material due to deletions of the long arm of chromosome 7, del(7q), is a consistent finding in all types of myeloid disorders, invariably associated with a poor prognosis. Two different segments, 7q22 and 7q32–q33, have been implicated as critical regions of gene loss associated with these disorders. In the present study, we used fluorescence in situ hybridization (FISH) to characterize the 7q22 breakpoint of an apparently balanced t(7;7)(p13;q22) in an acute myeloid leukemia patient. FISH analysis on bone marrow metaphases from this patient revealed that the sequence corresponding to a series of three ordered cosmids from 7q22 was deleted from one of the der(7) chromosomes. These cosmids contain the human homologue of the Drosophila homeobox gene cut (CUTL1) and span a region of approximately 150 kb. Although the proximal boundary of the deleted segment could not be exactly defined, we estimate the size of this deletion to be approximately 500 kb. Subsequently, we carried out FISH studies using the CUTL1 cosmids on a further 16 patients with deletions of 7q and myeloid disorders. The sequence corresponding to at least two of the cosmids was deleted from the del(7q) in 11 out of 14 cases with a proximal breakpoint within 7q22. Further detailed FISH mapping in this series of 17 patients has identified two other nonoverlapping commonly deleted segments at 7q31–q32 and 7q33, respectively. These data confirm and refine other studies, implying that several different genes on 7q may be involved in the pathogenesis of myeloid diseases. Genes Chromosomes Cancer 25:384–392, 1999.

Mutations of NFKBIA, encoding IκBα, are a recurrent finding in classical Hodgkin lymphoma but are not a unifying feature of non-EBV-associated cases

A consistent feature of the Hodgkin and Reed‐Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) is the constitutive activation of NF‐κB transcription factors. In Epstein‐Barr virus (EBV)‐associated cases of cHL, expression of viral antigens most probably leads to NF‐κB activation but for non‐EBV‐associated cases, the mechanism is not clear. Previous small studies have demonstrated deleterious mutations of NFKBIA, the gene encoding IκBα, in HRS cells. In the present study, we aimed to establish the frequency of NFKBIA mutation in cHL by investigating a larger series of cases and to determine whether these mutations are a characteristic feature of non‐EBV‐associated cHL. Single HRS cells from 20 cases of cHL were analysed by PCRs covering all 6 exons of the gene. Clonal deleterious mutations were detected in 3 cases and in 1 case both alleles of the gene were shown to harbour mutations. NFKBIA mutations were detected only in non‐EBV‐associated cases but the majority of these cases had wild‐type NFKBIA. It remains possible that defects in genes encoding other inhibitors of NF‐κB, such as TNFAIP3 (A20) and CYLD, are involved in the latter cases, as described for one case in this series.

Identification of new partner chromosomes involved in fusions with the ETV6 (TEL) gene in hematologic malignancies.

Several partner genes on different chromosomes have been reported to be fused with the ETV6 gene (located in chromosome band 12p13), with different breakpoints and different frequencies, in various hematologic malignancies, particularly acute myeloid and lymphoid leukemias and myelodysplastic syndromes. By using FISH and molecular analyses, we have analyzed five different pediatric and adult patients carrying cytogenetic abnormalities involving 12p13. Our findings demonstrate that ETV6 was rearranged in all the cases analyzed. In particular, ETV6 was disrupted by translocations with chromosomal bands 7q22, 7q36, 9q11, and 13q12, not previously described as partners of ETV6 in translocations, thus extending its promiscuity in rearranging with different partner genes. Genes Chromosomes Cancer 21:223–229, 1998.

Characterization of the human myeloid leukemia-derived cell line GF-D8 by multiplex fluorescence in situ hybridization, subtelomeric probes, and comparative genomic hybridization.

The human myeloid leukemia cell line GF‐D8 was established from the peripheral blood blasts of a patient with acute myeloid leukemia FAB subtype M1 (AML‐M1). The karyotype, which has not changed significantly over several years of culture, was described initially as 44,XY,‐5,del(7q),inv(7q),add(8q),add(11q),del(12p),‐15,‐17,+mar. With the advent of multicolor fluorescence in situ hybridization (FISH) techniques, the prospect of accurately characterizing this complex karyotype became feasible. In the present study, we applied 24‐color whole‐chromosome painting and analyzed the results using a filter‐based detection system and proprietary software for multiplex FISH (M‐FISH). This resulted in the refinement of the karyotype and the identification of hitherto unsuspected chromosome rearrangements. M‐FISH identified the origin of the add(8q) and add(11q) as well as the small marker chromosome. Both the del(7q) and del(12p) were redefined as unbalanced translocations and an apparently normal chromosome 11 was shown to be t(11;17). Importantly, the del(12p) was shown to be a der(12)t(7;12). Single‐color whole‐chromosome painting studies confirmed these findings, but also identified a cryptic t(Y;12) not seen in the original M‐FISH analysis. We then carried out a FISH screening assay using a complete set of chromosome‐specific subtelomeric probes. This allowed the identification of p and q subtelomeric regions involved in the translocations and indicated amplification of the 8q subtelomeric region. Comparative genomic hybridization (CGH) revealed a highly unbalanced karyotype, as deletions accompanied the majority of translocations, and identified the regions of amplification as 8q22.3‐qter and 11q21‐qter. Finally, conventional FISH with centromeric and unique sequence probes was necessary to elucidate all of the rearrangements. Genes Chromosomes Cancer 24:213–221, 1999.

Familial partial monosomy 7 and myelodysplasia: different parental origin of the monosomy 7 suggests action of a mutator gene.

Two sisters are reported, both with a myelodysplastic syndrome (MDS) associated with partial monosomy 7. A trisomy 8 was also present in one of them, who later developed an acute myeloid leukemia (AML) of the M0 FAB-type and died, whereas the other died with no evolution into AML. Besides FISH studies, microsatellite analysis was performed on both sisters to gather information on the parental origin of the chromosome 7 involved in partial monosomy and of the extra chromosome 8. The chromosomes 7 involved were of different parental origin in the two sisters, thus confirming that familial monosomy 7 is not explained by a germ-line mutation of a putative tumor-suppressor gene. Similar results were obtained in two other families out of the 12 reported in the literature. Noteworthy is the association with a mendelian disease in 3 out of 12 monosomy 7 families, which suggest that a mutator gene, capable of inducing both karyotype instability and a mendelian disorder, might act to induce chromosome 7 anomalies in the marrow. We postulate that, in fact, an inherited mutation in any of a group of mutator genes causes familial monosomy 7 also in the absence of a recognized mendelian disease, and that marrow chromosome 7 anomalies, in turn, lead to MDS/AML.

Heterogeneity of the 7q36 Breakpoints in the t(7;12) Involving ETV6 in Infant Leukemia

The t(7;12)(q36;p13) is a recurrent chromosome abnormality in infant leukemia. In these cases, the involvement of ETV6, with disruption of the gene consistently at its 5′ end, has been reported by several groups. A fusion transcript between ETV6 and HLXB9 has been detected in some, but not all, reported cases of t(7;12). We report here a study based on fluorescence in situ hybridization (FISH) mapping of the translocation breakpoints in seven patients and detailed molecular studies using Southern blotting on two of these patients. The FISH studies have shown a cluster of breakpoints within a cosmid contig proximal to the HLXB9 gene. Southern blotting analysis enabled us to define two distinct breakpoints within the area covered by the cosmid contig in two patients. The analysis of an unusual case of t(7;12)(q22;p13) [full karyotype: 46,XX,der(7)t(7;12)(q22;p13)del(7)(q22q36)] also revealed a break in 7q36, although in a region proximal to the overlapping cosmids. 5′ RACE PCR in one patient has shown a rearrangement involving the ETV6 allele not involved in the t(7;12), suggesting that no functional ETV6 allele might be present in this case. These data show some heterogeneity in the distribution of breakpoints in 7q36, indicating that the generation of a fusion gene might not be the mechanism responsible for leukemogenesis in the t(7;12), at least in some cases.

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

In the nuclei of human lymphocytes, chromosome territories are distributed according to the average gene density of each chromosome. However, chromosomes are very heterogeneous in size and base composition, and can contain both very gene-dense and very gene-poor regions. Thus, a precise analysis of chromosome organisation in the nuclei should consider also the distribution of DNA belonging to the chromosomal bands in each chromosome. To improve our understanding of the chromatin organisation, we localised chromosome 7 DNA regions, endowed with different gene densities, in the nuclei of human lymphocytes. Our results showed that this chromosome in cell nuclei is arranged radially with the gene-dense/GC-richest regions exposed towards the nuclear interior and the gene-poorest/GC-poorest ones located at the nuclear periphery. Moreover, we found that chromatin fibres from the 7p22.3 and the 7q22.1 bands are not confined to the territory of the bulk of this chromosome, protruding towards the inner part of the nucleus. Overall, our work demonstrates the radial arrangement of the territory of chromosome 7 in the lymphocyte nucleus and confirms that human genes occupy specific radial positions, presumably to enhance intra- and inter-chromosomal interaction among loci displaying a similar expression pattern, and/or similar replication timing.

Characterization of 6q abnormalities in childhood acute myeloid leukemia and identification of a novel t(6;11)(q24.1;p15.5) resulting in a NUP98–C6orf80 fusion in a case of acute megakaryoblastic leukemia

Chromosome abnormalities of 6q are not frequently observed in myeloid disorders. In this article, we report the incidence of these chromosome changes in childhood myeloid leukemia as 2%–4% based on the cytogenetic database of a single institution. We applied fluorescence in situ hybridization (FISH) to characterize precisely the types of 6q abnormalities in seven patients (six with acute myeloid leukemia and one with myelodysplastic syndrome). They carried various translocations involving different breakpoints in 6q, as confirmed by FISH using a whole‐chromosome‐6 paint. Four cases were reported as t(6;11), although the breakpoints varied. Among these, we identified a novel translocation, t(6;11)(q24.1;p15.5), in a patient with acute megakaryoblastic leukemia. Molecular cytogenetic studies using the PAC clone RP5‐1173K1 localized the genomic breakpoint on chromosome 11 to within the NUP98 gene. The breakpoint on chromosome 6 was narrowed down to a 500‐kb region between BAC clones RP11‐721P14 and RP11‐39H10. Reverse‐transcription PCR was performed using a forward primer specific for NUP98 and a reverse primer for the candidate gene in the 500‐kb interval in 6q. This experiment resulted in the identification of a new fusion between NUP98 and C6orf80. Further studies will aim to fully characterize C6orf80 and will elucidate the role of this new NUP98 fusion in myeloid leukemia.

Ectopic expression of the HLXB9 gene is associated with an altered nuclear position in t(7;12) leukaemias.

Ectopic expression of the HLXB9 gene is associated with an altered nuclear position in t(7;12) leukaemias

Graft versus leukemia effect after haploidentical HSCT in a MLL-negative infant AML with HLXB9/ETV6 rearrangement

Recently published data show an extremely poor survival of infants with AML and HLXB9/ETV6 rearrangement which is the fusion, resulting from the translocation t(7;12)(q36;p13). None of the patients reported survived a period of 3 years, including four patients who have received allogeneic hematopoietic stem cell transplantation (HSCT). Herein, we report the clinical course of an 8‐month‐old patient with acute myeloid leukemia, M2 subtype and with a HLXB9/TEL rearrangement. The patient received a haploidentical HSCT in relapse situation without any prior re‐induction. The patient became MRD‐negative over a period of 53 days after HSCT. This case reinforces the potential benefit of a graft‐versus‐leukemia effect in the haploidentical setting even in chemoresistant myeloid leukemias with poor‐prognosis molecular features. Pediatr Blood Cancer 2008;50:921–923.