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PAX5is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5alterations. We found PAX5internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5fusion genes displayed a simple karyotype. These data strongly suggest that PAX5fusion genes are early players in leukemogenesis. In addition, PAX5deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

The t(5;14)(q35;q32) chromosomal translocation is specifically observed in up to 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL). It affects the BCL11B/CTIP2locus on chromosome 14 and the RANBP17-TLX3/HOX11L2region on chromosome 5. It leads to ectopic activation of TLX3/HOX11L2.To investigate the reasons of the association between t(5;14) and T-ALL, we isolated the translocation breakpoints in 8 t(5;14) patients. Sequence analyses did not involve recombinase activity in the genesis of the translocation. We used DNAse1 hypersensitive experiments to locate transcriptional regulatory elements downstream of BCL11B.By transient transfection experiments, 2 of the 6 regions demonstrated cis-activation properties in T cells and were also effective on the TLX3promoter. Our data indicate that the basis of the specific association between t(5;14) and T-ALL lies on the juxtaposition of TLX3to long-range cis-activating regions active during T-cell differentiation.

CD4+, CD56+DC2 malignancies constitute a novel disease entity, which has recently been shown to arise from a transformed lymphoid-related plasmacytoid dendritic cell (DC2). Diagnosis is primarily based on a particular immunophenotype with tumor cells expressing CD4 and CD56 antigens in the absence of common lymphoid or myeloid lineage markers. Little is currently known about the cytogenetic features of this disease entity. In this setting, the Groupe Français de Cytogénétique Hématologique (GFCH) initiated a cytogenetic study of 18 adults and 3 children with CD4+, CD56+DC2 acute leukemia using conventional and fluorescence in situ hybridization/24-color karyotyping. Clonal, mostly complex chromosome aberrations were found in 14 patients (66%). Six major recurrent chromosomal targets were defined. These were 5q, 12p, 13q, 6q, 15q, and 9, which were involved in 72% (5q), 64% (12p and 13q), 50% (6q), 43% (15q), and 28% (monosomy 9) of cases, respectively. Cytogenetic features can be summarized as follows: (1) gross genomic imbalances (mostly losses) predominate, (2) no single anomaly can be considered as specific, whereas their combination/accumulation is, and (3) both lymphoid and myeloid lineage–associated rearrangements are observed in unusual combinations in the same cell. This is suggestive of complex multistep tumorigenic mechanisms and is supportive of the hypothesis that CD4+, CD56+DC2 acute leukemia may arise from an undifferentiated nonmyeloid nonlymphoid progenitor cell. In conclusion, the present study documents for the first time the existence of a characteristic cytogenetic profile for this novel disease entity.

Acute promyelocytic leukemia (APL) is typified by the t(15;17) translocation, which leads to the formation of thePML/RARAfusion gene and predicts a beneficial response to retinoids. However, approximately 10% of all APL cases lack the classic t(15;17). This group includes (1) cases with cryptic PML/RARAgene rearrangements and t(5;17) that leads to the NPM/RARAfusion gene, which are retinoid-responsive, and (2) cases with t(11;17)(q23;q21) that are associated with the PLZF/RARAfusion gene, which are retinoid-resistant. A key issue is how to rapidly distinguish subtypes of APL that demand distinct treatment approaches. To address this issue, a European workshop was held in Monza, Italy, during June 1997, and a morphologic, immunophenotypic, cytogenetic, and molecular review was undertaken in 60 cases of APL lacking t(15;17). This process led to the development of a novel morphologic classification system that takes into account the major nuclear and cytoplasmic features of APL. There were no major differences observed in morphology or immunophenotype between cases with the classic t(15;17) and those with the cryptic PML/RARAgene rearrangements. Auer rods were absent in the t(5;17) case expressing NPM/RARA. Interestingly, this classification system distinguished 9 cases with t(11;17)(q23;q21) and, in addition, successfully identified 2 cases lacking t(11;17), which were subsequently shown to have underlying PLZF/RARAfusions. The PLZF/RARAcases were characterized by a predominance of blasts with regular nuclei, an increased number of Pelger-like cells, and by expression of CD56 in 4 of 6 cases tested. Use of this classification system, combined with an analysis for CD56 expression, should allow early recognition of APL cases requiring tailored molecular investigations.

Tumor-associated chromosome translocations usually lead to the formation of two reciprocal fusion genes: one thought to be involved in the transformation process, the other the mechanical consequence of the translocation event. In the case of acute promyelocytic leukemia (APL) blasts, the 15; 17 chromosome translocation generates the putatively transforming PML/RARafusion gene and its reciprocal RARa/PML.We report APL cases with submicroscopic 15; 17 recombinations leading to the formation of nonreciprocal PML/RARaor RARa/PMLfusion genes. Therefore, each of the two reciprocal translocation products may be independently formed and selected by the leukemic phenotype, implying that both are involved in tumorigenesis.

In patients with an atypical stem-cell myeloproliferative disorder with lymphoma (B or T cell), myeloid hyperplasia, and eosinophilia, the chromosome 8p11-12 region is the site of a recurrent breakpoint that can be associated with three different partners, 6q27, 9q32-34, and 13q12. Rearrangements are supposed to affect a pluripotent stem cell capable of myeloid and lymphoid differentiation and to involve the same 8p11-12 gene. The t(8;13) translocation has recently been shown to result in a fusion between the FGFR1gene that encodes a tyrosine kinase receptor for fibroblast growth factors and a novel gene, FIM(also called RAMPor ZNF198), belonging to a novel family of zinc finger genes. In the present study, we have cloned the t(6;8)(q27;p11) translocation in two patients and found a fusion between FGFR1and a novel gene, FOP(FGFR1Oncogene Partner), located on chromosome band 6q27. This gene is alternatively spliced and ubiquitously expressed. It encodes a protein containing two regions of putative leucine-rich repeats putatively folding in α-helices and separated by a hydrophobic spacer. The two reciprocal fusion transcripts were evidenced by reverse transcription-polymerase chain reaction in the tumoral cells of the patients. The predicted chimeric FOP-FGFR1 protein contains the FOP N-terminus leucine-rich region fused to the catalytic domain of FGFR1. It may promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1.

Because activated T cells were previously shown to induce proliferation of human normal B-cell precursors (BCP) via the CD40 pathway, we investigated the effects of T cells on leukemic blasts isolated from patients with B-lineage acute lymphoblastic leukemia (BCP-ALL). An anti-CD3 activated human CD4+T-cell clone was found to induce significant cell proliferation in four of nine BCP-ALL samples analyzed. In one of these cases, the T-cell effect was clearly dependent on interaction between CD40 and its ligand. Accordingly, a more thorough analysis was performed on this particular leukemia (case 461, adult early pre-B-ALL, mBCR+, Philadelphia+, i(9q)+). Thus, autologous CD4+T cells isolated from the patient were also able to induce CD40-dependent proliferation of the leukemic blasts. Analysis of the phenotype after coculture showed that, among the CD19+cells, a proportion gradually lost expression of CD10 and CD34, whereas some cells acquired CD23. In addition, and in contrast with normal BCP, activated T cells promoted maturation of a subset of the case 461 leukemic cells into surface IgM+cells. The leukemic origin of the cycling and the maturing cells was assessed by the presence of i(9q), a chromosomal abnormality associated with this leukemia and evidenced by fluorescence in situ hybridization. Taken together, these results show that leukemic BCP can be activated via CD40 but that not all cases display detectable stimulation in response to T cells despite their expression of CD40. In addition, the present data suggest that CD4+T cells could potentially play a role in the physiology of BCP-ALL.