Pascaline Talmant

Myeloid cell differentiation arrest by miR-125b-1 in myelodysplasic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34+ cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

The clinical presentation and prognosis of diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC rearrangement

Background and Objectives Diffuse large B-cell lymphomas (DLBCL) are common lymphomas that have been classified into three subgroups on the basis of their patterns of gene expression. The aim of this study was to characterize the clinical, biological, immunophenotypic and cytogenetic features of DLBCL with concurrent t(14;18) and 8q24/c-MYC rearrangement. Design and Methods Sixteen cases of DLBCL with the dual translocation were identified between 1998 and January 2006. The clinical features of these cases were examined and morphological, immunohistochemical, flow cytometric and cytogenetic analyses were performed. Results All patients had aggressive features: B symptoms (81%), ECOG performance status >2 (81%), elevated lactate dehydrogenase (100%), stage IV disease (100%) with at least one extra-nodal localization (bone marrow, blood and central nervous system involvement in 93%, 50% and 50%, respectively) and age-adjusted IPI score of 3 in 81%. Despite intensive chemotherapy regimens (including allogeneic transplants), all patients died of disease progression. Progression-free and overall survival was 4 and 5 months, respectively. Immunophenotyping analysis (CD20, CD10, Bcl-6, Mum-1, Bcl-2 CD138, MIB1, CD19, CD5, CD38 and sIg) was performed and showed DLBCL with a germinal center (GC) profile. Ki-67 staining ranged from 70 to 90%. All cases assessed by cytogenetics analysis [conventional cytogenetic and/or fluorescence in situ hybridization (FISH)] had a complex karyotype. In one case, we identified a 8q24/c-MYC translocation variant never reported in DLBCL before: t(8;9)(q24;p13) and t(14;18)(q32;q21). The BCL-6 rearrangement was investigated by FISH and found to rearranged in four cases. Interpretation and Conclusions In conclusion, DLBCL with concurrent t(14;18) and 8q24/c-MYC rearrangement is a subgroup of GC-DLBCLwith poor outcome. It is worth searching for the coexistence of dual translocations in Bcl-2-positive DLBCL with unusual aggressive presentation.

Report of 34 patients with clonal chromosomal abnormalities in Philadelphia-negative cells during imatinib treatment of Philadelphia-positive chronic myeloid leukemia.

Imatinib mesylate (Gleevec®), an inhibitor of the BCR-ABL tyrosine kinase, was introduced recently into the therapy of chronic myeloid leukemia (CML). Several cases of emergence of clonal chromosomal abnormalities after therapy with imatinib have been reported, but their incidence, etiology and prognosis remain to be clarified. We report here a large series of 34 CML patients treated with imatinib who developed Philadelphia (Ph)-negative clones. Among 1001 patients with Ph-positive CML treated with imatinib, 34 (3.4%) developed clonal chromosomal abnormalities in Ph-negative cells. Three patients were treated with imatinib up-front. The most common cytogenetic abnormalities were trisomy 8 and monosomy 7 in twelve and seven patients, respectively. In 15 patients, fluorescent in situ hybridization with specific probes was performed in materials archived before the initiation of imatinib. The Ph-negative clone was related to previous therapy in three patients, and represented a minor pre-existing clone that expanded after the eradication of Ph-positive cells with imatinib in two others. However, in 11 patients, the new clonal chromosomal abnormalities were not detected and imatinib may have had a direct effect. No myelodysplasia was found in our cohort. With a median follow-up of 24 months, one patient showed CML acceleration and two relapsed.

High incidence of cryptic translocations involving the Ig heavy chain gene in multiple myeloma, as shown by fluorescence in situ hybridization.

Cytogenetic studies have shown rearrangements of the Ig heavy chain (IGH) gene at 14q32 in 10–60% of patients with multiple myeloma (MM) or primary plasma cell leukemia (PCL). Analysis of MM patients and human myeloma cell lines (HMCL) using interphase fluorescence in situ hybridization (FISH) and molecular techniques has shown IGH rearrangements in 75% of MM cases and in up to 100% of HMCL. A review of the literature revealed at least 18 different partner chromosomal regions. To investigate whether some of these translocations were recurrent and possibly to identify new partner regions, we developed a set of FISH probes to detect every IGH recombination. We analyzed 28 MM and 4 primary PCL patients with abnormal karyotypes and 12 HMCL. Whereas conventional cytogenetics detected a 14q32 abnormality in only 15% of the patients, FISH detected it in 47% of patients and in 75% of HMCL. The partner chromosome was identified in 10 of 15 patients with a 14q32 rearrangement. Interestingly, the same t(4;14)(p16;q32) was detected in five patients and three HMCL, i.e., 33% of patients and HMCL with an IGH rearrangement. New partner chromosomal regions have also been identified, i.e., 9p13, 12p11, 12p13, and Xq28, besides the previously reported 8q24, 11q13, 12q24, and 16q24 rearrangements. The genes involved in these new translocations are not known, except for 9p13, where PAX5 was shown to be the partner gene. We conclude that: 1) IGH recombinations are frequent but not constant in MM, 2) these rearrangements often occur through cryptic translocations, and 3) the t(4;14)(p16;q32) is one of the most frequent translocations, but many other chromosomal regions may be involved. Genes Chromosomes Cancer 24:9–15, 1999.

NUP98 rearrangements in hematopoietic malignancies: a study of the Groupe Francophone de Cytogénétique Hématologique.

The NUP98 gene is fused with 19 different partner genes in various human hematopoietic malignancies. In order to gain additional clinico-hematological data and to identify new partners of NUP98, the Groupe Francophone de Cytogénétique Hématologique (GFCH) collected cases of hematological malignancies where a 11p15 rearrangement was detected. Fluorescence in situ hybridization (FISH) analysis showed that 35% of these patients (23/66) carried a rearrangement of the NUP98 locus. Genes of the HOXA cluster and the nuclear-receptor set domain (NSD) genes were frequently fused to NUP98, mainly in de novo myeloid malignancies whereas the DDX10 and TOP1 genes were equally rearranged in de novo and in therapy-related myeloid proliferations. Involvement of ADD3 and C6ORF80 genes were detected, respectively, in myeloid disorders and in T-cell acute lymphoblastic leukemia (T-ALL), whereas the RAP1GDS1 gene was fused to NUP98 in T-ALL. Three new chromosomal breakpoints: 3q22.1, 7p15 (in a localization distinct from the HOXA locus) and Xq28 were detected in rearrangements with the NUP98 gene locus. The present study as well as a review of the 73 cases previously reported in the literature allowed us to delineate some chromosomal, clinical and molecular features of patients carrying a NUP98 gene rearrangements.

t(5;14)/HOX11L2-positive T-cell acute lymphoblastic leukemia. A collaborative study of the Groupe Francais de Cytogenetique Hematologique (GFCH)

To accurately estimate the incidence of HOX11L2 expression, and determine the associated cytogenetic features, in T-cell acute lymphoblastic leukemia (T-ALL), the Groupe Français de Cytogénétique Hématologique (GFCH) carried out a retrospective study of both childhood and adult patients. In total, 364 patients were included (211 children ⩽15 years and 153 adults), and 67 (18.5%) [47 children (22.4%) and 20 adults (13.1%)] were shown to either harbor the t(5;14)q35;q32) translocation or express the HOX11L2 gene or both. Most of the common hematological parameters did not show significant differences within positive and negative populations, whereas the incidence of CD1a+/CD10+ and cytoplasmic CD3+ patients was significantly higher in positive than in negative children. Out of the 63 positive patients investigated by conventional cytogenetics, 32 exhibited normal karyotype, whereas the others 31 showed clonal chromosome abnormalities, which did not include classical T-ALL specific translocations. Involvement of the RANBP17/HOX11L2 locus was ascertained by fluorescence in situ hybridization in six variant or alternative (three-way translocation or cytogenetic partner other than 14q32) translocations out of the 223 patients. Our results also show that HOX11L2 expression essentially occurs as a result of a 5q35 rearrangement, but is not associated with another identified T-ALL specific recurrent genetic abnormality, such as SIL-TAL fusion or HOX11 expression.

Wide diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogénétique Hématologique study

PAX5 is 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 PAX5 alterations. We found PAX5 internal 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 TAOK1 genes 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 PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, 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 PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

Detection of t(11;14) using interphase molecular cytogenetics in mantle cell lymphoma and atypical chronic lymphocytic leukemia

The chromosomal translocation t(11;14)(q13;q32) fuses the IGH and CCND1 genes and leads to cyclin D1 overexpression. This genetic abnormality is the hallmark of mantle cell lymphoma (MCL), but is also found in some cases of atypical chronic lymphocytic leukemia (CLL), characterized by a poor outcome. For an unequivocal assessment of this specific chromosomal rearrangement on interphase cells, we developed a set of probes for fluorescence in situ hybridization (FISH). Northern blotting was performed for analysis of the cyclin D1 expression in 18 patients. Thirty‐eight patients, with either a typical MCL leukemic phase (17 patients) or atypical CLL with an MCL‐type immunophenotype, i.e., CD19+, CD5+, CD23−/low, CD79b/sIgM(D)++, and FMC7+ (21 patients), were analyzed by dual‐color interphase FISH. We selected an IGH‐specific BAC probe (covering the JH and first constant regions) and a commercially available CCND1 probe. An IGH–CCND1 fusion was detected in 28 of the 38 patients (17 typical MCL and 11 cases with CLL). Cyclin D1 was not overexpressed in two patients with typical MCL and an IGH–CCND1 fusion. In view of the poor prognosis associated with MCL and t(11;14)‐positive CLL, we conclude that this set of probes is a valuable and reliable tool for a rapid diagnosis of these entities. Genes Chromosomes Cancer 23:175–182, 1998.

Fluorescence In Situ Hybridization on Peripheral-Blood Specimens Is a Reliable Method to Evaluate Cytogenetic Response in Chronic Myeloid Leukemia

PURPOSE To evaluate the usefulness of fluorescence in situ hybridization (FISH) on peripheral-blood specimens to evaluate the cytogenetic response to treatment in patients with chronic myeloid leukemia (CML). PATIENTS AND METHODS In a first attempt, we analyzed 62 bone marrow specimens using interphase FISH and compared the results with those of conventional cytogenetics. In a second step, we analyzed 60 paired sets of bone marrow and peripheral-blood specimens with interphase FISH. RESULTS The results of interphase FISH agreed with conventional cytogenetics on bone marrow for most patients, and only minor differences were found (r =.98). The comparison of interphase FISH on bone marrow versus peripheral-blood specimens showed a strong correlation between these two specimen sources (r =.97). CONCLUSION Our results confirmed that FISH is a sensitive technique for the evaluation of response to treatment in patients with CML. Moreover, our study suggests that follow-up of cytogenetic response to therapy can be evaluated on peripheral-blood specimens, thus enabling an easier and more frequent evaluation of patients. The next step will be to evaluate this technique in a large prospective trial to define the prognostic value of complete remissions evaluated by FISH.

Various types of rearrangements target TLX3 locus in T-cell acute lymphoblastic leukemia

Most chromosomal translocations observed in T‐cell acute lymphoblastic leukemia (T‐ALL) often produce transcriptional activation of transcription factor oncogenes. Ectopic expression of the TLX3 (also known as HOX11L2) gene has been shown to be associated with a cryptic t(5;14)(q35;q32) translocation specific for a subtype of T‐ALL. Here we report several examples of variant and alternative translocations resulting in expression of TLX3 in T‐ALL, and we describe three of these translocations in detail. In particular, the CDK6 gene was rearranged in two t(5;7)(q35;q21) translocations. In two additional instances, fusion of the BCL11B (also known as CTIP2) and RANBP17/TLX3 loci were shown to result from subtle genomic insertion/deletion within these loci. This study further underscores that TLX3 expression in T‐ALL is strongly associated with the presence of genomic rearrangements.