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.

High incidence of translocations t(11;14)(q13;q32) and t(4;14)(p16;q32) in patients with plasma cell malignancies.

Abnormalities involving the 14q32 region are recurrent chromosomal changes in plasma cell malignancies. Recent preliminary molecular analyses found IGH rearrangements in almost 100% of human myeloma cell lines and in 75% of patients. However, no systematic study analyzing the nature of the partner chromosomal regions have been reported thus far. To define the exact incidence of illegitimate IGH rearrangements and the respective incidence of partner genes cloned to date, we analyzed 141 patients with either multiple myeloma (MM, n = 127) or primary plasma cell leukemia (PCL, n = 14) using fluorescence in situ hybridization. The overall incidence of illegitimate recombinations was 57% (80 of 141 patients). Analysis of this incidence according to Durie and Salmon stage, patients' status, i.e., MM versus primary PCL and diagnosis versus relapse, immunoglobulin type and subtype, and beta2-microglobulin value, did not show any correlation. To analyze the nature of the partner chromosomal region, we selected probes specific for the following genes: FGFR3 (4p16), MYC (8q24), CCND1 (11q13), MAF (16q23), and BCL2 (18q21). These probes, combined with differentially labeled 14q32 probes, were used for dual-color fluorescence in situ hybridization on interphase plasma cells. Among the 80 patients with illegitimate IGH rearrangement, we identified 23 IGH-CCND1 fusion cases [i.e., t(11;14)], 17 IGH-FGFR3 fusion cases [i.e., t(4;14)], 3 IGH-MYC fusion cases [i.e., t(8;14)], and only one IGH-MAF fusion case. No IGH-BCL2 fusion case was detected. In 37 of 80 patients, none of these partner genes was involved. Analysis of cases with specific translocations according to their bioclinical features at diagnosis did not show any correlation. This study demonstrated that CCND1 and FGFR3 genes are involved together in about 50% of MM and primary PCL patients with illegitimate IGH rearrangements.

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.

Cytogenetic study of 75 erythroleukemias.

Chromosomal abnormalities of erythroleukemia (EL) are often described as complex and unspecific. A retrospective study of 75 EL defined following the WHO classification was performed by the Groupe Francophone de Cytogénétique Hématologique (GFCH) in order to reexamine the cytogenetics of this infrequent leukemia subtype. Clonal chromosomal abnormalities were found in 57 patients (76%), distributed in 4 subgroups according to their ploidy status: pseudodiploid (16%), hypodiploid (47%), hyperdiploid (19%), and 18% mixed cases associating 2 different clones (hypodiploid+hyperdiploid) or (pseudodiploid+hyperdiploid). Complex rearrangements and hypodiploid chromosome number were widely dominant (50%). Partial or entire monosomies represented 56% of abnormalities. Chromosomes 5 and 7 were the most frequently involved (41 and 33 times, respectively), followed by chromosomes 8, 16, and 21 (19 times each). Unbalanced abnormalities were more frequent than balanced. All these kinds of abnormalities were observed in de novo as well as in secondary EL. Four out of 7 cases of "pure erythroid" leukemia were associated with a BCR-ABL fusion. Lastly, no chromosome abnormality specific to EL could be established. However, the large overlap of chromosomal abnormality patterns of EL (pure erythroid form excepted) and refractory anemia with excess of blasts in transformation (RAEB-t) favors the hypothesis of similarities between these 2 hematologic disorders.

Amplification of AML1 gene is present in childhood acute lymphoblastic leukemia but not in adult, and is not associated with AML1 gene mutation.

The AML1/CBFA2/RUNX1 gene is the target of many recurrent translocations seen in different leukemia subtypes. The t(12;21)(p13;q22) is the most frequent translocation observed in childhood B acute lymphoblastic leukemia (ALL), occurring in 20% to 25% of cases. In adult ALL this rearrangement is scarce. Another route of AML1deregulation could be point mutations in the runt domain. We now report on AML1amplification in two cases of childhood ALL, found in a series of 107 consecutive children with B-lineage ALL analyzed by fluorescence in situ hybridization (FISH). A parallel analysis of 42 adult B-ALL failed to detect any AML1 rearrangement by FISH. The two patients with AML1 amplification were further analyzed using molecular techniques. SSCP analysis did not detect any mutation. Furthermore, direct sequencing of the cDNA did not reveal any mutation. In conclusion, AML1amplification seems to be observed only in childhood ALL and is not associated with AML1 gene mutation. Other mechanisms, such as gene dosage effects could be hypothesized.

Is t(14;18)(q32;q21) a constant finding in follicular lymphoma? An interphase FISH study on 63 patients.

The translocation (14;18)(q32;q21) is the hallmark of follicular lymphoma (FL). However, conventional cytogenetics and PCR techniques fail to detect it in at least 10% of cases. In order to evaluate the true incidence of this translocation in FL, we analyzed 63 patients with FL, and 17 patients with diffuse large cell lymphoma (DLCL) corresponding to suspected FL transformations using interphase fluorescence in situ hybridization (FISH). Colocalized signals related to the translocation were observed in 19-92% of cells (median = 51%), corresponding to positivity over the threshold in all (63/63) cases. Similarly, 16/17 possibly secondary DLCL displayed the translocation. Although some cytogenetic changes might be missed by this FISH assay (such as rare insertion, or translocations with other chromosomal partners), our results stress t(14;18)(q32;q21) as an almost constant finding in FL. Our sensitive interphase FISH assay should be of great value to define FL more accurately, namely in patients included into therapeutic trials. Furthermore, this approach could be of interest in (re)defining some types of FL, especially the grade 3 FL which frequently lack t(14;18).

Large deletions 5' to the ETO breakpoint are recurrent events in patients with t(8;21) acute myeloid leukemia.

Recurrent chromosomal rearrangements are observed in many leukemia subtypes. Recently, it has been shown that several of these translocations/inversions were associated with the loss of sequences located in the vicinity of the chromosomal breakpoints. So far, such deletions have not been described for the t(8;21) translocation. We have analyzed a series of 65 patients with t(8;21) using several probes specific for the ETO and AML1 regions. We have found six patients (9%) with deletion of the region 5' to ETO. In all six patients, the deletion encompassed at least 260 kb, and was even larger in two patients (up to 2 Mb). A similar analysis of the 21q22 region did not reveal any deletion of the 3'AML1 region. In conclusion, cytogenetically undetectable small deletions located immediately 5' to the ETO breakpoint were found to accompany the t(8;21) translocation in a significant percentage of cases. The clinical significance, if any, of these deletions remains to be determined.

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.

t(5;14)/HOX11L2-positive T-cell acute lymphoblastic leukemia. A collaborative study of the Groupe Français de Cytogénétique Hématologique (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

The MLL recombinome of acute leukemias in 2013.

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.

Hyperdiploid karyotypes in acute myeloid leukemia define a novel entity: a study of 38 patients from the Groupe Francophone de Cytogenetique Hematologique (GFCH).

A series of 38 patients with acute myeloblastic leukemia (AML) with 49 or more chromosomes and without structural abnormalities was selected within the Groupe Francophone de Cytogénétique Hématologique (GFCH) to better define their characteristics. The median age of the patients was 65 years, and all FAB subtypes were represented. Although all chromosomes were gained, some seems to prevail: chromosome 8 (68%), 21 (47%), 19 (37%), and 13 and 14 (34% each). Since MLL rearrangement leads patients in a group with an unfavorable prognosis, search for cryptic rearrangements of MLL was performed in 34 patients and showed abnormalities in 5 (15%). When we applied the most frequent definition of complex karyotypes (three or more abnormalities), all patients with high hyperdiploid AML fall in the unfavorable category. Among the 18 patients without MLL rearrangement receiving an induction therapy, 16 (89%) reached CR and 6 (33%) were still alive after a 31-month median follow-up (14-61 months). Although this study was retrospective, these results suggest that high hyperdiploid AML without chromosome rearrangement seems to be a subgroup of uncommon AML (less than 1%), and may be better classified in the intermediate prognostic group.

Acute myeloid leukaemia with 8p11 (MYST3) rearrangement: an integrated cytologic, cytogenetic and molecular study by the groupe francophone de cytogénétique hématologique.

Thirty cases of acute myeloid leukaemia (AML) with MYST histone acetyltransferase 3 (MYST3) rearrangement were collected in a retrospective study from 14 centres in France and Belgium. The mean age at diagnosis was 59.4 years and 67% of the patients were females. Most cases (77%) were secondary to solid cancer (57%), haematological malignancy (35%) or both (8%), and appeared 25 months after the primary disease. Clinically, cutaneous localization and disseminated intravascular coagulation were present in 30 and 40% of the cases, respectively. AMLs were myelomonocytic (7%) or monocytic (93%), with erythrophagocytosis (75%) and cytoplasmic vacuoles (75%). Immunophenotype showed no particularity compared with monocytic leukaemia without MYST3 abnormality. Twenty-eight cases carried t(8;16)(p11;p13) with MYST3-CREBBP fusion, one case carried a variant t(8;22)(p11;q13) and one case carried a t(8;19)(p11;q13). Type I (MYST3 exon 16-CREBBP exon 3) was the most frequent MYST3-CREBBP fusion transcript (65%). MYST3 rearrangement was associated with a poor prognosis, as 50% of patients deceased during the first 10 months. All those particular clinical, cytologic, cytogenetic, molecular and prognostic characteristics of AML with MYST3 rearrangement may have allowed an individualization into the World Health Organization classification.

Deletion of 5q31 is observed in megakaryocytic cells in patients with myelodysplastic syndromes and a del(5q), including the 5q- syndrome.

One of the most common structural rearrangements in myelodysplastic syndrome (MDS) is a deletion of the long arm of chromosome 5, del(5q). The 5q- syndrome is a distinct entity, that presents with specific morphologic abnormalities of the megakaryocytic lineage. Thus, we evaluated the presence or absence of the del(5q) in these cells. We performed fluorescence in situ hybridization analysis using unique sequence probes (one for 5q31, the other for the 5p telomeric band), and tested bone marrow specimens from 10 patients with MDS (including 6 patients with the 5q- syndrome) and a del(5q). Megakaryocytes were identified by nuclear morphology, size, and ploidy index. Our results demonstrate the presence of the del(5q) in the megakaryocytic lineage and, thus, the involvement of these cells in the disease process.

Childhood B-cell acute lymphoblastic leukemia with FAB-L1 morphology and a t(9;11) translocation involving the MLL gene.

The t(9;11) (p21-22;q23) translocation is frequently associated with acute monoblastic leukemia but may occasionally be seen in patients with acute lymphoblastic leukemia (ALL). We report a case of childhood ALL associated with t(9;11) (p21-22;q23) as the unique recurring chromosomal abnormality. A 3-month-old girl presented with "lymphomatous" ALL (renal enlargement), a high leukocyte count and central nervous system (CNS) involvement. Leukemic cell typing revealed a sIg+ B-cell immunophenotype without CD10 and CD34 antigenic expression while the blast cell morphology was of the FAB-L1 type. Splitting of a YAC encompassing the MLL gene was shown by fluorescence in situ hybridization (FISH) studies of the patient's metaphase chromosomes. Rearrangement of the MLL gene was confirmed by Southern blot analysis. Despite treatment with an hyperintensive polychemotherapeutic regimen, the patient achieved a complete remission but relapsed 9 months later. These results provide further evidence that the t(9;11) may be observed in ALL, involves the MLL gene and is associated with a poor outcome. Moreover, this observation clearly illustrates that sIg+ B-cell ALL is not necessarily associated with a Burkitt (L3) morphology.

[Chromosome 3 abnormalities with dysmegakaryocytopoiesis in in subacute transformation in chronic myeloid leukemia]. / Anomalies du chromosome 3 avec dysmégacaryocytopoïèse au cours d'une transformation subaiguë de leucémie myéloïde chronique.

A patient with Ph1 positive chronic myeloid leukemia developed accelerated phase with cytological abnormalities of platelets and megakaryocytes, and persistence of a high platelet count, after the failure of a course of intensive chemotherapy. During this phase, the karyotype analysis revealed two different Ph1 positive clones: one with a pericentric inversion of chromosome 3, the other with the same abnormality and a paracentric inversion of the second chromosome 3. Cases of acute leukemia with normal thrombopoiesis and abnormalities of chromosome 3, especially paracentric inversion, have already been reported. The significance of this association is discussed.

Comparative genomic hybridization is a powerful tool, complementary to cytogenetics, to identify chromosomal abnormalities in childhood acute lymphoblastic leukaemia.

Cytogenetics has a strong prognostic value in childhood acute lymphoblastic leukaemia (ALL), but results are often incomplete because of the poor chromosome morphology. To improve this analysis, we tested comparative genomic hybridization (CGH) for the detection of chromosomal imbalances. 72 children were retrospectively analysed using CGH. Only 53% of the patients had been fully banded by standard methods. With CGH, 36 patients retained a normal chromosomal profile and 36 had unbalanced abnormalities. No amplification was detected. Fluorescence in situ hybridization (FISH) with centromeric and unique sequence probes was used in those cases with discrepancies or unsuccessful karyotype to validate CGH results. CGH enabled clear identification of unbalanced chromosomal abnormalities, even in some cases which had a normal karyotype. In view of the strong prognostic value of hyperdiploidy in childhood ALL, CGH appears to be a powerful technique, complementary to conventional cytogenetics.