The complex karyotype in hematological malignancies: a comprehensive overview by the Francophone Group of Hematological Cytogenetics (GFCH).

Karyotype complexity has major prognostic value in many malignancies. There is no consensus on the definition of a complex karyotype, and the prognostic impact of karyotype complexity differs from one disease to another. Due to the importance of the complex karyotype in the prognosis and treatment of several hematological diseases, the Francophone Group of Hematological Cytogenetics (Groupe Francophone de Cytogénétique Hématologique, GFCH) has developed an up-to-date, practical document for helping cytogeneticists to assess complex karyotypes in these hematological disorders. The evaluation of karyotype complexity is challenging, and it would be useful to have a consensus method for counting the number of chromosomal abnormalities (CAs). Although it is not possible to establish a single prognostic threshold for the number of CAs in all malignancies, a specific consensus prognostic cut-off must be defined for each individual disease. In order to standardize current cytogenetic practices and apply a single denomination, we suggest defining a low complex karyotype as having 3 CAs, an intermediate complex karyotype as having 4 CAs, and a highly complex karyotype as having 5 or more CAs.

Copy-number analysis identified new prognostic marker in acute myeloid leukemia.

Recent advances in genomic technologies have revolutionized acute myeloid leukemia (AML) understanding by identifying potential novel actionable genomic alterations. Consequently, current risk stratification at diagnosis not only relies on cytogenetics, but also on the inclusion of several of these abnormalities. Despite this progress, AML remains a heterogeneous and complex malignancy with variable response to current therapy. Although copy-number alterations (CNAs) are accepted prognostic markers in cancers, large-scale genomic studies aiming at identifying specific prognostic CNA-based markers in AML are still lacking. Using 367 AML, we identified four recurrent CNA on chromosomes 11 and 21 that predicted outcome even after adjusting for standard prognostic risk factors and potentially delineated two new subclasses of AML with poor prognosis. ERG amplification, the most frequent CNA, was related to cytarabine resistance, a cornerstone drug of AML therapy. These findings were further validated in The Cancer Genome Atlas data. Our results demonstrate that specific CNA are of independent prognostic relevance, and provide new molecular information into the genomic basis of AML and cytarabine response. Finally, these CNA identified two potential novel risk groups of AML, which when confirmed prospectively, may improve the clinical risk stratification and potentially the AML outcome.

Evaluation of allogeneic hematopoietic SCT in younger adults with adverse karyotype AML.

To illustrate methodological issues, we compared donor vs no-donor to transplant vs no-transplant comparisons in a cohort of 107 patients aged 50 years with adverse karyotype AML in first CR. Adverse karyotypes were defined as -7, del(7q), -5, del(5q), t(9;22), 11q23, 3q26 or complex abnormalities. Mantel-Byar estimations and hematopoietic SCT (HSCT) as a time-dependent variable were used to compare transplant vs no-transplant cumulative incidence of relapse (CIR), relapse-free survival (RFS) and OS. In all, 52 patients had a sibling donor, but only 35 of them were transplanted in first CR, whereas 9 patients received HSCT from alternative stem cell sources. Donor-based analysis showed lower CIR in the donor group, not translating in prolonged RFS or OS. Conversely, transplant-based analysis showed that HSCT in the first CR improved the three CIR (multivariate hazard ratio (HR), 0.31; P<0.001), RFS (multivariate HR, 0.57; P=0.047) and OS (multivariate HR, 0.54; P=0.03) endpoints. At 5 years, OS was estimated at 33% in transplanted vs 18% in non-transplanted patients. The positive effect of HSCT was more pronounced in patients aged 35 years and/or in those transplanted in the more recent years. These results confirm that HSCT is likely the best curative option in younger patients with adverse karyotype AML.

Prognostic significance of DNA methyltransferase 3A mutations in cytogenetically normal acute myeloid leukemia: a study by the Acute Leukemia French Association.

Recently, DNA methyltransferase 3A (DNMT3A) mutations have been identified in acute myeloid leukemia (AML), the highest frequency being found within cytogenetically normal (CN) AML. In this study, diagnostic samples from 123 adults younger than 60 years with primary CN-AML homogeneously treated in the Acute Leukemia French Association-9801 and -9802 trials were screened for mutations in DNMT3A-conserved domains by direct sequencing. Patients were also assessed for the presence of FLT3 (fms-like tyrosine kinase receptor-3), NPM1 (nucleophosmin), CEBPA, WT1 (Wilms tumor 1), IDH1 (isocitrate dehydrogenase 1) and IDH2 mutations. Thirty-eight mutations were detected in 36 patients (29%): 36 nucleotide substitutions, mostly affecting amino-acid residue R882 and two frameshift deletions. DNMT3A mutations were significantly associated with the French-American-British subtypes M4/M5 and the presence of NPM1 mutations. In the whole cohort, DNMT3A mutated patients had a shorter event-free survival (5-year EFS: 13% vs 32%, P = 0.02) and overall survival (5-year OS: 23% vs 45%, P = 0.02) compared with DNMT3A wild-type patients. In multivariate analysis including age, white blood cell count, NPM1/FLT3-internal tandem duplication/CEBPA risk group and DNMT3A mutational status, the presence of a DNMT3A mutation remained an independent adverse prognostic factor for EFS and OS, suggesting that testing for DNMT3A mutations could help further improve risk stratification in CN-AML.

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.

Fluorescence in situ hybridization analysis of 110 hematopoietic disorders with chromosome 5 abnormalities: do de novo and therapy-related myelodysplastic syndrome-acute myeloid leukemia actually differ?

A retrospective cytogenetic study of acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) was conducted by the Groupe Francophone de Cytogénétique Hématologique (GFCH) to evaluate the structural abnormalities of chromosome 5 associated with other chromosomal abnormalities, in particular of chromosome 7, in these pathologies. In all, 110 cases of AML/MDS were recruited based on the presence of chromosome 5 abnormalities under conventional cytogenetics and supplemented by a systematic fluorescence in situ hybridization study of chromosomes 5 and 7. The abnormalities of the long arm of chromosome 5 (5q) were deletions of various sizes and sometimes cryptic. The 5q abnormalities were associated with translocations in 54% of cases and were simple deletions in 46%. In 68% of cases, 5q deletions were associated with chromosome 7 abnormalities, and 90% of these presented a complex karyotype. Of the 110 patients, 28 had a hematopoietic disorder secondary to chemotherapy, radiotherapy, or both. Among 82 patients with de novo AML/MDS, 63 were older than 60 years. Chromosomal abnormalities often associated hypodiploidy and chromosome 5 and 7 abnormalities in complex karyotypes, features resembling those of secondary hemopathies. Systematic investigation of the exposure to mutagens and oncogenes is thus essential to specify the factors potentially involved in MDS/AML with 5q abnormalities.

Effect of priming with granulocyte-macrophage colony-stimulating factor in younger adults with newly diagnosed acute myeloid leukemia: a trial by the Acute Leukemia French Association (ALFA) Group.

In a multicenter trial, 259 young adults (15-49 years) with newly diagnosed acute myeloid leukemia (AML) were first randomized to receive a timed-sequential induction regimen given either alone (135 patients) or concomitantly with granulocyte-macrophage colony-stimulating factor (GM-CSF) (124 patients). Patients reaching complete remission (CR) were then randomized to compare a timed-sequential consolidation to a postremission chemotherapy including four cycles of high-dose cytarabine followed by maintenance courses. In the appropriate arm, GM-CSF was given concurrently with chemotherapy during all cycles of consolidation. CR rates were significantly better in the GM-CSF arm (88 vs 78%, P<0.04), but did not differ after salvage. Patients receiving GM-CSF had a higher 3-year event-free survival (EFS) estimate (42 vs 34%), but GM-CSF did not impact on overall survival. Patients with intermediate-risk cytogenetics benefited more from GM-CSF therapy (P=0.05) in terms of EFS than patients with other cytogenetics. This was also confirmed when considering only patients following the second randomization, or subgroups defined by a prognostic index based on cytogenetics and the number of courses required for achieving CR. Priming of leukemic cells with hematopoietic growth factors is a means of enhancing the efficacy of chemotherapy in younger adults with AML.

Clinical, cytogenetic and molecular characteristics of 14 T-ALL patients carrying the TCRbeta-HOXA rearrangement: a study of the Groupe Francophone de Cytogénétique Hématologique.

Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

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.

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.

BCR/ABL fusion gene detected on 9q34 by fluorescence in situ hybridization in an acute leukemia with two BCR/ABL positive clones, one Ph-negative and one Ph-positive.

We report cytogenetic, fluorescence in situ hybridization (FISH), and molecular analyses in the first reported case of an acute leukemia with two BCR-positive clones: one cell Ph-positive and all others Ph-negative. A BCR/ABL fusion gene on 9q34 was detected only with a BCR/ABL dual color translocation probe. These FISH interphase signals must be confirmed on a metaphase to avoid an erroneous interpretation. This observation appears to indicate a 2-step mechanism for this aberrant fusion gene localization: first, a classical t(9;22), and then the transfer of the fusion gene formed on chromosome 22 to chromosome 9 by a second translocation between the long arms of the derivative chromosomes 9q+ and 22q-, masking the first chromosome exchange.

Three cases of typical aplastic anaemia associated with a Philadelphia chromosome.

We report three cases of typical aplastic anaemia (AA) associated with a Philadelphia chromosome. This translocation was detected at the time of diagnosis of AA (one patient) and when overt leukaemia was diagnosed (two patients: one chronic myeloid leukaemia and one acute lymphoblastic leukaemia) after AA therapy and recovery of blood counts. We discuss the literature arguments about considering some cases of AA as preleukaemic disorders and suggest that our cases illustrate the association of AA with a clonal malignant disorder. We conclude that cytogenetic analysis is necessary at diagnosis of AA or after recovery of blood counts.

Cytogenetic, interphase, and multicolor fluorescence in situ hybridization analyses in primary plasma cell leukemia: a study of 40 patients at diagnosis, on behalf of the Intergroupe Francophone du Myélome and the Groupe Français de Cytogénétique Hématologique.

Primary plasma cell leukemia (PCL) is a rare plasma cell malignancy. Consequently, few large reports have been published. Presented is a cytogenetic analysis of 40 patients with primary PCL compared with 247 newly diagnosed patients with stage III multiple myeloma (MM). Cytogenetic abnormalities were observed in 23 of 34 patients, with usually complex hypodiploid or pseudodiploid karyotypes. Analysis of rearrangements of the 14q32 region revealed significant differences with high cell mass MM-a higher incidence of t(11;14) (33% vs 16%; P <.025) and of t(14;16) (13% vs 1%; P <.002) though incidences of t(4;14) were identical and a higher incidence of monosomy 13 (68% vs 42%; P =.005). Hypodiploid karyotypes and monosomy 13 may explain, at least in part, the poorer prognosis of primary PCL. In contrast, significantly longer survival was observed in patients displaying t(11;14) in comparison with those lacking this translocation (P =.001).

A case of chronic neutrophilic leukemia with deletion (11)(q23).

We report a case of chronic neutrophilic leukemia (CNL) in a 68-year-old man. Karyotype showed a clonal abnormality, never described before in CNL: 46,XY,del(11)(q23). Southern blot analysis of the MLL gene did not reveal any rearrangement, and reverse transcriptase polymerase chain reaction (RT-PCR) analysis did not show any fusion of BCR-ABL. Treatment with hydroxyurea and cytosine arabisonide was ineffective.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: relevance to diseases of the nervous system.

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl-lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado-Joseph disease (SCA3), or dentato-rubral pallidoluysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl-lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.