Paola Agostini

A His-155 to Tyr Polymorphism Confers Gain-of-Function to the Human P2X7 Receptor of Human Leukemic Lymphocytes

The P2X7R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X7R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X7R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X7R function was monitored by measuring ATP-induced Ca2+ influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X7 allelic variant. Ca2+ influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca2+ flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X7R confirmed an increased ATP-dependent activation of the P2X7 489T mutant with respect to the wild type receptor, as assessed by both by [Ca2+]i influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X7R.

Chronic lymphocytic leukemia with 6q- shows distinct hematological features and intermediate prognosis

Cytogenetic and fluorescence in situ hybridization studies were successfully performed in 217 chronic lymphocytic leukemia (CLL). In all, 13 patients with 6q21 deletion were identified and characterized in comparison with 92 patients with ‘favourable’ karyotype (normal or 13q−), 69 cases with ‘intermediate risk’ (1–2 anomalies) and 43 cases with ‘unfavourable’ karyotype (complex, 11q− or 17p−). Six out of 13 cases with 6q− showed an excess of atypical lymphocytes, a finding confirmed at the histologic level; >20% CD38+ cells were seen in 5/6 cases. IGVH mutational status revealed >98% homology to the germline sequence in 4/10 cases. When compared with the ‘favourable’ group, patients with 6q− showed a higher white blood cell (WBC) count, frequent splenomegaly, atypical morphology, CD38+ and short time from diagnosis to first treatment and short survival. A higher median WBC count was found in the 6q− group vs the intermediate-risk group; survival was shorter in the unfavourable group. To ascertain if the 6q− anomaly was an independent factor predicting for an inferior outcome among those patients with ‘favourable’ cytogenetics, we performed an analysis of prognostic factors in 105 patients (92 ‘favourable’ plus 13 with 6q−), showing that the 6q− chromosome maintained its prognostic significance at multivariate analysis (P=0.02) along with stage (P=0.01). We conclude that CLL with 6q− is characterized by a high incidence of atypical morphology, classical immunophenotype with CD38 positivity and intermediate incidence of IGVH somatic hypermutation. Clinicobiological features and outcome show that this cytogenetic subset of CLL should be allocated in an intermediate-risk category.

Clinical importance of interphase cytogenetics detecting occult chromosome lesions in myelodysplastic syndromes with normal karyotype

At diagnosis, approximately half of myelodysplastic (MDS) patients presents a normal karyotype by conventional cytogenetic analysis (CCA). Fluorescent in situhybridization (FISH) is more sensitive than CCA allowing for the detection of minor clones and of submicroscopic lesions. We have analyzed by FISH 101 MDS patients with normal karyotype for the occurrence of the abnormalities which are most frequently observed in MDS (ie −5/5q−, −7/7q−, +8, 17p−). In 18 patients, 15 to 32% of interphase cells were found to carry one FISH abnormality. Six patients presented trisomy 8, five had del(5)(q31), five del(7)(q31), one monosomy 7 and one del(17)(p13). FISH abnormalities were more frequently observed among patients with an increased percentage of bone marrow blasts (P = 0.001). FISH abnormalities were also associated with a higher rate of progression into AML (13/18 vs 12/83, P < 0.001) and were predictive for a worse prognosis (P < 0.001). Multivariate analysis indicated that FISH positivity and IPSS risk group were independent predictors for a poor survival (P = 0.0057 and 0.0123, respectively) and for leukemic transformation (P = 0.0006 and 0.035, respectively). Leukemic transformation in FISH-positive patients was associated in all cases with an expansion of the abnormal clone. Our data demonstrated that a significant proportion of MDS patients with normal karyotype presented, if analyzed by FISH, clones of cytogenetically abnormal cells which played a determinant role in the progression of the disease. The presence of FISH abnormalities identified a group of MDS patients with normal karyotype characterized by an inferior prognosis.

Exposure to myelotoxic agents and myelodysplasia: case–control study and correlation with clinicobiological findings

To better define the role of exposure to myelotoxic agents in the genesis of myelodysplastic syndrome (MDS), we carried out (a) a case–control study for the determination of the relative risk (RR) of developing MDS, including 178 consecutive patients and 178 sex‐ and age‐matched controls; (b) a study of clinicobiological features in MDS arising after occupational exposure to myelotoxic agents and in MDS in ‘non‐exposed’ patients. The definition of the ‘exposure’ status was based on a predetermined questionnaire, with calculation of an ‘exposure’ index (hours/day × days/year × years). Cumulative exposure to pesticides or to organic solvents, for >2400 h, was recorded in 48 and 25 MDS patients, respectively, compared to 27 and four controls (P < 0.00001; RR 3.74; 95% confidence interval 2.02–5.37). Older age and an excess of refractory anaemia with ringed sideroblasts and refractory anaemia with excess of blasts was noted among ‘exposed’ MDS‐patients (group 1), compared to non‐exposed MDS‐patients (group 2). 68.3% patients in group 1 had clonal chromosome changes, compared with 43.2% patients in group 2. Complex karyotypes, −7/7q−, −5/5q−, +8, 7p and 17p aberrations were seen more frequently in group 1, whereas a normal karyotype, isolated 5q− or 20q− occurred more frequently in group 2. The association of exposure to myelotoxic agents with older age at presentation and with unfavourable chromosome changes accounted for the shorter survival observed in ‘exposed’ patients. These data show that occupational exposure to pesticides and organic solvents in our region resulted in an increased RR of developing MDS and that a distinct cytogenetic profile was associated with MDS in ‘exposed’ patients. These findings provide strong indirect evidence that these agents may play a role in the pathogenesis of MDS, preferentially targeting some of the chromosome regions which are frequently involved in therapy‐related myeloid neoplasias.

Acquired Chromosome 11q Deletion Involving the Ataxia Teleangiectasia Locus in B-Cell Non-Hodgkin’s Lymphoma: Correlation With Clinicobiologic Features

PURPOSE To study the clinicobiologic significance of acquired 11q deletions involving the ataxia teleangiectasia locus (ATM+/-) in B-cell non-Hodgkins lymphomas (NHL). PATIENTS AND METHODS Fifty-three indolent lymphomas and 82 aggressive lymphomas were studied by conventional cytogenetic analysis and by fluorescence in situ hybridization using an 11q22-23 probe recognizing ATM sequences. Pertinent clinical data were collected. RESULTS A hemizygous ATM deletion was seen in 44% to 88% of the interphase cells in 15 cases (11.1%); four patients had an indolent lymphoma (follicular center cell lymphoma), and 11 patients had an aggressive lymphoma (five mantle-cell lymphomas [MCLs] and six diffuse large-cell lymphomas). Dual-color hybridization studies showed ATM deletion to be possibly a secondary aberration in three patients with MCL. Ten out of 15 ATM+/- patients had a complex karyotype, 11 out of 15 had more than 90% abnormal metaphases (AA karyotype status), and +12, 13q14 deletion, and 17p13 deletion were seen in seven, four, and five cases, respectively. Patients with ATM+/- more frequently had a complex karyotype (P =.01) and the AA karyotype (P =.04) compared with patients without ATM+/-. With the exception of a poor performance status (P =.001), no correlation was found between ATM+/-, initial clinical variables, and complete remission rate; whereas a highly significant association was found with shorter survival (P <.0001). This cytogenetic lesion maintained its prognostic importance in multivariate analysis (P =.0004), along with performance status (P =.0006), serum lactate dehydrogenase level (P =.03), splenomegaly (P =.01), and histologic grade (P =.03). When analyzing indolent lymphomas and aggressive lymphomas separately, ATM+/- maintained its prognostic importance as an independent variable in both histologic groups (P =.0001 and P =.016, respectively). CONCLUSION Though possibly not representing a primary genetic lesion in the majority of cases, the acquired ATM+/- status has clinicobiologic importance in NHL, possibly representing a major cytogenetic determinant of outcome.

Incidence and significance of cryptic chromosome aberrations detected by fluorescence in situ hybridization in acute myeloid leukemia with normal karyotype

To better define the incidence and significance of cryptic chromosome lesions in acute myeloid leukemia (AML), fluorescence in situ hybridization (FISH) studies were performed in interphase cells and, when appropriate, in metaphase cells and in morphologically intact BM smears. Fifty-five adult de novo AML (group A) and 27 elderly AML or AML after myelodysplastic syndrome (AML-MDS) (group B) were tested using probes detecting the following anomalies: −5, −7, +8, deletions of 5q31, 7q31, 12p13/ETV6, 17p13/p53, 20q11. All the patients had a normal karyotype in more than 20 cells and tested negative for the common AML-associated fusion genes. No patient in group A was found to carry occult chromosome anomalies, whereas 8/27 patients in group B (P < 0.0001) showed 5q31 or 7q31 deletion (three cases each), a 17p13/p53deletion or trisomy 8 (one case each) in 33–60% interphase cells. Metaphase cells showed only one hybridization signal at 5q31 (three cases) and 7q31 (one case), whereas two normal signals at 7q31 and chromosome 8 centromeres were seen in two patients with 7q deletion and trisomy 8 in interphase cells. The majority of blast cells (76–94%) carried the chromosome anomaly in all cases; erythroid involvement in a minority of cells was seen in three patients. In group B, the presence of occult chromosome anomalies was associated with exposure to myelotoxic agents in the workplace (5/8 cases vs 3/19, P = 0.026) and with a lower complete remission rate (0/6 patients vs 7/12, P = 0.024). We arrived at the following conclusions: (1) cryptic chromosome deletions in the order of a few hundred kb magnitude may be found in a fraction of elderly AML or MDS-related AML and not in de novo adult AML with normal karyotype; (2) these chromosome lesions are usually represented by submicroscopic rearrangements; (3) they display a specific pattern of cell-lineage involvement arguing in favor of their role in the outgrowth of the leukemic blast cells; (4) they are associated with a history of exposure to myelotoxic agents in the workplace and, possibly, with resistance to induction treatment.

Secondary Chromosome Changes in Mantle Cell Lymphoma: Cytogenetic and Fluorescence in situ Hybridization Studies

To better define the incidence and nature of secondary chromosome anomalies in mantle cell lymphoma (MCL) carrying the t(11;14)/BCL1 rearrangement, cytogenetic and fluorescence in situ hybridization studies (FISH) were performed in 42 patients (39 classical histology, 3 blastoid variant), using 6q21, 9p21/p16, 13q14, 17p13/p53 and chromosome-12-specific probes. Karyotypes from 89 cases published in 5 recent series including patients diagnosed in a homogeneous fashion were reviewed. In our series, FISH confirmed the interpretation of the karyotype in all cases and disclosed cryptic chromosome deletions in a sizeable fraction of cases. One patient (2,4% of total) was found with a cryptic 9p21 deletion by FISH. Two cases (4,8%) had a 6q21 deletion at CCA and at FISH; +12 was found in three cases by CCA plus nine by FISH (28.6%); 13q14 deletion was found in six cases by CCA plus 16 by FISH (52.4%), 17p13 deletion in three cases by CCA plus 8 by FISH (26.2%). In 131 patients (42 present series plus 89 in the literature) secondary chromosome aberrations seen by conventional cytogenetic analysis in more than 5 cases included deletions/translocations (del/t) 6q15–23 [15 cases]; -13 [14 cases]; del/t 1p21–31 [12 cases]; +3q [11 cases]; del/t 17p [9 cases]; 8p translocations and del(Y) [8 cases each]; -20 [7 cases]; 13q14 deletion, del/t 11q22–23, del/t 9q, del(10)(q22q24), -20, -21, -22 and -X [6 cases each]. We arrived at the following conclusions: i) though no secondary anomaly is specific for MCL, there is a distinct profile of recurrent chromosome lesions in MCL with 1 p21–31 deletions, 8p translocations, 11q22–23 anomalies having a strong association with CD5+ B-cell lymphomas of low-to-intermediate grade histology; ii) FISH enabled the detection of cryptic chromosome 12, 13q and 17p rearrangements in a sizeable fraction of cases; iii) 9p21/p16 deletions did not occur at a high incidence in this series, possibly because of the low number of cases with blastoid variant.

Abnormalities of chromosomes 1p34-36, 4p16, 4q35, 9q11-32 and +7 represent novel recurrent cytogenetic rearrangements in chronic lymphocytic leukemia.

Karyotypes were studied in over 250 cases of CLL seen at our Institution and 12 cases with a previously undescribed chromosome abnormality were identified. Cytogenetic and clinicobiological features in these patients are described. Fluorescence in situ hybridization using probes for the detection of +12 and deletions of 13q14, 17p13, and 11q22-23 was performed. Hematologic and clinical data were reviewed and a review of the literature was performed. Twelve patients were found carrying the following aberrations in the stemline: abnormalities at 1p34 (n = 2), 4p16 (n = 2), 4q35 (n = 2), 9q11-32 (n = 4) and +7 (n = 2). Trisomy 12 was found in 3 cases, whereas no case carried 13q-, 11q-, 17p-. Our data showed that (i) aberrations involving 1p34 and 4p16 as isolated chromosome anomalies were preferentially associated with early stage disease; (ii) 4q35 anomalies were associated with a relatively aggressive disease, atypical morphology and with monoclonal gammopathy; (iii) rearrangements of 9q were characterized by atypical morphology and aggressive disease with splenic involvement; (iv) +7 be may associated with +12. 1p34-36; 4p16; 4q35; 9q and chromosome 7 represent novel recurrent rearranged sites in CLL, with a 0.5-3% incidence. Transformation in these patients seemingly occured through a cytogenetic route not involving the classical CLL-associated chromosome regions. These chromosome rearrangements may be associated with peculiar hematologic features.

Frequency of ETV6/AML1 fusion in adult acute lymphoblastic leukemia.

Leukemia L Fouillard Department of Hematology, hôpital M Bensidhoum2 Saint-Antoine, AP-HP, Paris, France; D Bories3 2Laboratoire de recherche sur la thérapie H Bonte4 cellulaire, LTCRA/UPRES EA1638, M Lopez Université Paris VI (Pierre et Marie Curie), A-M Moseley5 Association Claude Bernard et Institut de A Smith5 Protection et de Sureté Nucléaire (IPSN), S Lesage Paris, France; Service d’hématologie F Beaujean6 biologique, hôpital Henri-Mondor, AP-HP, D Thierry2 Créteil, France; 4Laboratoire d’anatomoP Gourmelon2 pathologie, hôpital Saint-Antoine, AP-HP, A Najman Paris, France; Osiris Therapeutics, N-C Gorin1,2 Baltimore, MD, USA; 6EFS hôpital HenriMondor, AP-HP, Créteil, France