Antonella Bardi

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.

Chromosome 14q32 translocations involving the immunoglobulin heavy chain locus in chronic lymphocytic leukaemia identify a disease subset with poor prognosis

Immunophenotypic studies, fluorescence in situ hybridization (FISH) and conventional karyotyping were used to define the clinicobiological significance of 14q32 translocations involving the immunoglobulin gene locus (14q32/IGH) in 252 chronic lymphocytic leukaemia (CLL) patients. The following regions were studied: 13q14, centromere 12, 6q21; 11q22/ATM; 17p13/TP53, 14q32/IGH. Patients were classified as group 1 (favourable, i.e. 13q‐single or normal), group 2 (intermediate risk, i.e. +12, 6q‐, 1–2 anomalies), group 3 (unfavourable, i.e. 17p‐, 11q‐, complex karyotype), or group 4 (14q32/IGH translocation). Endpoints were treatment‐free survival (TFS) and overall survival (OS). One hundred and ten patients were included in group 1, 99 in group 2, 25 in group 3 and 18 in group 4. 14q32/IGH translocation partners were identified in eight cases (BCL2 in five cases, BCL11A, CCND3 and CDK6 in one case each). group 4 showed shorter TFS versus groups 2 and 1 (25% patients treated at 2 months vs. 12 (P = 0·02) and 20 months (P = 0·002), respectively) and shorter OS (25% patients dead at 18 months versus 50 (P = 0·0003) and >60 months (P < 0·0001) respectively. The 14q32/IGH translocation maintained prognostic significance at multivariate analysis on TFS (P = 0·025) and OS (P < 0·001), along with advanced stage and CD38+. These findings show that the 14q32/IGH translocation predicts for an unfavourable outcome in CLL and that this cytogenetic subset might be included as a separate entity in a hierarchical cytogenetic classification of CLL.

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.

Chromosome aberrations in atypical chronic lymphocytic leukemia : a cytogenetic and interphase cytogenetic study

To define better the chromosomal profile of atypical chronic lymphocytic leukemia (aCLL), cytogenetic and interphase cytogenetic studies were performed in 43 cases, using mitogen-stimulated cultures and DNA probes detecting the two most frequently occurring aberrations in CLL, ie +12 and 13q14 deletions. All cases showed monoclonal CD5/CD19-positive lymphocytosis, with more than 10% large lymphocytes and/or prolymphocytes in peripheral blood smears and reactivity with FMC7, or bright expression of surface immunoglobulins in a fraction of the cases. Karyotype aberrations were detected in 27 of 43 cases (62.8%). Recurrent chromosome changes were +12 (nine cases), 13q14 aberrations (five cases), 11q anomalies (three cases), 6q21-q23 abnormalities and 4q anomalies with different breakpoints (two cases each). Additional chromosome changes were seen in four cases with +12, in three cases with 13q14 anomalies, in two cases with 11q anomalies, in one case with 6q and 4q anomalies. Trisomy 12 was associated with 13q14 anomalies in three cases, one of which also had an 11q abnormality; other associations, found in one case each, were: 13q14 deletion with a 6q anomaly, 11q anomaly with 13q− and 7q−, a 6q anomaly with 7q− and +12. Interphase cytogenetics confirmed the results of chromosome banding analysis and showed that six patients with normal karyotype or no mitosis in fact had concomitant +12 and 13q14 deletion in four cases and isolated +12 or 13q14 deletion in one case each, with a resultant 76% overall incidence of cytogenetic abnormalities. The presence of +12, 13q14 deletions, 11q, and 6q21-q23 anomalies in 19 cases was associated with a 2-month median interval between diagnosis and start of treatment, as compared with a 24-month median interval in 14 cases with normal karyotype or non-recurrent chromosome changes (P = 0.003). We conclude that aCLL is characterized by a relatively high incidence of chromosome anomalies, with recurrent chromosome changes, involving chromosomes 12, 13q14, 6q21q23, 11q, and, possibly, 4q. The presence of complex karyotypes, with concomitant abnormalities of 13q, +12, 6q, 11q, suggests that the development of sequential chromosome changes, rather than any single specific anomaly, may underlie leukemogenesis in this cytologic subset of CLL, partially accounting for the relatively aggressive clinical course.

Fluorescence in situ hybridization for the detection and monitoring of the Ph-positive clone in chronic myelogenous leukemia: comparison with metaphase banding analysis

In order to analyze the efficiency of interphase FISH for the detection and monitoring of Ph+ cells in chronic myelogenous leukemia (CML) under interferon (IFN) treatment, the following experiments were performed: (1) 98 specimens derived from 32 patients were analyzed in parallel by dual-color FISH and by conventional chromosome analysis (CCA). A 300/200 kb BCR/ABL probe was used in all tests and a smaller 35.5/39 kb probe was tested in parallel in 22 BM samples; (2) 30 BM samples were prepared by direct harvest and by 24-h culture and were analyzed in parallel; (3) PB and BM samples obtained simultaneously from 11 patients were analyzed. The cut-off point for the recognition of BCR/ABL fusion was set at 2.4%, calculated as the mean percent of false positivity in 11 controls plus 3 s.d. A very close correlation was observed (r = 0.994, r2 = 0.988, P < 0.0001) between the percentages of ph+ cells as assessed by CCA and by interphase FISH in 98 samples (26 at diagnosis). There was a moderate overestimation of the frequency of Ph+ cells by FISH with respect to CCA, that was more evident at low-to-medium values of Ph positivity. Seven specimens without Ph+ metaphases (17–50 cells analyzed) were shown to carry 2.5–8% interphase cells with BCR/ABL fusion. Similar percentages of BCR/ABL+ nuclei were recorded in 22 samples hybridized using the 300/200 kb and the 35.5/39 kb probe-sets (variation range: 0–5%, mean 2.3%). A very good correlation between the frequency of Ph+ interphase cells was observed when analyzing in parallel BM preparations after direct harvest and after 24-h culture. Underestimation of the percentage of BCR/ABL+ cells was noted to occur in 2/11 PB samples, compared to BM samples, the remaining nine cases showing superimposable results at either sites. We arrived at the following conclusions: (1) dual-color FISH enables an accurate detection and monitoring of the size of the Ph-positive clone in CML at diagnosis and after IFN-therapy; (2) FISH is more accurate than CCA, especially at low levels of Ph-positive cells; (3) testing of directly harvested BM samples is feasible and accurate, giving the opportunity to perform centralized FISH analysis in the context of multicentre trials; (4) the percentage of BCR/ABL+ PB cells usually, though not invariably, reflects the frequency of mutated cells in the BM.

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.

Flow cytometry measurement of GM-CSF receptors in acute leukemic blasts, and normal hemopoietic cells

A quantitative analysis of expression levels of GM-CSF receptors was performed by flow cytometry in different disease categories, ie AML (n = 72), ALL (n = 18), and MDS (n = 12), as well as 12 healthy volunteers, using three different unconjugated GM-CSF/R monoclonal antibodies (McAbs) (HGM-CSFR (CD116), M5D12, 4B5F5), and appropriate standards. By using the reference HGM-CSFR McAb, in healthy subjects we found detectable levels of GM-CSF/R on blood monocytes (mean MESF (molecules of equivalent soluble fluorochrome)/cell: 36.1 × 103), neutrophils (mean MESF/cell: 7.4 × 103), bone marrow (BM) myelo-monocytic precursors (MESF range for the myeloid component, ie promyelocytes, myelocytes, metamyelocytes: 11.7–40.5 × 103, and for the monocytic lineage: 25.7–69.2 × 103), and in two distinct subsets of BM CD34+ progenitor cells (GM-CSF/R dim: 2.5 × 103 MESF/cell, GM-CSF/R bright (10% of the total number of CD34 cells: 22.0 × 103 MESF/cell). In these subjects, there was no correlation between the expression levels of GM-CSF/R and CFU (CFU-GM, CFU-GEMM, BFU-E) colony production. Among the AML samples, M5D12 McAb was positive in 33%, 4B5F5 McAb in 90%, and HGM-CSF/R McAb in 78% of the cases examined (range of MESF/cell for the HGM-CSFR McAb: 0.9 × 103–106.7 × 103). The highest MESF values were seen in the M5 FAB subvariety (mean: 39.4 × 103), where all the patients tested (n = 20) showed a strong positivity for the HGM-CSFR McAb. On the contrary, all ALL samples were GM-CSF/R negative except in two patients, who displayed a dim GM-CSF/R positivity (My+ALL: 1.3 × 103 MESF/cell; pro-B ALL: 1.0 × 103 MESF/cell). In most (>70%) M1 FAB subtypes, GM-CSF/R+ blasts co-expressed CD34low, HLA-DRhigh, CD33, CD38 antigens, and had little or no capacity to form CFU-GM colonies. GM-CSF/R+ blasts from the M5 FAB category were also positive for CD14, CD11c, CD33 and CD87. Furthermore, the number of GM-CSF/R expressed by leukemic cells from five out of 72 (7%) AML patients was above the highest values seen in normal samples (>69.2 × 103 MESF/cell), allowing the possibility of using this marker for the monitoring of the minimal residual disease (MRD) in a subset of AML. Cell culture studies aimed at evaluating GM-CSF receptor modulation following AML blast exposure to rhGM-CSF showed two distinct patterns of response; in the first group (6/10 cases) rhGM-CSF down-modulated GM-CSF receptors, whereas in the second group (4/10 cases), rhGM-CSF treatment was associated with either an increase or no change in the number of GM-CSF/R. In conclusion, cellular GM-CSF/R expression was variable and ranged from undetectable (ALL and a minority of AML) to very high intensities in M5 AML, and were also documented in some M0 AML, thus suggesting the concept that GM-CSF/R detection may be of help in lineage assignment of undifferentiated forms. Since the number of GM-CSF/R on AML blasts may be modulated after GM-CSF treatment, it can be postulated that the clinical use of GM-CSF in this disease may be optimized by a dynamic analysis of the number and the affinity status of GM-CSF-R in blasts and normal hemopoietic cells.