Heterogeneity of lineage involvement by trisomy 8 in myelodysplastic syndrome. A multiparameter analysis combining conventional cytogenetics, DNA in situ hybridization, and bone marrow culture studies.

To better understand the role of trisomy 8 in myelodysplastic syndrome (MDS), we performed a multiparameter analysis combining conventional chromosome studies (CCS), fluorescence in situ hybridization (FISH), and bone marrow (BM) culture studies in two patients with MDS evolving into acute myeloid leukemia (AML). A mosaicism of a cytogenetically normal clone and a clone with trisomy 8 was detected in both patients throughout the course of the disease, a finding confirmed by FISH on BM cells. The relative size of the trisomic clone increased from 52% to 71% (p < 0.0001) and from 53% to 69% (p = 0.001) of all BM cells at the time of the leukemic switch in patients 1 and 2, respectively. Combined FISH and immunophenotyping of BM cells showed involvement of the granulomonocytic lineage in patient 1 and involvement of erythroid cells as well as of the granulomonocytic lineage in patient 2. Only disomic lymphocytes were detected in both patients. FISH on single hemopoietic colonies grown in semisolid media detected trisomic CFU-GM and disomic BFU-E in patient 1, whereas a proportion of CFU-GM and BFU-E deriving from the trisomic clone was detected in patient 2. However, the percent of trisomic colonies was lower than the percent of involved granulomonocyte precursors and involved erythroblasts, as detected by combined FISH and immunophenotyping on fresh BM samples. We have thus shown heterogeneity of lineage involvement by trisomy 8 in MDS undergoing transformation into AML. Although preferential growth of disomic clones may occur in vitro, the finding of an increased size of the trisomic clone at the time of leukemic switch suggests that these cells had proliferative advantage in vivo over cells without trisomy 8.

Involvement of erythrocytic and granulomonocytic lineages by trisomy 11 in two cases of acute myelomonocytic leukemia with trilineage myelodysplasia. An interphase cytogenetic study.

To study the cytologic profile and lineage involvement in acute myeloid leukemia (AML) with trisomy 11, cytologic, cytogenetic, and interphase cytogenetic studies were performed at presentation in two cases of acute myelomonocytic leukemia (AML-M4). Patient 1 had +11 as the sole chromosome aberration in 16/20 karyotypes whereas two related clones with +11 in all abnormal metaphases (14/18) were detected in patient 2. A proportion of interphase cells with three signals, comparable to the proportion of abnormal metaphases, was detected by fluorescent in situ hybridization (FISH) in these patients. Morphologic aberrations of the nonblast cell population affecting multiple cell lineages, along with a circulating minor megakaryoblastic component, were observed at diagnosis in both patients. By separation of bone marrow cells over a density gradient of Percoll two cell fractions were obtained, the former containing more than 80% erythroid precursors (collected at a density of 1065-1075 mg/ml), the latter containing more than 78% blast cells plus granulomonocytic precursors (collected at a density of 1060-1055 mg/ml). FISH documented the presence of a majority of interphase nuclei with three signals in the erythroblast-enriched cell fraction and in the blast-enriched cell fraction. It is concluded that cytologic features, as well as interphase cytogenetic findings on enriched cell fractions, suggest the occurrence of multipotent stem cell involvement in AML-M4 with +11.

Trisomy 12 in chronic lymphocytic leukemia and hairy cell leukemia: a cytogenetic and interphase cytogenetic study.

Fluorescent in situ hybridization (FISH) with a chromosome 12-specific pericentromeric probe was performed in 42 patients with B-cell chronic lymphocytic leukemia (CLL) and in 10 patients with hairy cell leukemia (HCL). In all cases, a normal karyotype in more than 10 metaphase cells was obtained by conventional chromosome study. FISH documented that 6/42 patients with CLL in fact had trisomy 12 in 15-49% interphase cells. Sequential FISH studies were performed in 2 cases, showing an increase of percentage of trisomic cells over a 2-month to 4-year period. Two out of 10 patients with HCL, one of whom had morphologic features consistent with a diagnosis of HCL variant, showed 5.5 and 10% interphase nuclei with three fluorescent signals, a finding suggestive of the presence of trisomy 12. Combined immunophenotyping and FISH staining in these patients with HCL documented that trisomic cells were CD11c-positive, CD13-negative, and CD2-negative. We conclude that FISH is a sensitive technique allowing for the detection of trisomy 12 in a fraction of cytogenetically normal patients affected with CLL and HCL.

Minor myeloid component in Ph chromosome-positive acute lymphoblastic leukaemia: correlation with cytogenetic pattern and implication for poor response to therapy.

Morphological, immunological and cytogenetic features were studied in 27 adults presenting with Ph chromosome-positive acute lymphoblastic leukaemia (ALL), in correlation with clinical outcome. Twenty patients (group 1) were diagnosed as having typical ALL according to the FAB criteria supported by immunological findings. Less than 1% blast cells with azurophilic granules were detected in all cases. Myeloid cytochemistry, i.e. peroxidase and Sudan black-B stain, was negative in all cases. A minor phenotype deviation consisting of the expression of the CD13 myeloid-associated marker was detected in two patients. In seven patients (group 2) a diagnosis of ALL with a minor myeloid component was made because of the presence of a majority of lymphoid blasts and of 5-15% blast cells with morphological cytochemical and immunological features of the myeloid lineage. Abnormal metaphases were found in 6/20 (30%) patients in group 1, compared with 7/7 (100%) patients in group 2. All patients were treated by antilymphoid regimens; however, complete remission was achieved in 17/20 (85%) patients in group 1 versus 1/7 (14.3%) patients in group 2. Median survival was 16 months, range < 1-120+ in group 1 and 9 months, range < 1-15 in group 2. It is concluded that morphological, immunological and cytogenetic studies allow for the recognition of two cytological subsets of Ph+ ALL. The presence of a minor myeloid component in otherwise typical Ph chromosome-positive ALL may be associated with a distinct cytogenetic pattern and poor responses to antilymphoid therapy.

Lineage switch and multilineage involvement in two cases of pH chromosome-positive acute leukemia: evidence for a stem cell disease.

Philadelphia chromosome-positive acute leukemias (Ph+ AL) show variable cytologic features, possibly reflecting heterogeneous stem cell involvement. Morphologic, immunologic and cytogenetic studies were performed in two cases of Ph+ acute lymphoblastic leukemia (ALL) in order to better delineate the clinicobiological features of this cytogenetic subset of AL. Sequential cytoimmunologic studies in patient 1 documented a lineage switch from pro-B ALL with a minor myeloid component at diagnosis to minimally differentiated acute myeloid leukemia (AML) at relapse. In this patient the major breakpoint cluster region (M-bcr) was in a rearranged configuration and all metaphase cells showed t(9;22)(q34;q11), both at diagnosis and at relapse. In patient 2 a diagnosis of Ph+ early T-cell ALL with minor myeloid component was made. In this patient the M-bcr was in a germline configuration. Cytogenetic studies documented the presence of the Ph chromosome in all metaphases from a lymphoid cell population obtained by fine-needle aspiration of an enlarged lymph node, and from a bone marrow cell fraction enriched in granulocyte precursors. This finding suggests multilineage involvement in this patient. Lineage switch and multilineage involvement in two patients suggest that a pluripotent stem cell may be affected rather frequently in patients with Ph+ AL. These findings show that biologically Ph+ AL may resemble chronic myelogenous leukemia blast crisis, since it may originate from an undifferentiated stem cell carrying the t(9;22) translocation.

Total loss of p53 DNA sequences in acute myeloid leukemia.

Mutations of the p53 tumour suppressor gene on chromosome 17p are a common genetic change in the malignant progression of many cancers. Here we report a case of a 71-year-old man with haematological, cytofluorimetric and cytochemical findings consistent with a 'de novo' M2 acute myeloid leukaemia (AML). A complex karyotype including a whole chromosome 17 and a t(17;?) (p11;?) was present in 8 of 10 metaphases of bone marrow cells. Southern blot analysis of the bone marrow DNA showed a specific loss of p53 gene in the AML cells. As far as we know, this is the first report of a deletion of both p53 alleles in leukaemia. The effect of the loss of p53 on the course of AML is discussed.

Chromosome aberrations in CD34-positive acute myeloid leukemia. Correlation with clinicopathologic features.

Morphologic, immunologic, and cytogenetic features were studied in 30 newly diagnosed patients with CD34-positive (CD34+) de novo acute myeloid leukemia (AML) in comparison with 30 patients with CD34-negative (CD34-) AML. Karyotype at diagnosis was abnormal in 25/30 CD34+ AML patients, of which nine had major karyotype aberrations (MAKA). Clonal chromosome changes were detected in 9/30 patients with CD34- AML. The most frequent chromosome aberration in CD34+ patients was -5/5q-, an aberration showing a strong association with the M2 FAB subtype of AML. Other recurring chromosome changes involved chromosome 16q (four cases) and chromosome 17p (three cases). Total or partial monosomy 7q was detected in three cases. Among CD34- AML, two patients had the classical t(15;17) and two had structural aberrations of 6q. Among patients with CD34+ AML, nine had MAKA in association with trilineage myelodysplasia (TMDS). TMDS was infrequent in CD34+ AML without MAKA and in CD34- AML. Complete remission (CR) was achieved in 8/30 CD34+ AML (26%), as compared with 22/30 CD34- AML (73%), and median survival was 2 months in the former group and 8 months in the latter. No patient with CD34+ AML and MAKA achieved CR, whereas 8/21 CD34+ AML without complex chromosome changes or with normal karyotype achieved CR. In conclusion, a distinct cytogenetic profile may be associated with CD34+ AML. Cytogenetic findings in CD34+ AML may be clinically relevant in that they may disclose a subset of patients with MAKA with a low CR rate.

Clinical review on features and cytogenetic patterns in adult acute myeloid leukemia with lymphoid markers.

Cytogenetic patterns in correlation with cytologic, biomolecular and clinical findings were studied in 45 adult patients with AML expressing at least one of the following lymphoid associated markers (LM): CD2, CD7, CD10, CD19, CD22, TdT. Four cytogenetic groups were recognized: group I, including 8 patients with 11q23 rearrangements; group II including 5 patients with the Ph chromosome; group III, with 19 patients and aberrations of the "myeloid type" including 4 cases with aberrations of chromosome 13, 3 cases with 1q and 7q anomalies, 2 cases with trisomy 11q; group IV, including 13 patients with normal karyotype. Patients showing extensive lineage infidelity were encountered more frequently in cytogenetic groups I and II than in groups III and IV. Two of 4 cases with aberrations of chromosome 13 showed two or more lymphoid features either at immunophenotyping or at biomolecular analysis of the configuration of lg and TCR genes. Patients with 11q23 rearrangements and with the Ph chromosome were generally young, presented with high WBC count and had low complete remission rate. Survival in Ph chromosome positive patients was uniformly short. We conclude that, although there is no cytogenetic anomaly specific for AML with LM, chromosome findings may be clinically relevant in AML with LM. A morphologic, immunologic and cytogenetic classification of AML with LM may constitute a working basis for future studies aimed at a better definition of clinicopathological features and optimal treatment strategy for these leukemias.

Morphologic, immunologic and cytogenetic studies in acute myeloid leukemia following occupational exposure to pesticides and organic solvents.

In order to analyze the correlation between environmental exposure and the clinicopathological picture in acute myeloid leukemia (AML), cytogenetic, cyto-immunologic and clinical studies were performed in 70 newly diagnosed AML patients, 30 of which were anamnestically exposed to pesticides (21 cases) or to organic solvents (9 cases). Clonal chromosome aberrations, with involvement of chromosome 5 and/or 7 were more frequently encountered among exposed patients. While the classical t(15;17), t(8;21) and t(9;11) were detected more frequently among non-exposed patients, other recurring chromosome changes in the exposed group were: rearrangements leading to total or partial monosomy 17p (5 cases), structural aberrations involving the band 16q22 (4 cases), trisomy 11q (2 cases), breaks involving bands 6p23, 7p14, 11q13 (2 cases each). Cytologically, trilineage myelodysplasia was observed in 21 exposed patients, whereas morphologic aberrations of the non-blast cell population were confined to a minority of cells in most patients non-exposed. Immunologic studies revealed positivity for the CD34 stem cell marker in 80% exposed patients vs 22% in the non-exposed group. Conventional chemotherapy achieved complete remission in 3/21 patients exposed and in 16/32 patients non-exposed. Median survival was 2 months in the former group and 9 months in the latter group. These findings show that AML following occupational exposure to pesticides and organic solvents may represent a distinct cytogenetic and clinicopathological entity.

Distinct cytogenetic and clinicopathologic features in acute myeloid leukemia after occupational exposure to pesticides and organic solvents.

BACKGROUND: To study the correlation of environmental exposure to potentially mutagenic agents and the clinicopathologic picture in acute myeloid leukemia (AML), clinical features, morphologic characteristics, immunophenotype, and cytogenetics were studied in 59 patients with newly diagnosed AML. METHODS: Based on interviews on occupational hazards and hobbies showing prolonged contact with pesticides (18 patients) and organic solvents (7 patients), 25 patients were categorized as "exposed". Thirty-four patients were categorized as "unexposed,", based on anamnestic findings. RESULTS: Light microscopic studies showed myelodysplasia involving multiple cell lineages in all assessable patients with professional exposure to pesticides and organic solvents, whereas morphologic aberrations of the non-blast cell population were confined to a minority of cells in unexposed patients. These findings were confirmed by electron microscopic studies in 31 patients. Immunologic analysis showed the presence of a minor megakaryoblastic component in six exposed patients and showed positive findings for the CD34 stem cell marker in 85% of exposed patients, a figure significantly higher as compared with that for unexposed subjects. Cytogenetic studies confirmed the frequent occurrence of 5q and/or 7q aberrations in patients occupationally exposed (10 of 25 cases). Other recurring chromosome aberrations in the exposed group were 17p-, trisomy 11q, and translocation of 16q, 6p, 7p, and 11p, whereas the classic AML-specific translocations (i.e., t[15;17]; t[8;21]) were detected only in unexposed subjects. Conventional chemotherapy achieved complete remission in 1 of 19 exposed patients, as opposed to 14 of 29 unexposed patients, with a median survival of 2 months in the former group and 8 months in the latter. CONCLUSIONS: Taken together, these findings document that AML in patients professionally exposed to toxic substances may represent a distinct cytogenetic and clinicopathologic entity. The clinicobiologic characteristics in these exposed patients are similar to the features of AML arising in patients with prior chemotherapy for another tumor, thus suggesting that similar transformation pathways may underlie leukemogenesis induced by cytotoxic drugs and by environmental exposure to some pesticides or organic solvents.

Non-radioactive in situ hybridization for the detection and monitoring of trisomy 12 in B-cell chronic lymphocytic leukaemia.

Non-radioactive in situ hybridization (NISH) with a chromosome 12-specific alpha satellite probe was performed on 20 patients with chronic lymphocytic leukaemia (CLL) with normal karyotype (15 cases) or with inadequate mitotic yield (5 cases) from mitogen-stimulated cultures. All patients had over 70% lymphocytes coexpressing the CD5/CD23 antigens. While less than 1% interphase nuclei showed three fluorescent spots in 16/20 patients, evidence of trisomy 12 in 15-25% interphase cells was detected in four patients. According to the FAB classification the diagnosis in these patients was typical B-CLL, stage III (Rai's staging system) in one case, CLL/PLL, stage II and III in two cases, PLL, stage III in one case. In order to confirm these results, NISH was repeated after 1 month in one patient and after 2 years in three patients. All patients had been treated with chemotherapy in the period between the two NISH experiments. In all cases a 1.8-3-fold increase of percentage of trisomic interphase cells was detected. These findings suggest that in B-CLL clones with trisomy 12 may have proliferative advantage over clonal B-lymphocyte without +12 and, possibly, that they may be more resistant to chemotherapy. We conclude that NISH is a sensitive technique allowing for the detection and monitoring of trisomy 12 in a fraction of B-CLL patients with normal karyotype or with no analysable mitoses despite employment of polyclonal B-cell mitogens.

Lymphoblastic lymphoma with primary splenic involvement and the classic 14;18 translocation.

Clinicopathologic features of a case of lymphoblastic lymphoma (LyL) with the classic 14;18 translocation are described in this article. The patient had prominent splenomegaly with numerous splenic nodules, exhibiting a homogeneous blast cell infiltrate and occasional cells with cleft nuclei, a picture suggestive of high-grade non-Hodgkin lymphoma (NHL) possibly lymphoblastic. Early B-cell features were detected immunologically, thus confirming the diagnosis of LyL. The presence of primary splenic involvement and of the t(14;18)(q32;q21) are unusual in this histologic subset of B-cell NHL, these cytogenetic and clinicopathologic characteristics being typically associated with low- or intermediate-grade NHL of follicle center origin. These features, along with the presence of some centrocytelike cells in the biopsy sections, suggest that an unusual pattern of histologic evolution from a follicle center cell NHL may have occurred in this case of LyL.

Chromosome studies of enriched blast cell fractions in myelodysplastic syndromes terminating in acute myeloid leukemia.

For a better understanding of the karyotype evolution of different marrow cell populations in the course of MDS, 6 patients who eventually developed overt leukemia, belonging to a series of 46 MDS referred to our Institution, were studied ad diagnosis and at leukemic progression. In each case the blast cells were separated from the maturing precursors of the erythroid and granulocytic lineage by centrifugation on a Percoll density gradient. Parallel chromosome investigations were performed in each cell fraction. Cytogenetic analysis performed at presentation did not reveal distinctive karyotype features in metaphases arising in the blast enriched cell fraction, as compared with those obtained from the fraction containing erythroblasts and promyelocytes--myelocytes. These findings suggest that in the initial phase of MDS blast cells may lack distinctive cytogenetic features and may thus represent part of a clonal preleukemic proliferation. At the time of leukemia onset, clonal aberrations [trisomy 21 and del(11)(q23)] showing a restricted pattern of distribution within the blast cell enriched fractions were detected in two patients, whereas one patient showed an increase in size of the abnormal clone carrying monosomy 7, an aberration detected in metaphases obtained from both cell fractions. Thus, some evolutive steps in the natural history of these disorders can be heralded by the acquisition of chromosome aberrations more readily detectable in blast enriched cell fractions. In some cases, partial loss of differentiative capability by the abnormal clone may account for the detection, at leukemia onset, of chromosome aberrations involving both the blast cell fraction and the erythroblast-promyelocyte enriched cell fraction.

Immunophenotypic, cytogenetic and molecular investigations in two cases of CALLA positive acute myeloid leukemia.

Clinicopathological and cytogenetic features of two patients with acute myelogenous leukemia (AML) whose blast cells coexpressed myeloid-associated antigens and CALLA are described. Leukemia cells revealed myelomonocytic (FAB-M4) and monocytic (FAB-M5) features, while the nonblast cell population exhibited trilineage myelodysplasia in both cases, a finding suggestive of multiple-cell-lineage involvement. Cytogenetically, a deletion of the long arm of chromosome 6 was found in one patient, and normal metaphases were detected in the other. Molecular studies disclosed a rearrangement of the IgH locus in one patient. Clinically, these patients were unresponsive to antimyeloid regimens including Daunorubicin and Cytarabine, two agents normally also effective on lymphoblastic leukemias, possibly indicating the need for alternative protocols for the treatment of CALLA positive AML.