Gianluigi Scapoli

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.

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, immuno‐phenotype, and cytogenetics were studied in 59 patients with newly diagnosed AML.

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.

Acute myelomonocytic leukemia terminating in histiocytic medullary reticulosis.Cytochemical, cytogenetic and electron microscopic studies

A case of acute myelomonocytic leukemia terminating in histiocytic medullary reticulosis is reported. The evolution of a single cellular clone presenting with progressive change of the morphological features of the leukemic cells towards more anaplastic elements endowed with prominent phagocytic properties is suggested on the basis of both cytochemical and chromosomal data. The histiocytic nature of the malignant proliferating cells and platelet phagocytosis has been confirmed by electron microscopic investigation. The main pathogenetic explanations of the evolutionary patterns of the disease are discussed with relation to: a) involvement of a common stem cell giving rise to different proliferative patterns of cells in a multiphasic sequence; b) release of dysplastic platelets and defective erythrocytes with massive sequestration by histioid phagocytic cells; and c) coexistence of two different disorders.

Flow cytochemical analysis of peripheral lymphocytes in chronic B-lymphocytic leukemia. Prognostic role of the blast count determined by the H∗1 system and its correlation with morphologic features

Peripheral blood samples from 148 previously untreated patients with chronic B-lymphocytic leukemia (B-CLL) were analyzed with the Technicon H*1 flow cytometer. The absolute number and the percentage values of both LUCs (large unstained cells) and blasts were correlated with survival, as well as with well-known prognostic factors including morphological subtypes of lymphoid cells. Results showed that patients at the most advanced clinical stages (Rai: III and IV; Binet: C) had the highest percentage and count of both LUCs and blasts. Furthermore, the proportion of LUC positively correlated with the following prognostic factors: peripheral lymphocytosis (greater than 50 x 10(9)/l); marked splenomegaly (greater than 10 cm UCM); % of circulating prolymphocytes, % immunoblasts, and % LGL. Our data analysis further revealed that chemotherapy produced a greater reduction of both the LUCs and of the blast count than of that of small lymphocytes. An increase in LUC count was found to coincide with deterioration of clinical status (progressive changes in the clinical stages, occurrence of prolymphocytoid transformation). A rapid increase in blast count was found to occur in concomitance with the development of Richters syndrome, and correlated positively with the number of peripheral immunoblasts determined by light microscopy. Moreover, a blast percentage higher than 7% had the strongest predictive relation to survival rate when compared with other hematological parameters (lymphocytosis greater than 50 x 10(9)/l, % of LUCs greater than 12%, LUC to lymphocyte ratio greater than 16%, LUCs count greater than 2.2 x 10(9)/l). In the light of these findings, it may be suggested that the presence both of larger proportions of LUCs and of blasts measured with the flow cytometry may be considered unfavorable prognostic factors in B-CLL. However, based on morphological and multivariate statistical analyses, the blast count proved to be the most important prognostic parameter determined by the H*1 system in B-CLL.

Chromosomal abnormalities in angio-immunoblastic lymphadenopathy

SummaryChromosomal studies have been performed in 2 patients with angio-immunoblastic lymphadenopathy. In both the cases the presence of abnormal cell lines characterized by marker chromosomes has been detected. Application of banding techniques allowed to detect the structural composition of the marker chromosome in 1 of the cases and to show a clonal evolutive pattern of the rearranged chromosomal set. In the same patient a consistent Y loss observed in the major fraction of the investigated metaphases did not appear to be related to any defined rearrangement of the karyotype. Longitudinal chromosomal studies are stressed in order to better correlate the cytogenetic abnormalities and the immunoreactive picture of the lymph nodes along the course of the disease.

Coexistence of Factor V G1691A and Factor II G20210A Gene Mutations in a Thrombotic Family Is Associated with Recurrence and Early Onset of Venous Thrombosis

Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject – the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations – showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus’ uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus’ two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.

Multiple chromosomal associations and paracentromeric region instability in a case of acute leukemia

SummaryA case of acute myelomonocytic leukemia is described, which was characterized cytogenetically by the presence of centromeric elongations, somatic crossovers, selective endoreduplication figures, and multiple chromosomal clusters. The demonstration of these phenomena by selective staining techniques for the chromosome bands (Q, C, G and S) and the nucleolar areas (acridine-orange, amido black B 10) raises some biological aspects involved in the proliferation of leukemic cells, such as nucleolar persistence during the metaphase and the non-separation of chromatids in the clusters during the anaphase. These structural abnormalities may represent the background for the explanation of the appearance of subclones in neoplastic disorders.

Nuclear projections in tumour cells and large chromosome markers

The application of banding techniques on cytological smears from pleural effusion in a case of histiocytic sarcoma has provided direct evidence for correspondence between nuclear projections in tumor cells and extra large chromosome markers observed in the neoplastic karyotype obtained by direct preparations.

Association of Multiple Haematological Disorders (Acute Myeloblastic Leukaemia, Paraproteinaemia, and Thalassaemia) in a 46, XX/46, XXqi Female

In a thalassaemic female carrying a 46, XX/46, XXqi mosaic, the occurrence of acute myeloblastic leukaemia was observed. Karyotype analysis of several bone marrow aspirates disclosed, besides both nor