An unusual case of untreated chronic lymphocytic leukemia patient presenting with Rhodococcus equi bacteriemia.
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Actinomycetales Infections↗
Biomedical subjects
G Castoldi
Publications and source records attributed to G Castoldi.
p53 exon 5 mutations in two cases of leukemic mantle cell lymphoma.
Although p53 mutations have been described frequently in high-grade B-cell non-Hodgkin's lymphoma (NHL), they have only been reported occasionally in low-grade NHL. We therefore describe clincobiologic and molecular genetic findings in two patients with p53 mutations and leukemic mantle cell lymphoma featuring an unusually aggressive course. Circulating malignant cells showed irregularity of nuclear outline with frequent deep clefts in both cases. Immunologic studies of neoplastic cells from peripheral blood samples and from cells obtained from an involved lymph node showed a mantle B-cell phenotype (CD5+, CD19+, CD22+, CD23- or weakly+ and bright expression for surface immunoglobulins). Malignant cells were shown to be hyperdiploid by cytofluorimetric study of DNA content and the presence of the t(11;14)(q13q32) was documented in one case. An altered electrophoretic mobility of p53 exon 5 was seen in both cases, with a missense mutation at codon 158 present in one case and a CAG to TAG mutation resulting in a 167-stop codon present in the second case. The percent of reactive cells with the 1801 monoclonal antibody detecting an epitope of the p53 was 37% in one case and 1% in the second case, supporting the notion that immunologic overexpression cannot be used for a selection criterion for the detection of p53 mutations. From these findings and from data available in the literature the conclusion can be drawn that p53 gene mutations at codons 158 and 167 may be associated with lymphoproliferative disorders and that low- or intermediate-grade NHL, including leukemic mantle cell lymphoma, may frequently carry this genetic change.
Aged↗
Cytogenetic and clinicobiological features of acute leukemia with stem cell phenotype: study of nine cases.
Morphologic, immunologic, cytogenetic, and clinical features were studied in 9 cases of acute undifferentiated leukemia (AUL). These patients were unclassifiable by FAB criteria, they were CD34+ and did not express myeloid- or lymphoid-associated antigens (CD13, CD33, CD14, CD15, CD61, CD19, CD10, CD22, CD7, CD2, CD5, CD3). Clonal abnormalities were seen in 8 of 9 cases. Del(5q) as the sole anomaly was observed in 3 cases; +13 was the primary change in 3 cases, and isolated trisomy 12 was found in 1 patient. A complex karyotype with trisomy 12q, in association with del 17p and trisomy 21q was detected in 1 case. One patient with 5q- relapsed with refractory anemia with excess of blasts; the presence of dysgranulopoiesis and a few blasts with possible monocytoid morphology in the remaining 2 patients point to a "myeloid nature" of these leukemias. Analysis of cytologic features in our 3 patients with +13, in combination with previously reported cases, suggests the occurrence of immature stem cell involvement with limited differentiation potential, possibly more along the myeloid than the lymphoid lineage. The significance of trisomy 12q in this subset of leukemia remains elusive; some clues of minimal differentiation towards the myeloid lineage in our cases are provided by positivity for the CD117 (c-kit) antigen and by relapse with acute myeloid leukemia without maturation (M1) in one patient. We conclude that, with presently available diagnostic techniques, AUL is a rare subset of leukemia, in which cytogenetic changes are confined to a few chromosomes, with prevalent involvement of 5q and of chromosomes 13 and 12. Chromosome findings may be of value in clinical practice, especially in those cases with "myeloid-oriented" karyotype.
Acute Disease↗
Interleukin-3 plus interleukin-6 may improve chromosomal analysis of multiple myeloma: cytologic and cytogenetic evidence in thirty-four patients.
To better define the role of interleukin-3 (IL-3) and IL-6 in the cytogenetic analysis of multiple myeloma (MM), we performed concomitant chromosome and cytologic studies in 34 patients. In each case, 10-30 x 10(6) bone marrow cells were incubated in two independent cultures consisting of conventional cytogenetic medium with and without IL-3 plus IL-6 added for 72 hours. 1-ml aliquots of each culture were aspirated at 24, 48, and 72 hours and exposed to colcemid for 6 hours. Cytospin preparations were then made and mitotic cells were counted and identified as plasma cells or as nonmalignant cells based on their reactivity with an appropriate anti kappa/lambda serum. Slides for conventional cytogenetic analysis were prepared at 72 hours. A greater than two-fold increase of mitotic plasma cells was observed in cytospin preparations from stimulated cultures versus unstimulated cultures in 15 of 34 cases, whereas a less than 2-fold increase, no variation or no mitosis was recorded in 19 cases. Comparison of the number of mitotic plasma cells in stimulated cultures at 24, 48, and 72 hours showed a decreased mitotic activity at 72 hours. Clonal abnormalities were detected by conventional cytogenetic analysis in 19 of 34 cases (55.8%). Recurrent clonal aberrations involved chromosome 13 (4 cases), chromosomes 1p, and 14q (3 cases); chromosomes 3p, 6q, 7q, and 9q (2 cases). We conclude that IL-3 + IL-6 may increase the number of dividing plasma cells in cytogenetic cultures and that a 2-day culture with these cytokines may facilitate the detection of chromosome abnormalities in MM.
Bone Marrow↗
Special cytological subtypes of acute myeloid leukaemias and myelodysplastic syndromes.
The recognition of distinct cytoimmunological subsets of pre-leukaemia and overt AML has been accomplished by morphological, immunological and ultrastructural studies. In many cases, a strong association has been documented between distinctive cytological features and specific chromosome changes. The primary genetic event underlying malignant transformation was also elucidated in a number of acute leukaemias and, as a matter of fact, assessment of these biological parameters has now an established role in the diagnostic work-up and in the monitoring of residual disease. On a more general basis, biological research in MDS is gradually clarifying the fundamental pathophysiological mechanisms of altered cell growth, and differentiation and therapeutic decision making in leukaemia is becoming increasingly dependent on the precise characterization of blast cells. Further refinement of the cytoimmunological classification of acute leukaemias and MDS is warranted in order to provide the physician with an updated framework of reference for the categorization of these heterogeneous haematological disorders and to improve the reproducibility of current morphological diagnosis among different centres (Castoldi et al, 1993).
Acute Disease↗
Richter's syndrome in a case of atypical chronic lymphocytic leukaemia with the t(11;14)(q13;q32): role for a p53 exon 7 gene mutation.
Clinicobiological, histological, cytogenetic and molecular genetic studies were performed in a case of atypical B-cell chronic lymphocytic leukaemia (B-CLL) with the t(11;14)(q13;q32) evolving into Richter's syndrome (RS) in order (a) to determine the clonal relationship between the cell of origin for B-CLL and RS, and (b) to analyse genetic events underlying the disease progression in this patient. After 4 years following diagnosis, a rapid deterioration of the clinical picture occurred, concomitant with the appearance of large lymphoid blasts in peripheral blood (PB), bone marrow (BM) and ascites samples. A diagnosis of RS was made and cytogenetic analysis revealed karyotype evolution with trisomy 7 and del(17p) in addition to t(11;14). Fluorescence in situ hybridization showed 78% lymphoid blast cells obtained from ascites sample to be trisomic using a chromosome-7-specific pericentromeric probe. Whereas no rearrangement of the c-myc proto-oncogene was detected at disease progression, direct sequencing of p53 gene exon 5-9 revealed an exon 7 missense point mutation. This abnormality was not present in the CLL phase. Immunological staining with the monoclonal antibody PAb-1801, detecting the p53 protein product, revealed a negative pattern in the CLL phase, whereas 24% positivity was documented in representative samples obtained at RS. It is concluded that RS was cytogenetically related with B-CLL in this patient, suggesting the occurrence of a bona fide transformation and that the mutation of p53 exon 7, in association with the development of 17p deletion, possibly played a role in the development of RS.
Aged↗
Classification of acute leukemias: reply from EGIL to Dr van Dongen.
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Acute Disease↗
Photomultiplier voltage setting: possible important source of variability in molecular equivalents of soluble fluorochrome (MESF) calculation?
We evaluated the effect of flow cytometer photomultiplier (PMT) voltage setting on molecular equivalents of soluble fluorochrome (MESF) calculation by using two different quantitative microbead calibrating kits as calibrating standards and a third one as an unknown testing sample of stable intensity of fluorescence. Based on the analysis of residual values derived from linear regression and on the determinations of the resolution index, we demonstrated that, on a FACScan instrument, the window of photomultiplier setting for fluorescein isothiocyanate (FITC) fluorescence determinations is 590-670 V. However, the best region was in the range of 610-650 V. Within the 590-670 PMT voltage region, the evaluation of the same testing sample at different PMT voltages, at 590 V, gave MESF percentage differences of 15% and 13% on the two lower unknown standards of intensity compared to the values obtained at the nearby 630 V setting. In the analyzed PMT region, logarithmic amplifier response was globally maintained, whereas evaluation of the same unknown testing sample in both linear (Lin) and logarithmic (Log) amplification demonstrated (at the higher intensities of fluorescence) Log-Lin minimal differences within the 610-650 PMT voltage range. According to our data, it can be stated that PMT voltages between 610 and 650 provide the best instrument performances and that PMT setting may be proposed as a source of variability in MESF calculation that deserves a careful evaluation in quality-control trials.
Calibration↗
A novel t(9;11)(p22;q23) with ALL-1 gene rearrangement associated with progression of a myeloproliferative disorder to acute myeloid leukemia.
We have analyzed genomic DNAs from a patient who developed acute myeloid leukemia 1 year after a myeloproliferative disorder was diagnosed. The development of the acute leukemia was associated with the acquisition of a t(9;11)(p22;q23) chromosome translocation. ALL-1 gene rearrangement, on chromosome 11, was present at the onset of the acute phase, but not during the chronic phase of the myeloproliferative disorder. The genomic rearrangement on chromosome 9 was within an unidentified region. By the use of polymerase chain reaction, we were able to determine that the chromosomal rearrangement was completely absent during the chronic phase of the myeloproliferative disorder, indicating that the ALL-1 gene rearrangement was causally related to the development of the acute phase. The rapid progression into the acute phase suggests that this case might be therapy related. This work provides a clear example of association of a molecular defect with the development of a specific clinical leukemic stage, and supports the indication that ALL-1 gene rearrangement is associated with poor clinical outcome in adult leukemias.
Adult↗
Modulation of cell kinetics and cell cycle status by treating CD34+ chronic myeloid leukaemia cells with p53 antisense phosphorothioate oligonucleotides.
Mutations of the p53 tumour suppressor gene occur in 20% of chronic myeloid leukaemia (CML) patients in blastic crisis, but it is still uncertain whether this inactivation plays a role in the pathogenesis of blastic transformation or in maintaining the leukaemic proliferation in CML, as it does in several solid tumours. We have previously shown that more than 50% of both normal and CML CD34+ cells express the p53 protein. However, haemopoietic cells at different phases of the cell cycle express p53 with different conformations, suggesting that the function of p53 may be closely regulated during the cell cycle. In order to elucidate the mechanism by which p53 suppresses cell proliferation, we evaluated the effects of inhibiting p53 expression on cell cycle and cell kinetics of chronic phase CML (n = 12) and normal (n = 7) bone marrow light-density cells and purified CD34+ progenitors by using an 18-mer modified antisense oligonucleotide which targets the region covering the six base pairs immediately before the first codon and the first four coding codons of p53. We found that the number of cells positive for the cell cycle-specific nuclear antigen Ki67 and for the BrdU monoclonal antibody (McAb) was significantly increased after p53 antisense olignucleotide treatment. At the same time, p53 protein expression was completely abrogated in both light-density and CD34+ cells. In addition, DNA analysis by flow cytometry demonstrated that the number of cells in quiescent phases of the cell cycle (G0-G1) was significantly decreased after exposure of light-density cells to p53 antisense oligomers, whereas the number of cells in S or G2-M phases was increased. Furthermore, the longer the incubation time the higher the increase in cell proliferation. Treatment of CML, cells with p53 antisense oligomers also resulted in significantly increased numbers of CFU-GM colonies. Our data suggest that p53 is a negative regulator of cell proliferation and its action is mediated through changes in cell cycle kinetics, mainly before the S phase. We can further speculate that the loss of p53 function, at the time of blastic crisis of CML, may play a role, in combination with other genetic changes (p210 BCR/ABL, Rb gene abnormality, others to be defined), in inducing disturbances in cell proliferation, differentiation, and apoptosis.
Adult↗
CD34+ leukemic cells assessed by different CD34 monoclonal antibodies.
CD34 monoclonal antibodies (McAbs) are widely used to identify and isolate hemopoietic progenitors and to classify acute and chronic leukemias. We assessed the reactivity of 17 CD34 McAbs from the 5th International Workshop on Leukocyte Differentiation Antigens with a variety of cells types: normal bone marrow hemopoietic progenitors, 10 AML, 6 ALL, 11 CML. The reactivity for these McAbs was compared with that of reference CD34 McAbs (Q-Bend 10 and 8G12). For each cell population the % of McAb binding cells, the peak channel and the mean fluorescence intensity (MFI) of the positive cells was evaluated. The peak channel, the MFI and the number of positive cells varied significantly from case to case, depending on the McAb and the type of leukemia. According to the spectrum of reactivity three groups of McAbs were defined; however, 7 McAbs do not belong to any of these subgroups. These groups were not entirely in accordance with McAb classification based on enzyme cleavage that identified three epitopes of the CD34 molecule. Some reagents were found to be more specific for AML, other for ALL, CML or normal CD34+ cells. Normal bone marrow light density cells showed a significantly higher percentage of positive cells for 43A1 and MD34.2 McAbs compared to that documented for the remaining McAbs. AML cells showed the most variable pattern of expression for the CD34 McAbs. In leukemic samples, MESF (molecular equivalents of soluble fluorochrome) values ranged from 18,200 to 322,000 and the number of binding sites per cells was 5,000-81,000.(ABSTRACT TRUNCATED AT 250 WORDS)
Antibodies, Monoclonal↗
CD34+/CD33+ blast cells: correlation with FAB subtypes.
The reproducibility of FAB morpho-cytochemical classification in a multicenter trial was recently assessed as 78.1%. The relevance of immunophenotyping in AML is debated because of the lack of a clear prognostic impact and the necessity of uniform diagnostic criteria and standardised methodologies. Several authors, studying the morpho-immunological characteristics of AML blasts by flow cytometry, suggested the necessity for the definition of clusters of cells with similar morphological patterns and intensity of expression. In this respect we reviewed blast CD34 and CD33 intensity of expression in 65 AMLs classified according to the FAB criteria. Four morphological groups could be identified: 1) low side scatter (SSC) and forward scatter (FSC); 2) intermediate SSC and FSC; 3) high SSC and FSC; 4) combination of pattern 2 plus 3. Five immunological patterns were defined: a) high CD34 expression with negative or weak CD33; b) high CD34 and CD33 expression; c) weak to negative CD34 with high CD33 expression; d) coexistence of 2 different subpopulations; e) negative to weak CD34 and CD33 expression. Based on this morpho-immunological analysis we were able to subdivide AML patients into 5 homogeneous subgroups and a comparison with FAB classification showed a concordance of 73.9%. Regarding CD34 and CD33 intensity of expression, a correlation with prognosis was demonstrated among all M1-M2 patients and in the M2 and M4 subgroups. In conclusion even if immunophenotype cannot substitute the FAB approach to AML, we feel that a flow cytometry morpho-immunological analysis could be helpful in achieving a greater understanding and agreement between different Institutions and assist in the definition of more precise prognostic subgroups.
Antigens, CD↗
Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL).
Criteria for the immunological classification of acute leukemias are proposed by a recently established European group designated EGIL. The main aims of EGIL are to establish guidelines for the characterization of acute leukemias based on marker expression and provide a uniform basis for the diagnosis of the various types of these hemopoietic malignancies which should be helpful for future multinational clinical and laboratory investigations. Within the two major types (B and T cell lineage) of acute lymphoblastic leukemia (ALL), several groups are delineated according to the degree of cell differentiation. Within the acute myeloid leukemias (AML), only three subtypes as defined by the FAB classification: M0-AML, M6-AML and M7-AML, can be unequivocally defined by immunological markers; prospective studies are undertaken to see whether characteristic immunological profiles are associated with particular AML subtypes defined by specific cytogenetic abnormalities. Criteria for the definition of biphenotypic acute leukemia (BAL) are devised and a scoring system is outlined aimed to distinguish BAL from those acute leukemias with expression of a marker from another lineage. In addition, an uncommon subset of acute leukemias with no evidence of lymphoid or myeloid differentiation is recognized and the useful panel of markers to investigate and establish the cell nature of the acute leukemias is outlined. EGIL will focus in the future on testing the reproducibility of the proposed guidelines, particularly those for BAL, assessing their clinical value within a framework of multicentric trials and setting up uniform methodological criteria.
Acute Disease↗
Effects of adenosine-receptor agonists on renin release in anaesthetized rats.
OBJECTIVE: To investigate the effects of the interaction between adenosine receptors and renal nerves on renin release. MATERIALS AND METHODS: The effects on renin secretion of A1 (2-chloro-N6-cyclopentiladenosine) and A2 (2-hexynil-5'-N-ethyl-carboxamido-adenosine) adenosine-receptor agonists were studied in two groups of anaesthetized rats, each with one kidney surgically denervated. Arterial blood pressure and the renal blood flow of innervated and denervated kidneys were continuously recorded. Cannulae were inserted into both renal veins through femoral veins. After 1h of rest, A1 and A2 agonists were intravenously infused for 30 min in the two groups of rats. Plasma renin activity was measured by radioimmunoassay in blood samples drawn simultaneously from both renal veins and the femoral artery before and after the drug infusion. RESULTS: Infusions of A1 and A2 agonists produced comparable hypotensive effects. During A1 agonist administration, the heart rate decreased significantly, but it did not change after A2 agonist treatment. Renal blood flow was reduced by administration of the A1 agonist in both kidneys, while A2 agonist administration significantly reduced the renal blood flow of the innervated kidney only. The veno-arterial difference in plasma renin activity decreased after the A1 agonist infusion in both kidneys, but after the A2 agonist infusion it increased significantly in the innervated kidney only. CONCLUSIONS: Renal nerves do not influence the inhibition of renin release mediated by A1 adenosine receptors. In vivo, A2-receptor agonist administration can stimulate renin release only in the presence of intact renal nerves.
Adenosine↗
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.
Adult↗
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.
Aged↗
Prognostic value of immunophenotypic characteristics of blast cells in acute myeloid leukemia.
In order to elucidate the prognostic role of cytofluorimetry analysis of leukemic cells in AML, the immunophenotypic characteristics of blast cells obtained from 66 AML patients belonging to M0-M2 and M4-M5 FAB subtypes have been investigated by flow cytometry using a large panel of monoclonal antibodies (McAbs) utilized in single, double, and triple fluorescence experiments. On a univariate analysis, four different immunophenotypic blast cell characteristics were found to be associated with a poor prognosis: expression of CD34 "bright" (ratio > 10 between fluorescence emission of positive cells and that of negative (isotypic) control-P/N ratio: mean MESF value: 265,000) in > 15% blast cells, co-expression of CD34 and CD33 in > 60% blast cells, expression of CD14 in > 30% leukemic cells, the MDR+ ("multiple drug resistant") phenotype. In contrast, the duration of remission, and overall survival of AML patients showing a "dim" CD34 expression (P/N ratio: 3-10: mean MESF value: 49,000) was similar to that of CD34- AML patients, irrespective of the percentage of positivity for CD34, which was, however, a predictive factor of survival in patients with higher CD34 fluorescence intensities in their blastic population. No correlation between FAB subtypes, prognosis and immunophenotype was found. The multivariate regression analysis showed that, besides age, only the combined expression of CD34 and CD33 had independent prognostic meaning. Indeed, in each FAB subtypes the CD34+/CD33+ phenotype was associated with a shorter survival and a lower mitotic rate. These data may contribute to the understanding of the discrepancies so far observed in the literature regarding the prognostic role played by the CD34 expression on leukemic AML blasts.
Antigens, CD↗
Reduced expression of macrophage-associated antigens on alveolar mononuclear phagocytes from acquired immunodeficiency syndrome.
In this study we evaluated the phenotype of alveolar mononuclear phagocytes recovered from the bronchoalveolar lavage fluid of 24 patients with human immunodeficiency virus infection (AIDS-related complex 8 patients. AIDS 16 patients) and 8 healthy individuals by using a panel of monoclonal antibodies known to react with tissue macrophages, in combination with a flow cytometer. The results showed that 90% of patients with AIDS present a marked reduction in the expression of several antigenic determinants (in decreasing order: CD68, CD36, CR1, CD11c, HLA-DR). The levels of antigen expression by flow cytometry seem to decline with disease progression, showing the most dramatic perturbations in patients with full-blown AIDS associated with pulmonary infections (especially Pneumocystis carinii pneumonia) and lower peripheral CD4 lymphocyte counts. In contrast, patients with AIDS-related complex or AIDS without histological or cultural evidence of pulmonary involvement showed, respectively, only minimal or medium antigenic decreases. However, only a minor proportion (16%, 20%, 20%, 25%, and 25% respectively) of human immunodeficiency virus infected patients (mostly with AIDS) had a significant reduction of the levels of CD4, CD14, CD45R, CD11b, and CD16 antigens in the alveolar macrophages. Since macrophages play a central role in the pathogenesis of AIDS, it may be postulated that the loss of various phenotypic markers on alveolar mononuclear phagocytes (some of them known for their important immunoregulatory actions) could have an important part in the pathogenesis of human immunodeficiency virus induced immunosuppression, and thereby condition the abnormal susceptibility to pulmonary diseases typical of human immunodeficiency virus-infected patients.
Acquired Immunodeficiency Syndrome↗