Maria Christina Paixão Maioli

Cytogenetic and immunophenotypic evidence of independent clonal origins of concomitant chronic lymphocytic leukaemia and acute myeloid leukaemia

To the Editor: Although chronic B-cell leukaemia (B-CLL) is the most common leukaemia in the Western world, the association of CLL and acute leukaemia (AL) is a rare event. The presentation of acute myeloid leukaemia (AML) concomitantly with CLL is unusual and, so far, only 16 cases have been described (1). In the most of these cases the characterization of the diseases has been made by morphology and immunophenotyping studies (1–4), and only in two cases was a cytogenetic study carried out (5, 6). We report a case with the simultaneous occurrence of CLL and AML without previous exposure to a cytotoxic agent or irradiation. Immunophenotyping and cytogenetic studies were performed, and the clonal origins of the diseases are discussed. A 70-yr-old Portuguese woman was hospitalized with a one-month history of daily fever (38–39 uC), gum bleeding, an episode of epistaxis and weight loss. Physical examination was significant for cervical and inguinal lymphadenopathy, gum hypertrophy, and an enlarged liver and spleen (6 and 3 cm, respectively). The patient also had high arterial blood pressure and congestive heart failure. The leukocyte count was 61,600 cells/mm with 31% blast cells and 59% lymphocytes. Five months before this admission her haemogram had been normal. A bone marrow (BM) aspirate revealed approximately 27% blast cells and 49% apparently mature lymphocytes. Cytochemical analysis revealed the blastic cells to be positive Sudan black. The great majority of cells were negative to PAS, and few cells were weakly positive; a-naphtyl butyrate was also negative. LDH measured 5261 UI. Immunophenotyping studies showed the presence of two cell populations with different light-scatter in BM. Large cells which corresponded to blast cells had an immature myeloid immunophenotype, (HLA-Dr, CD13, CD33, CD34), and small cells, corresponding to a mature B cell population, displayed a typical B-cell phenotype (CD19, CD5, HLA-DR, weak SMIg and K). ANLL-M2 was diagnosed. Hydroxyurea 1.5 g/d was started. The number of blast cells was reduced in subsequent days, although the total leukocyte count had increased due to an increase in the absolute number of lymphocytes (93,000 cells/mm in 5 d with 93% lymphocytes and 5% blasts). The patient died on the tenth day of hospitalization due to complications of both metabolic functions and infection. A pathologic leg fracture was also report-ed. The karyotype of bone marrow cells was obtained after cultures in RPMI 1640 with 20% foetal calf serum (Gibco) and pokeweed mitogen at 37 uC for 72 h. Cell cultures were pulsed with colcemid (0.06 mg/ml) in the last hour of incubation. Cells were subsequently harvested by standard procedures (hypotonic shock with 0.075 M KCl) and fixed in methanol–acetic acid (3:1). GTG banding was per-formed as described by Seabright (7), and chromosomes were identified and arranged according to the International System for Cytogenetic Nomenclature (8). The cytogenetic analysis showed the presence of two abnormal clones: 47,XX,+12 [10]/46,XX,del(5)(q31),t(8;13) (q22;q21) [4]/46,XX [6]. Although other cases of CLL associated with AML have been reported (1–6), the present study showed some peculiarities. There were no data suggesting the presence of AML after the diagnosis of CLL, since the patient had received no prior chemotherapy or radiotherapy and the haemogram obtained 5 months before had been normal. The cytogenetic study showed two abnormal clones, and as yet only two cases reporting concomitant CLL and AML included cytogenetic studies (5, 6). The patient described by Lima et al. (5) had chromosome aberrations commonly associated with CLL and AML in the same metaphases, suggesting that both diseases might be derived from a single cell clone. The cytogenetic analysis for the case reported by Mateu Eur J Haematol 2001: 66: 281–283 Printed in UK. All rights reserved Copyright # Munksgaard 2001

Relationship between pulmonary and cardiac abnormalities in sickle cell disease: implications for the management of patients

Objective To evaluate the association between clinical, pulmonary, and cardiovascular findings in patients with sickle cell disease and, secondarily, to compare these findings between sickle cell anemia patients and those with other sickle cell diseases. Methods Fifty-nine adults were included in this cross-sectional study; 47 had sickle cell anemia, and 12 had other sickle cell diseases. All patients underwent pulmonary function tests, chest computed tomography, and echocardiography. Results Abnormalities on computed tomography, echocardiography, and pulmonary function tests were observed in 93.5%, 75.0%; and 70.2% of patients, respectively. A higher frequency of restrictive abnormalities was observed in patients with a history of acute chest syndrome (85% vs. 21.6%; p-value < 0.0001) and among patients with increased left ventricle size (48.2% vs. 22.2%; p-value = 0.036), and a higher frequency of reduced respiratory muscle strength was observed in patients with a ground-glass pattern (33.3% vs. 4.3%; p-value = 0.016). Moreover, a higher frequency of mosaic attenuation was observed in patients with elevated tricuspid regurgitation velocity (61.1% vs. 24%; p-value = 0.014). Compared to patients with other sickle cell diseases, sickle cell anemia patients had suffered increased frequencies of acute pain episodes, and acute chest syndrome, and exhibited mosaic attenuation on computed tomography, and abnormalities on echocardiography. Conclusion A significant interrelation between abnormalities of the pulmonary and cardiovascular systems was observed in sickle cell disease patients. Furthermore, the severity of the cardiopulmonary parameters among patients with sickle cell anemia was greater than that of patients with other sickle cell diseases.

Changes seen on computed tomography of the chest in mildly symptomatic adult patients with sickle cell disease

Objective To describe and quantify the main changes seen on computed tomography of the chest in mildly symptomatic adult patients with sickle cell disease, as well as to evaluate the radiologist accuracy in determining the type of hemoglobinopathy. Materials and Methods A prospective study involving 44 adult patients with sickle cell disease who underwent inspiration and expiration computed tomography of the chest. The frequency of tomography findings and the extent of involvement are reported. We also calculated radiologist accuracy in determining the type of hemoglobinopathy by analyzing the pulmonary alterations and morphology of the spleen. Results The changes found on computed tomography scans, in descending order of frequency, were as follows: fibrotic opacities (81.8%); mosaic attenuation (56.8%); architectural distortion (31.8%); cardiomegaly (25.0%); lobar volume reduction (18.2%); and increased caliber of peripheral pulmonary arteries (9.1%). For most of the findings, the involvement was considered mild, five or fewer lung segments being affected. The accuracy in determining the type of hemoglobinopathy (HbSS group versus not HbSS group) was 72.7%. Conclusion In adult patients with sickle cell disease, the main tomography findings reflect fibrotic changes. In addition, computed tomography can be helpful in differentiating among hemoglobinopathies.

Complex karyotype and N-RAS point mutation in a case of acute megakaryoblastic leukemia (M7) following a myelodysplastic syndrome.

The development of acute megakaryoblastic leukemia (ANLL-M7) following myelodysplastic syndrome (MDS) has been described only in a few reports, and the mutations necessary for this transformation are still unknown. In this study, we describe a case of ANLL-M7 with a previous history of MDS presenting a complex karyotype 46,XX, t(4;11)(q21;q23),del(5)(q13q33),t(12;13)(p13;q21) and N-RAS point mutation. During MDS, the patient showed a hypercellular myelogram with dysplasia of the three myeloid lineages and the clinical symptoms characteristic of the 5q- syndrome. During the follow-up, we observed the appearance of two additional subclones, one with an isochromosome 17q and another with polyploidy. The presence of an isochromosome 17q in one subclone and polyploidy in another is probably due to the genetic instability generated by the malignant transformation.

Complex karyotype including ring chromosome 11 in a patient with acute myeloid leukemia: case report

CONTEXT Complex karyotypes in acute myeloid leukemia (AML) are characterized by an overall low response rate with frequent relapses after clinical treatment. CASE REPORT Here, we describe the case of a 61-year-old obese female with clinically diagnosed AML who presented a complex karyotype involving an uncommon abnormality: ring chromosome 11. Immunophenotypic analysis confirmed the diagnosis. Classical and molecular cytogenetic analyses, using GTG banding and FISH (fluorescence in situ hybridization), revealed the presence of complex structural rearrangement involving r(11), add(12)(p13), der(5) and der(13). CONCLUSIONS Molecular cytogenetic analysis is suitable for better identification and characterization of chromosomal rearrangements in AML. Case reports like this, as well as population-based studies, are necessary for understanding the karyotypic changes that occur in humans.

Respiratory resistance and reactance in adults with sickle cell anemia: Correlation with functional exercise capacity and diagnostic use

Background The improvement in sickle cell anemia (SCA) care resulted in the emergence of a large population of adults living with this disease. The mechanisms of lung injury in this new population are largely unknown. The forced oscillation technique (FOT) represents the current state-of-the-art in the assessment of lung function. The present work uses the FOT to improve our knowledge about the respiratory abnormalities in SCA, evaluates the associations of FOT with the functional exercise capacity and investigates the early detection of respiratory abnormalities. Methodology/Principal findings Spirometric classification of restrictive abnormalities resulted in three categories: controls (n = 23), patients with a normal exam (n = 21) and presenting pulmonary restriction (n = 24). FOT analysis showed that, besides restrictive changes (reduced compliance; p<0.001), there is also an increase in respiratory resistance (p<0.001) and ventilation heterogeneity (p<0.01). FOT parameters are associated with functional exercise capacity (R = -0.38), pulmonary diffusion (R = 0.66), respiratory muscle performance (R = 0.41), pulmonary volumes (R = 0.56) and airway obstruction (R = 0.54). The diagnostic accuracy was evaluated by investigating the area under the receiver operating characteristic curve (AUC). A combination of FOT and machine learning (ML) classifiers showed adequate diagnostic accuracy in the detection of early respiratory abnormalities (AUC = 0.82). Conclusions In this study, the use of FOT showed that adults with SCA develop a mixed pattern of respiratory disease. Changes in FOT parameters are associated with functional exercise capacity decline, abnormal pulmonary mechanics and diffusion. FOT associated with ML methods accurately diagnosed early respiratory abnormalities. This suggested the potential utility of the FOT and ML clinical decision support systems in the identification of respiratory abnormalities in patients with SCA.

Protector effect of α-thalassaemia on cholecystitis and cholecystectomy in sickle cell disease

ABSTRACT Objectives: Cholecystitis is one of the complications of symptomatic cholelithiasis responsible for high levels of morbidity of sickle cell disease (SCD) patients. Here, we investigated the possible protective role of single gene deletions of α-thalassaemia in the occurrence of cholelithiasis and cholecystitis in SCD patients, as well as the cholecystectomy requirements. Methods: The α-globin genotype was determined in 83 SCD patients using the multiplex-polymerase chain reaction and compared with clinical events. Results: Overall, in 23% of patients, -α3.7 deletion was found. α-Thalassaemia concomitant to SCD was an independent protective factor to cholecystitis (OR = 0.07; 95% CI: 0.01–0.66; p = 0.020) and cholecystectomy requirement (OR = 0.14; 95% CI: 0.03–0.60; p = 0.008). The risk of cholelithiasis was not affected by the α-thalassaemia concomitance. Conclusions: To the best our knowledge, our study is the first to show the protective effect of α-thalassaemia on cholecystitis and cholecystectomy requirements in SCD, which may be due to an improved splenic function.

Screening of mutations in the additional sex combs like 1, transcriptional regulator, tumor protein p53, and KRAS proto-oncogene, GTPase/NRAS proto-oncogene, GTPase genes of patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal bone marrow disorders characterized by ineffective hematopoiesis, different degrees of cellular dysplasia, and increased risk of progression to acute myeloid leukemia. International Prognostic Scoring System is the gold standard for MDS classification; however, patients exhibiting different clinical behaviors often coexist in the same group, indicating that the currently available scoring systems are insufficient. The genes that have recently been identified as mutated in MDS, including additional sex combs like 1, transcriptional regulator (ASXL1), tumor protein p53 (TP53), and KRAS proto-oncogene and GTPase (KRAS)/NRAS proto-oncogene, GTPase (NRAS), may contribute to a more comprehensive classification, as well as to the prognosis and progression of the disease. In the present study, the mutations in the ASXL1, TP53 and NRAS/KRAS genes in 50 patients were evaluated by sequencing genomic bone marrow DNA. Nine patients (18%) presented with at least one type of mutation. Mutations in TP53 were the most frequent in six patients (12%), followed by ASXL1 in two patients (4%) and NRAS in one patient (2%). The nine mutations were detected in patients with low- and high-risk MDS. The screening of mutations in MDS cases contributes to the application of personalized medicine.

Flow cytometry as a diagnostic support tool in juvenile myelomonocytic leukemia.

Th e diagnosis of juvenile myelomonocytic leukemia (JMML) follows a diffi cult question in pediatric hematology [1]. No consistently recurring cytogenetic abnormalities are reported in JMML, and normal karyotypes are found in 40 – 70% of patients. Monosomy 7, del(7q) or other abnormalities of chromosome 7 have been reported in ∼ 25% of cases [2,3]. Mutations in NRAS, NF1 and PTPN11 genes are found in ∼ 20 – 35% of JMML patients and are mutually exclusive; thus, overall, nearly 75% of patients with JMML have one of these abnormalities [3,4]. Although these molecular abnormalities are of help in the diagnostic workup of JMML, they are not pathognomonic, since these abnormalities are also found in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and others myelodysplastic syndrome/myeloproliferative disorders (MDS/ MPL) [3]. Moreover, these genetic tests are not widely available for clinical use. In addition, no specifi c phenotypic abnormalities have been reported in JMML to date [3]. Based on this, new diagnostic tools are urgently required to improve patient care. For several decades now, fl ow cytometry (FCM) immunophenotyping has been shown to be essential for rapid diagnosis, classifi cation and monitoring of most hematological malignancies [5,6] and also holds promise for the diagnosis of pediatric solid tumors [7]. However, the role of FCM in the diagnosis of JMML has been restricted to blast cell compartment analysis. Our aim was to identify abnormalities in the relative distribution and phenotypic characteristics of the diff erent compartments of hematopoietic cells that could contribute to the diagnosis of JMML. Here, we describe recurrent immunophenotypic abnormalities in three children ( � 22 months of age) with JMML that were referred to our hospital to investigate intermittent fever due to recurrent episodes of severe infections. Th e study was approved by the Ethical Committee from IPPMG/UFRJ and is in accordance with the Helsinki Declaration. Clinical and laboratory fi ndings are summarized in Table I. In all cases, fi nal diagnosis of JMML was established based on World Health Organization (WHO) 2008 criteria. Th e identifi cation, quantifi cation and characterization of hematopoietic cells were done by FCM on bone marrows (BM, all cases) and also on peripheral blood (PB) from case#1 at diagnosis and evolution. Briefl y, all samples were stained with the following three-color combinations of antibodies (all from BD Biosciences) conjugated with fl uorescein isothiocyanate (FITC)/phycoerythrin (PE)/perid inclorophyll proteincyanine 5.5 (PerCP-Cy5.5): CD19/CD10/CD45; CD34/ HLA-DR/CD45; CD15/CD34/CD45; CD7/CD34/CD45; CD7/CD117/CD45; CD13/CD11b/CD45; Cy MPO/CD13/ CD45; CD36/CD64/CD45; CD14/CD33/CD45; CD14/ HLA-DR/CD45; CD4/CD33/CD45, CD4/CD13/CD45. Th e samples (50 μ l per tube) were incubated for 15 min at room temperature in the dark, in the presence of saturating amounts of each of the above-mentioned monoclonal antibodies (MAb). Afterward, 2 ml of FACS lysing solution (BD) diluted 1:10 (v/v) in distilled water was added and the samples were incubated for another 10 min under the same conditions as those mentioned above. Th en, cells were centrifuged (5 min at 540 g) and the cell pellet was washed with 2 ml of PBS BSA 0.5%. Finally, cells were resuspended in 0.4 ml of PBS BSA 0.5%. Stained cells were acquired at low speed in a FACSCalibur fl ow cytometer (BD) using the

Left ventricular structural and functional changes evaluated by echocardiography and two-dimensional strain in patients with sickle cell disease

Introduction Patients with sickle cell disease have increased left ventricular size, which is not usually accompanied by changes in systolic function indexes. We assessed echocardiographic abnormalities present in patients with sickle cell anemia (SCA) and compared echocardiographic parameters to other sickle cell diseases (OSCD). Material and methods A blind cross-sectional study with 60 patients with SCA and 16 patients with OSCD who underwent transthoracic echocardiography was performed. Results Echocardiographic findings were: left atrial volume index 47.7 ±11.5 ml/m² in SCA group and 31.7 ±8.42 ml/m² in OSCD group (p < 0.001); left ventricular diastolic diameter index 3.47 ±0.37 cm/m² in SCA group and 2.97 ±0.41 cm/m² in OSCD group (p < 0.001); left ventricular systolic diameter index 2.12 ±0.31 cm/m² in SCA group and 1.86 ±0.28 cm/m² in OSCD group (p < 0.001). There were no differences in the left ventricular ejection fraction: 68.2 ±6.69% in SCA group and 67.1 ±6.21% in OSCD group (p = 0.527). The ratio between mitral E wave and mean mitral annulus e’ wave velocities was higher in the SCA group (7.72 ±1.54 vs. 6.70 ±1.65; p = 0.047). Mitral A wave correlated significantly with hemoglobin levels (r = –0.340; p = 0.032). Conclusions There was an increase of left ventricular and left atrial sizes in patients with SCA, compared to patients with OSCD, without changes in systolic or diastolic function in both groups. This could be due to the hyperkinetic state due to the more severe anemia in the SCA subjects.