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The TP73 gene transcript is alternatively spliced and translated into the transcriptionally active (TAp73) or inactive (ΔNp73) isoforms, with opposite effects on the expression of p53 target genes and on apoptosis induction. The imbalance between ΔNp73 and TAp73 may contribute to tumorigenesis and resistance to chemotherapy in human cancers, including hematologic malignancies. In acute promyelocytic leukemia (APL), both isoforms are expressed, but their relevance in determining response to therapy and contribution to leukemogenesis remains unknown. Here, we provide the first evidence that a higher ΔNp73/TAp73 RNA expression ratio is associated with lower survival, lower disease-free survival, and higher risk of relapse in patients with APL homogeneously treated with all-trans retinoic acid and anthracycline-based chemotherapy, according to the International Consortium on Acute Promyelocytic Leukemia (IC-APL) study. Cox proportional hazards modeling showed that a high ΔNp73/TAp73 ratio was independently associated with shorter overall survival (hazard ratio, 4.47; 95% confidence interval, 1.64-12.2; P = .0035). Our data support the hypothesis that the ΔNp73/TAp73 ratio is an important determinant of clinical response in APL and may offer a therapeutic target for enhancing chemosensitivity in blast cells., (© 2015 by The American Society of Hematology.)

Some myeloproliferative neoplasm (MPN) patients harbor JAK2(V617F) mutation, and CALR mutations were recently discovered in wild type (WT) JAK2(V617F). We evaluated the frequency and type of CALR mutations, and clinical and hematological characteristics in WT JAK2(V617F) and MPL(W515K/L) MPN patients. Sixty-five patients were included: 21 with primary myelofibrosis (PMF), 21 with myelofibrosis post-essential thrombocythemia (MPET) and 23 with essential thrombocythemia (ET). Screening for JAK2(V617F) and MPL(W515K/L) were performed using real-time PCR, while CALR mutations were analyzed by fragment analysis and Sanger sequencing. JAK2(V617F) was the most frequent mutation (54.5%) and one patient (1.5%) harbored MPL(W515L). CALR mutations were present in 38.1% of PMF, 12.5% of ET and 33.3% of MPET patients. Five types of CALR mutations were detected, among which type 1 (32.1%) and type 2 (21.4%) were found to be the most common. A novel CALR mutation in a PMF patient was found. Patients carrying CALR mutations had higher platelet count and less presence of splenomegaly than JAK2(V617F), while triple negatives had higher C-reactive protein levels than CALR mutant carriers. Screening for CALR mutations and its correlation with clinical features could be useful for the characterization of MPN patients and result in its incorporation into a new prognostic score., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Genetic variations in membrane transporters may contribute to imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML). Objective To investigate the relationship between SLCO1B3, SLCO1A2, and ABCA3 polymorphisms and IM response in CML patients., Methods: Patients in chronic phase CML (N = 118) were studied. All patients were treated with a standard dose of IM (400 mg/day) and classified into one of the two groups according to their responses. Major molecular response (MMR) and complete molecular response (CMR) were evaluated. Criteria for response failure were established according to European LeukemiaNet (2009). Analysis of the SLCO1B3 c.334T > G (rs4149117) and c.699G > A (rs7311358), SLCO1A2 c.516A > C (rs11568563) and c.-62-361G > A (rs3764043), and ABCA3 c.1755C > G (rs323043) and c.4548-191C > A (rs150929) polymorphisms was carried out by real-time polymerase chain reaction., Results: SLCO1A2 and ABCA3 polymorphisms have similar frequencies between responders and non-responders. SLCO1B3 699GG and 344TT genotypes were more frequent in the responder group (63.8%) than in the non-responder group (44.7%, P = 0.042). Furthermore, carriers of 699GA/AA and 334TG/GG genotypes presented a higher probability of not responding to the standard dose of IM (odds ratio: 2.17; 95% confidence interval: 1.02-4.64, P = 0.04). Poor CMR for ABCA3 4548-91C > A was observed in patients with the CC/CA genotype when compared to AA carriers in the responder group (P = 0.014)., Conclusions: SLCO1B3 699GG and 344TT genotypes are associated with non-response to IM, while ABCA3 4548-91 CC/CA genotypes are related to poor CMR in CML patients treated with standard-dose imatinib.

Objective: Myeloid neoplasms are heterogeneous diseases that are more incident in the elderly. The goals of this study were to aggregate a geriatric approach to the patient assessment, to show the impact of gender, age, hemoglobin concentration and comorbidities on the functionality of elderly with myeloid neoplasms and to better understand how the instruments of functional assessment work according to the aggressiveness of the disease., Methods: Elderly patients (≥60 years old) with myeloid neoplasms were assessed using the Karnofsky scale, Eastern Cooperative Oncologic Group scale, and basic and instrumental activities of daily living scales. The hematopoietic cell transplantation-comorbidity index assessed the comorbidities. A mixed logistical regression model was fitted to estimate the impact of gender, age, hemoglobin concentration and the hematopoietic cell transplantation-comorbidity index on patients' functionality., Results: Eighty-two patients with a mean age of 72.8 years (range: 60-92 years) were evaluated. Eighty percent had good Karnofsky and Eastern Cooperative Oncologic Group scales and 39% were independent according to the daily living activity scales. All of the patients with poor Karnofsky and Eastern Cooperative Oncologic Group scales were classified as dependent by the daily living activity scales. The mixed logistic regression models showed that age, gender, hemoglobin concentration and the comorbidity index impacted on the daily living activity scales. Karnofsky and Eastern Cooperative Oncologic Group scales were affected by hemoglobin and the comorbidity index. The model hypothesized the hemoglobin concentration at which there was a higher risk of poor Karnofsky and Eastern Cooperative Oncologic Group scales. This hemoglobin concentration depended on comorbidities and on the aggressiveness of the myeloid neoplasm., Conclusion: The geriatric approach improved the sensitivity and specificity of the patients' assessment. Hemoglobin concentration associated to the risk of poor Karnofsky and Eastern Cooperative Oncologic Group scales depended on the comorbidity score and on the disease aggressiveness. The Karnofsky and Eastern Cooperative Oncologic Group scales had higher sensitivity in patients with more aggressive diseases., (Copyright © 2015 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights reserved.)

Objective: To standardize the single nucleotide polymorphism array (SNPa) method in acute myeloid leukemia/myelodysplastic syndromes, and to identify the similarities and differences between the results of this method and karyotyping., Methods: Twenty-two patients diagnosed with acute myeloid leukemia and three with myelodysplastic syndromes were studied. The G-banding karyotyping and single nucleotide polymorphism array analysis (CytoScan(®) HD) were performed using cells from bone marrow, DNA extracted from mononuclear cells from bone marrow and buccal cells (BC)., Results: The mean age of the patients studied was 54 years old, and the median age was 55 years (range: 28-93). Twelve (48%) were male and 13 (52%) female. Ten patients showed abnormal karyotypes (40.0%), 11 normal (44.0%) and four had no mitosis (16.0%). Regarding the results of bone marrow single nucleotide polymorphism array analysis: 17 were abnormal (68.0%) and eight were normal (32.0%). Comparing the two methods, karyotyping identified a total of 17 alterations (8 deletions/losses, 7 trissomies/gains, and 2 translocations) and single nucleotide polymorphism array analysis identified a total of 42 alterations (17 losses, 16 gains and 9 copy-neutral loss of heterozygosity)., Conclusion: It is possible to standardize single nucleotide polymorphism array analysis in acute myeloid leukemia/myelodysplastic syndromes and compare the results with the abnormalities detected by karyotyping. Single nucleotide polymorphism array analysis increased the detection rate of abnormalities compared to karyotyping and also identified a new set of abnormalities that deserve further investigation in future studies., (Copyright © 2014 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights reserved.)

Background: Chronic myeloid leukemia is a myeloproliferative disorder characterized by the Philadelphia chromosome or t(9;22)(q34.1;q11.2), resulting in the break-point cluster region-Abelson tyrosine kinase fusion gene, which encodes a constitutively active tyrosine kinase protein. The Philadelphia chromosome is detected by karyotyping in around 90% of chronic myeloid leukemia patients, but 5-10% may have variant types. Variant Philadelphia chromosomes are characterized by the involvement of another chromosome in addition to chromosome 9 or 22. It can be a simple type of variant when one other chromosome is involved, or complex, in which two or more chromosomes take part in the translocation. Few studies have reported the incidence of variant Philadelphia chromosomes or the breakpoints involved among Brazilian chronic myeloid leukemia patients., Objective: The aim of this report is to describe the diversity of the variant Philadelphia chromosomes found and highlight some interesting breakpoint candidates for further studies., Methods: the Cytogenetics Section Database was searched for all cases with diagnoses of chronic myeloid leukemia during a 12-year period and all the variant Philadelphia chromosomes were listed., Results: Fifty (5.17%) cases out of 1071 Philadelphia-positive chronic myeloid leukemia were variants. The most frequently involved chromosome was 17, followed by chromosomes: 1, 20, 6, 11, 2, 10, 12 and 15., Conclusion: Among all the breakpoints seen in this survey, six had previously been described: 11p15, 14q32, 15q11.2, 16p13.1, 17p13 and 17q21. The fact that some regions get more frequently involved in such rare rearrangements calls attention to possible predisposition that should be further studied. Nevertheless, the pathological implication of these variants remains unclear., (Copyright © 2014 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights reserved.)

Activating internal tandem duplication (ITD) mutations in the fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor outcome in acute myeloid leukemia, but their prognostic impact in acute promyelocytic leukemia (APL) remains controversial. Here, we screened for FLT3-ITD mutations in 171 APL patients, treated with all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy. We identified FLT3-ITD mutations in 35 patients (20 %). FLT3-ITD mutations were associated with higher white blood cell counts (P < 0.0001), relapse-risk score (P = 0.0007), higher hemoglobin levels (P = 0.0004), higher frequency of the microgranular morphology (M3v) subtype (P = 0.03), and the short PML/RARA (BCR3) isoform (P < 0.0001). After a median follow-up of 38 months, FLT3-ITD(positive) patients had a lower 3-year overall survival rate (62 %) compared with FLT3-ITD(negative) patients (82 %) (P = 0.006). The prognostic impact of FLT3-ITD on survival was retained in multivariable analysis (hazard ratio: 2.39, 95 % confidence interval [CI] 1.17-4.89; P = 0.017). Nevertheless, complete remission (P = 0.07), disease-free survival (P = 0.24), and the cumulative incidence of relapse (P = 0.94) rates were not significantly different between groups. We can conclude that FLT3-ITD mutations are associated with several hematologic features in APL, in particular with high white blood cell counts. In addition, FLT3-ITD may independently predict a shorter survival in patients with APL treated with ATRA and anthracycline-based chemotherapy.

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12&#95;JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12&#95;JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12&#95;JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN.

Background: Quantification of bone marrow (BM) blasts by cytomorphology is essential for the diagnosis of myelodysplastic syndromes (MDS). Owing to its subjectivity and the potential impact of dysplastic features on accurate identification of blast cells, more objective approaches are required, multiparameter flow cytometry (MFC) being a particularly promising approach in this regard. However, no consensus exists about the optimal combination of markers and strategy to be used., Methods: BM blast counts from 74 MDS patients were evaluated by morphology versus four different MFC phenotypic criteria: "CD34⁺", "CD34⁺ and/or CD117⁺", "CD34⁺, and/or CD117⁺ HLA-DR⁺", and "CD34⁺ and CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-/lo) " cells. For each criterium, the percentage of blasts was calculated using either all BM nucleated cells or non-erythroid CD45⁺ cells as denominator. RESULTS.: The number of "CD34⁺ and/or CD117⁺ HLA-DR⁺"cells showed the highest correlation and agreement with morphological counts, only a minor proportion of cases being misclassified by MFC vs. morphology for the >5% and >10% classification thresholds. In turn, a CD34⁺ phenotype was insufficient to correctly identify and quantify blasts. Conversely, usage of non-erythroid BM cells as denominator, or inclusion of "CD34⁺ and/or CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-lo") cells were both associated with overestimated blast counts., Conclusions: Quantification of "CD34⁺ and/or CD117⁺ HLA-DR⁺" cells (from all nucleated BM cells) by MFC is an efficient method for the enumeration of blasts in MDS. However, caution should be taken with replacing morphology by MFC blast counts; its combined use may rather provide complementary information increasing the accuracy and reproducibility of BM blast cell counts in these patients., (Copyright © 2013 International Clinical Cytometry Society.)

Thanks to modern treatment with all-trans retinoic acid and chemotherapy, acute promyelocytic leukemia (APL) is now the most curable type of leukemia. However, this progress has not yielded equivalent benefit in developing countries. The International Consortium on Acute Promyelocytic Leukemia (IC-APL) was established to create a network of institutions in developing countries that would exchange experience and data and receive support from well-established US and European cooperative groups. The IC-APL formulated expeditious diagnostic, treatment, and supportive guidelines that were adapted to local circumstances. APL was chosen as a model disease because of the potential impact on improved diagnosis and treatment. The project included 4 national coordinators and reference laboratories, common clinical record forms, 5 subcommittees, and laboratory and data management training programs. In addition, participating institutions held regular virtual and face-to-face meetings. Complete hematological remission was achieved in 153/180 (85%) patients and 27 (15%) died during induction. After a median follow-up of 28 months, the 2-year cumulative incidence of relapse, overall survival (OS), and disease-free survival (DFS) were 4.5%, 80%, and 91%, respectively. The establishment of the IC-APL network resulted in a decrease of almost 50% in early mortality and an improvement in OS of almost 30% compared with historical controls, resulting in OS and DFS similar to those reported in developed countries.

Acute myeloid leukemia (AML) is a group of clonal diseases, resulting from two classes of mutation. Investigation for additional abnormalities associated with a well-recognized subtype, core-binding factor AML (CBF-AML) can provide further understanding and discrimination to this special group of leukemia. In order to better define genetic alterations in CBF-AML and identify possible cooperating lesions, a single-nucleotide polymorphism-array (SNP-array) analysis was performed, combined to KIT mutation screening, in a set of cases. Validation of SNP-array results was done by array comparative genomic hybridization and FISH. Fifteen cases were analyzed. Three cases had microscopic lesions better delineated by arrays. One case had +22 not identified by arrays. Submicroscopic abnormalities were mostly non-recurrent between samples. Of relevance, four regions were more frequently affected: 4q28, 9p11, 16q22.1, and 16q23. One case had an uncovered unbalanced inv(16) due to submicroscopic deletion of 5´MYH11 and 3´CBFB. Telomeric and large copy number neutral loss of heterozygosity (CNN-LOH) regions (>25 Mb), likely representing uniparental disomy, were detected in four out of fifteen cases. Only three cases had mutation on KIT gene, enhancing the role of abnormalities by SNP-array as presumptive cooperating alterations. Molecular karyotyping can add valuable information to metaphase karyotype analysis, emerging as an important tool to uncover and characterize microscopic, submicroscopic genomic alterations, and CNN-LOH events in the search for cooperating lesions.

Improved outcome of acute myeloid leukemia (AML) depends on the better differentiation of subtypes to predict treatment response and the identification of new target for treatment. In this study, array comparative genomic hybridization (aCGH) was used to distinguish eight cases of AML cases. Validation was performed by FISH and quantitative genomic PCR. The aCGH revealed new large and small recurrent genomic imbalances, such as gains of 1p36, 10q26, 11p15, 20q13, 22q23, harboring many proto-oncogenes. These results better define genetically the studied cases and could be used to understand the multiple phenomena involved in leukemogenesis.

Background: Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes., Design and Methods: We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated., Results: Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n = 5), CD42a (n = 1) and CD61 (n = 2), together with reactivity for CD34 (n = 1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n = 16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival., Conclusions: Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders.

Objectives: Several clinical trials conducted in Europe and US reported favorable outcomes of patients with APL treated with the combination of all trans retinoic acid (ATRA) and anthracyclines. Nevertheless, the results observed in developing countries with the same regimen was poorer, mainly due to high early mortality mainly due bleeding. The International Consortium on Acute Promyelocytic Leukemia (IC-APL) is an initiative of the International Members Committee of the ASH and the project aims to reduce this gap through the establishment of international network, which was launched in Brazil, Mexico and Uruguay., Methods: The IC-APL treatment protocol is similar to the PETHEMA 2005, but changing idarubicin to daunorubicin. All patients with a suspected diagnosis of APL were immediately started on ATRA, while bone marrow samples were shipped to a national central lab where genetic verification of the diagnosis was performed. The immunofluorescence using an anti-PML antibody allowed a rapid confirmation of the diagnosis and, the importance of supportive measures was reinforced., Results: The interim analysis of 97 patients enrolled in the IC-APL protocol showed that complete remission (CR) rate was 83% and the 2-year overall survival and disease-free survival were 80% and 90%, respectively. Of note, the early mortality rate was reduced to 7.5%., Discussion: The results of IC-APL demonstrate the impact of educational programs and networking on the improvement of the leukemia treatment outcome in developing countries.

To evaluate the grading of fibrosis and immunohistochemical expression of MPL in bone marrow biopsies of ET and PMF. Fibrosis in bone marrow biopsies (BMBs) was evaluated according to the European Consensus for grading of fibrosis, according to reticulin proliferation. Immunohistochemical analysis was performed in samples from 18 ET and 38 PMF patients: 19 were classified as pre-fibrotic and 19 were classified as fibrotic according to the World Health Organization (WHO) criteria, by means of the MPL antibody. Six bone marrow donors' biopsies were used as controls. Average reticulin (p<0.003) and MPL (p<0.008) values differed significantly between the ET group and the pre-fibrotic stage PMF group. The MPL immunohistochemical expression may represent a new marker for differential diagnosis between ET and pre-fibrotic stage PMF., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chronic myeloproliferative diseases without the Philadelphia chromosome marker (Ph-), although first described 60 years ago, only became the subject of interest after the turn of the millennium. In 2001, the World Health Organization (WHO) defined the classification of this group of diseases and in 2008 they were renamed myeloproliferative neoplasms based on morphological, cytogenetic and molecular features. In 2005, the identification of a recurrent molecular abnormality characterized by a gain of function with a mutation in the gene encoding Janus kinase 2 (JAK2) paved the way for greater knowledge of the pathophysiology of myeloproliferative neoplasms. The JAK2 mutation is found in 90-98% of polycythemia vera and in about 50% essential thrombocytosis and primary myelofibrosis. In addition to the JAK2 mutation, other mutations involving TET2 (ten-eleven translocation), LNK (a membrane-bound adaptor protein); IDH1/2 (isocitrate dehydrogenase 1/2 enzyme); ASXL1 (additional sex combs-like 1) genes were found in myeloproliferative neoplasms thus showing the importance of identifying molecular genetic alterations to confirm diagnosis, guide treatment and improve our understanding of the biology of these diseases. Currently, polycythemia vera, essential thrombocytosis, myelofibrosis, chronic neutrophilic leukemia, chronic eosinophilic leukemia and mastocytosis are included in this group of myeloproliferative neoplasms, but are considered different situations with individualized diagnostic methods and treatment. This review updates pathogenic aspects, molecular genetic alterations, the fundamental criteria for diagnosis and the best approach for each of these entities.

Background: The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms., Objectives: THE AIM OF THIS STUDY WAS TO DETECT THE FOLLOWING MUTATIONS: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases., Methods: Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities., Results: Chromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L-positive patient with primary myelofibrosis had more severe anemia than other patients with primary myelofibrosis., Conclusions: This study demonstrates that karyotyping for JAK2 and MPL mutations is useful in the diagnosis of myeloproliferative neoplasms. The precise pathogenetic contribution of these alterations is still unclear. However, this study adds more information about the pathophysiology of polycythemia vera, essential thrombocythemia and primary myelofibrosis.

Objective: To describe chromosomal abnormalities in spontaneous abortion material., Methods: A retrospective compilation of karyotype analysis of slides stained with Band G was carried out by optical microscopy with materials of 428 abortion products referred for study., Results: There were 145 normal results (33.9%) and 237 abnormal results (55.4%). In 46 samples there was no cell growth (10.7%). Numerical abnormalities were the most frequent, especially trisomy 16 (41 cases), triplodia (27 cases), monosomy X (26 cases), tetraploidy (13 cases) and trisomy 15 (13 cases)., Conclusion: Cytogenetic alterations are an important cause of pregnancy loss and their detection helps the genetic counseling to the couple. Trisomy 16 is the most often found change.

Recent investigations postulate a participation of the interferon regulatory factor-1 (IRF-1) in the development of myelodysplasia (MDS) and in the pathogenesis of autoimmune manifestations (AIMs) in patients with this disease. The aim of this prospective study was to compare the IRF-1 immunoexpression in MDS patients with or without AIMs and to investigate its prognostic relevance. Fifty consecutive MDS patients entered this prospective study. There was no difference in overall survival between patients with or without autoimmune manifestations. In a multivariate Cox regression "IRF-1 expression in immature myeloid cells", Hb, and the IPSS risk group stratification were independent prognostic parameters. Bootstrap resampling confirmed these data. In a multivariate logistic regression older patients with, higher platelet count and increased IRF-1 expression had a higher risk to develop autoimmune-like phenomena. Thus our study shows that IRF-1 plays an ambiguous role in MDS patients. Whereas high levels of IRF-1 in myeloid cells are a favorable prognostic factor for overall survival, they increase the probability of the manifestation of autoimmune phenomena, with a diminished quality of life.

Context and Objective: Non-myeloablative hematopoietic stem cell transplantation (NMA-HSCT) is performed in onco-hematological patients who cannot tolerate ablative conditioning because of older age or comorbidities. This approach does not completely eliminate host cells and initially results in mixed chimerism. Long-term persistence of mixed chimerism results in graft rejection and relapse. Involvement of graft-versus-host disease is concomitant with complete chimerism and graft-versus-tumor effect. The aim of this study was to evaluate the prevalence of chimerism in onco-hematological patients who underwent NMA-HSCT., Design and Setting: Observational clinical study on chimerism status after human leukocyte antigen-identical NMA-HSCT at the Discipline of Hematology and Hemotherapy of Universidade Federal de São Paulo., Methods: We sequentially analyzed the amplification of APO-B, D1S80, DxS52, FVW, 33.6, YNZ-2 and H-ras primers using variable number of tandem repeats (VNTR) on 17 pairs and fluorescent in situ hybridization (FISH) with the XY probe and SRY primer on 13 sex-unmatched pairs., Results: The informativeness of the primers using VNTR was 60% for APO-B, 75% D1S80, 36% DxS52, 14% FVW, 40% YNZ-22 and 16% H-ras. The SRY primer was informative in female receptors with male donors. The XY-FISH method was informative in 100% of the sex-unmatched pairs., Conclusion: These methods were sensitive and informative. In VNTR, the association of APO-B with D1S80 showed 88% informativeness. The quantitative FISH method was more sensitive, but had the disadvantage of only being used for sex-unmatched pairs.

Purpose: To evaluate the expression of NF-kappaB pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival., Material and Methods: Expression of eight genes related to NF-kappaB pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples., Results: The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p=0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p=0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p=0.04)., Conclusion: We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.

Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM). Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients. We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 46 upregulated genes in the MM library. Ten upregulated genes were selected for further investigation. Differential expression was validated by quantitative real-time PCR in purified plasma cells of 31 patients and three controls. P53CSV, DDX5, MAPKAPK2 and RANBP2 were found to be upregulated in at least 50% of the MM cases tested. All of them were also found upregulated in MM when compared to normal plasma cells in a meta-analysis using ONCOMINE microarray database. Antibodies specific to DDX5, RANBP2 and MAPKAPK2 were used in a TMA containing 57 MM cases and confirmed the expression of these proteins in 74%, 96%, and 21% of the MM samples, respectively. Analysis of differential expression using SAGE could identify genes important for myeloma tumorigenesis (P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.

Context and Objective: The mechanism involved in leukemogenesis remains unclear and more information about the disruption of the cell proliferation, cell differentiation and apoptosis of neoplastic cells is required., Design and Setting: Cross-sectional prevalence study at the Discipline of Hematology, Hospital São Paulo, Universidade Federal de São Paulo., Methods: We investigated FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD+) in 40 adult patients with de novo acute myeloid leukemia (AML), categorized according to cytogenetic results, from September 2001 to May 2005., Results: Thirteen patients (32.5%) were classified as presenting the favorable karyotype, 11 patients (27.5%) as an intermediate group, 7 patients (17%) as an undefined group and 9 patients (22.5%) as the unfavorable group. FLT3/ITD+ was found in 10 patients (25%): 3 with FLT3/ITD+ and favorable karyotype; 4 with FLT3/ITD+ and intermediate karyotype; 2 with FLT3/ITD+ and undefined karyotype; and only 1 with FLT3/ITD+ and unfavorable karyotype. Among the patients without FLT3/ITD+, 10 presented favorable karyotype, 8 intermediate, 4 undefined and 8 unfavorable karyotype. The cytogenetic results showed no correlations between FLT3/ITD presence and the prognostic groups (P = 0.13). We found that 2 patients were still alive more than 24 months later, FLT3/ITD+ did not influence the patients' survival rate., Conclusion: We found the same frequency of AML with FLT3/ITD+ in both the favorable and intermediate prognosis groups. Only one patient presented AML, FLT3/ITD+ and unfavorable karyotype (the hypothetical worst clinical situation). Therefore, the prognostic advantage of favorable cytogenetics among patients with FLT3/ITD+ remains to be elucidated, for it to be better understood.

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and risk for evolving to acute leukemia. Some molecular abnormalities related to acute myeloid leukemia (AML) transformation have been reported, such as FLT3 (FMS-like tyrosine kinase 3) mutations. FLT3, a member of the class 3 receptor tyrosine kinase family, mediates stem cell proliferation and differentiation, and its mutations, internal tandem duplication (ITD) and Asp835, have been reported in rare MDS patients. We studied FLT3 ITD, prospectively, in 50 MDS patients at diagnosis, at 6 and 12 months follow-up, and at any other time-point if AML transformation was detected. FLT3 ITD was not observed at diagnosis, but during follow-up the mutation was present in 2 of 50 patients (4%). Of these, one case exhibited FLT3 ITD at the end of the 6 months of follow-up in approximately 8% of bone marrow cells; this case evolved into AML at 8 months, at which time FLT3 ITD was present in approximately 85% of bone marrow cells. The other case exhibited FLT3 ITD in 68% of bone marrow cells at 7 months, precisely at the time of AML transformation. Although rare in MDS, FLT3 ITD is associated with a high probability of evolution to AML., ((c) 2008 Elsevier Inc.)

Poor outcomes of some chronic myeloid leukemia ((CML) patients have been associated with submicroscopic der(9q) deletions, particularly the 5'ABL region. Deletion profiles of 120 BCR/ABL+ CML patients were studied using the dual-fusion fluorescence in situ hybridization probe. Poor prognosis was associated with 5'ABL deletion but not with 3'BCR deletion. Overall survival (OS) and chronic phase duration (CPD) were significantly shorter for 5'ABL deletion than for those without deletions (OS time: 27 vs. 61 months, P = 0.02; CPD: 17 vs. 56 months, P = 0.02). In addition, when isolated 5'ABL deletion patients were compared to those without it, a greater impact on prognosis was detected (OS time: 18 vs. 59 months, P = 0.0008; CPD: 7 vs. 54 months, P = 0.0003). Isolated 5'ABL deletion seems to have a greater impact on survival than does concomitant 5'ABL and 3'BCR deletion, although the difference was not statistically significant in this aspect (OS time: 18 vs. 28 months, P = 0.08).

The aim of this report is to present a case of Myelodysplastic syndrome (MDS) who presented, during AML transformation, a step-wise genetic progression that corroborates the two hit model of leukemogenesis. A RCDM-RS (WHO)/RARS (FAB) patient with normal karyotype at diagnosis, evolved into AML after six months of follow up. At transformation, AML/ETO fusion was detected, although marrow blast cells were not increased until 21 days later, when FLT3-ITD was also demonstrated pointing out that the overgrowth of the FLT3/ITD clone was concomitant with the outburst of marrow blasts. These findings corroborates the two hit model of leukemogenesis in which one class of mutations (Class I) (FLT3/ITD) confers a proliferative or survival advantage to cells, and a second class of mutations (Class II) (AML/ETO) interferes with hematopoietic differentiation.

Objective: To develop a scoring system based on clinical findings to assist pediatricians in the diagnosis of William syndrome and to delineate when the fluorescent in-situ hybridization test to detect the microdeletion at 7q11.23 may be needed., Methods: The fluorescent in-situ hybridization test was performed on 20 patients presenting William syndrome suggestive clinical features. Eleven studies were selected from the literature in which there were 2 groups: patients with positive or negative fluorescent in-situ hybridization tests. Forty-two clinical characteristics were compared to those reported in the literature to determine which ones were associated with the affected patients (ie, bearing deletions) using meta-analysis. The 2-tailed Fisher exact test were used so that the frequency of findings observed in fluorescent in-situ hybridization positive and fluorescent in-situ hybridization negative patients could be compared in the present study together with the patients from the literature. We developed a scoring system based on clinical findings and their significant associations with patients with positive fluorescent in-situ hybridization tests. From the mean and standard-deviation values of the data from our patients, we determined the cut-off score that that indicated the need for a fluorescent in-situ hybridization test to confirm diagnosis., Results: Seventeen patients were fluorescent in-situ hybridization positive, and 3 were fluorescent in-situ hybridization negative. The more discriminative findings among fluorescent in-situ hybridization positive patients were the following: typical facies, low birth weight, feeding difficulties, constipation, supravalvar aortic stenosis, mental retardation, and friendly personality. The distribution of the points among the 20 patients ranged from 19 to 28 points with a mean value of 23.3 out of a possible total of 31 points. The cut-off score that indicated the need for a fluorescent in-situ hybridization test was 20., Conclusions: Our scoring system enables physicians to differentiate between those individuals who can be reliably diagnosed as having Williams syndrome solely from the clinical findings and those who need to undergo fluorescent in-situ hybridization testing for a correct diagnosis.

This study aims to investigate the prevalence of posttraumatic stress disorder (PTSD) symptoms, anxiety, and depression in patients with hematological malignancies, and to investigate the possible relationship between these symptoms and variables such as demographic data, social support, and quality of life (QOL). We studied 107 patients: 54 with non-Hodgkin's lymphoma (NHL), 18 acute myelogenous leukaemia (AML), 10 acute lymphoblastic leukaemia (ALL), and 25 multiple myeloma (MM). Demographic data were collected, and three standardized instruments were applied to this group of patients: Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire of QOL. The results showed a significant percentage of patients presenting with symptoms: 13% had high levels of intrusive thoughts, 20.5% had high levels of anxiety, and 16.8% had high levels of depression. Patients with MM had the lowest QOL scores in the EORTC physical functioning subscale. Patients under intravenous chemotherapy treatment had a higher level of anxiety than the monitoring patients. Patients with recent diagnosis had a level of intrusion symptoms (IES) relevantly higher than the others. The unemployed patients and those with lower social support had levels of stress, anxiety, and depression significantly higher than the others. Our results confirm the high incidence of intrusion, avoidance, anxiety, and depression in patients with hematological malignancies and highlight the importance of a multidisciplinary staff to complement the treatment of these patients, including psychosocial assistance.

Myelodysplastic syndrome is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis, peripheral cytopenias and an additional risk to evolve to acute leukemia in up to 30% of the cases. Autoimmune manifestations as vasculitis, pyoderma gangrenosum, hemolytic anemia, immune thrombocytopenia, rheumatoid arthritis as well as positive anti-nuclear factor and rheumatoid factor have been reported in 13-30% of MDS patients. The aim of this report is to present three patients with 5q- syndrome who presented different autoimmune serological and clinical phenomena and review the literature. Patient 1 showed a focal and segmental glomerulosclerosis (FSGE) in the course of a MDS. Renal involvement in MDS as autoimmune phenomenon is rare and few reports have documented different forms of glomerular diseases in adults with MDS. Patients 2 and 3 showed a rheumatoid factor of 1/140 and the direct Coomb's test positive (3+), respectively, but without evidence of clinical autoimmune manifestation. In conclusion, patients with the 5q- syndrome experience a relative benign disease course extending over several years. We believe that careful follow-up of patients with autoimmune manifestations as here reported is important to detect any unexpected outcome.

The description of the association between the use of amphotericin-B (amB) and the development of systemic arterial hypertension was only anecdotal so far. We describe the case of a 19-year-old female patient who had acute lymphoblastic leukemia and developed prolonged neutropenia after reinduction chemotherapy. Candida parapsilosis was isolated from blood cultures, and amB was started. Sustained severe arterial hypertension developed shortly after amB administration and continued for several hours after the infusion. Aldosterone, blood urea nitrogen, and creatinine levels were normal. After clinical improvement, amB was replaced by fluconazole, and blood pressure normalized. Severe hypertension may be an adverse event associated with AmB treatment that requires intensive treatment.

Balanced complex chromosome rearrangements (CCR) are extremely rare in humans. They are usually ascertained either by abnormal phenotype or reproductive failure in carriers. These abnormalities are attributed to disruption of genes at the breakpoints, position effect or cryptic imbalances in the genome. However, little is known about possible imbalances at the junction points. We report here a patient with a CCR involving three chromosomes (2;10;11) and eight breakpoints. The patient presented with behavioural problems as the sole phenotypic abnormality. The rearrangement, which is apparently balanced in G-banding and multicolour FISH, was shown by genomic array analysis to include a deletion of 0.15-1.5 Mb associated with one of the breakpoints. To explain the formation of this rearrangement through the smallest possible number of breakage-and-reunion events, one has to assume that the breaks have not occurred simultaneously, but in a temporal order within the span of a single cell division. We demonstrate that array comparative genomic hybridisation (CGH) is a useful complementary tool to cytogenetic analysis for detecting and mapping cryptic imbalances associated with chromosome rearrangement.

Immunosuppressed renal recipients are at an increased risk of developing cancer. Leukemias are less frequent than other hematopoietic tumours and development of CML after immunosuppression is rare. We describe a 37-year-old male who presented with left-shifted leukocytosis, hypercellular bone marrow 32 months after the kidney transplant. G-banding karyotype revealed 46,XY,t(9;22)(q34;q11) and the diagnosis of chronic myeloid leukemia was made. This is the 13th case of CML after kidney transplant reported. Whether this CML appeared as a random phenomenon or chemically induced is a matter of debate. Some individuals might have an increased susceptibility to the effects of azathioprine.