ABSTRACT Introduction: Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eightto ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology. Objective and Method: In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels. Results: Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification. Conclusion: Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

ABSTRACT Introduction: The incidence of grade 3-4 anemia was reported to be 3% with imatinib therapy for newly diagnosed chronic myeloid leukemia (CML) in the chronic phase (CP). However, there are few data regarding the causes and the development of anemia after long-term treatment. This study aimed to evaluate the incidence of anemia after at least two years of imatinib treatment of CML patients in the CP and to identify other contributing causes of anemia in this population. Patients and methods: We performed a retrospective analysis of 97 CML patients in the CP treated with imatinib for at least two years. We analyzed the hemoglobin (Hb) levels of CML patients at diagnosis, upon initiation of treatment with imatinib and after two years of imatinib treatment, and investigated other causes of anemia in this population. Results: Most of the patients presented Hb levels below the normal range (80.4%) after the second year of treatment, 17.9% grade 2 and 1.3% grade 3. In 13 cases (16.7%), anemia was attributed to resistance and in 13 cases (16.7%) the following causes were identified: iron deficiency (n = 5), hypothyroidism (n = 2), vitamin B12 deficiency (n = 3), acquired immune deficiency syndrome (AIDS) (n = 1), pulmonary tuberculosis (n = 1) and renal toxicity (n = 1). In 52 patients (66.6%), there were no other factors contributing to anemia, except imatinib treatment. Conclusion: Regular follow-up is required to identify the causes of anemia not related to CML or imatinib toxicity. The importance of investigating secondary causes of anemia should be emphasized, especially in patients with good adherence to treatment and satisfactory therapeutic response.

ABSTRACT We analyzed the management and outcomes of pregnancies of patients with chronic myeloid leukemia at a single center over fifteen years. Among the 203 CML female patients, there were ten pregnancies in seven women, all of them not planned. In three cases, the chronic myeloid leukemia diagnosis was made during pregnancy. Five patients received tyrosine kinase inhibitors in the first weeks of pregnancy and the drug was interrupted until delivery. One patient lost complete cytogenetic response, and two patients lost the hematological response. A patient with a stable major molecular response had two successful pregnancies without loss of response. There were four premature births. There were no maternal adverse events, fetal malformation or death. All patients received Interferon-alpha during gestation, and two received hydroxyurea for a short period. Leukapheresis was performed in two patients for hyperleukocytosis control. One patient with sickle cell disease died from disease progression six months after delivery. Conclusions: The tyrosine kinase inhibitors ministration should be interrupted during pregnancy. Patients should be advised to achieve a stable and deep molecular response if they plan to conceive, to avoid the risk of disease progression.

ABSTRACT Chronic lymphocytic leukemia is characterized by clonal proliferation and progressive accumulation of B-cell lymphocytes that typically express CD19+, CD5+ and CD23+. The lymphocytes usually infiltrate the bone marrow, peripheral blood, lymph nodes, and spleen. The diagnosis is established by immunophenotyping circulating B-lymphocytes, and prognosis is defined by two staging systems (Rai and Binet) established by physical examination and blood counts, as well as by several biological and genetic markers. In this update, we present the recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia for the diagnosis and treatment of chronic lymphocytic leukemia. The following recommendations are based on an extensive literature review with the aim of contributing to more uniform patient care in Brazil and possibly in other countries with a similar social-economic profile.

OBJECTIVE: To evaluate hematological, cytogenetic and molecular responses as well as the overall, progression-free and event-free survivals of chronic myeloid leukemia patients treated with a third tyrosine kinase inhibitor after failing to respond to imatinib and nilotinib/dasatinib. METHODS: Bone marrow karyotyping and real-time quantitative polymerase chain reaction were performed at baseline and at 3, 6, 12 and 18 months after the initiation of treatment with a third tyrosine kinase inhibitor. Hematologic, cytogenetic and molecular responses were defined according to the European LeukemiaNet recommendations. BCR-ABL1 mutations were analyzed by Sanger sequencing. RESULTS: We evaluated 25 chronic myeloid leukemia patients who had been previously treated with imatinib and a second tyrosine kinase inhibitor. Nine patients were switched to dasatinib, and 16 patients were switched to nilotinib as a third-line therapy. Of the chronic phase patients (n=18), 89% achieved a complete hematologic response, 13% achieved a complete cytogenetic response and 24% achieved a major molecular response. The following BCR-ABL1 mutations were detected in 6/14 (43%) chronic phase patients: E255V, Y253H, M244V, F317L (2) and F359V. M351T mutation was found in one patient in the accelerated phase of the disease. The five-year overall, progression-free and event-free survivals were 86, 54 and 22% (p<0.0001), respectively, for chronic phase patients and 66%, 66% and 0% (p<0.0001), respectively, for accelerated phase patients. All blast crisis patients died within 6 months of treatment. Fifty-six percent of the chronic phase patients lost their hematologic response within a median of 23 months. CONCLUSIONS: Although the responses achieved by the third tyrosine kinase inhibitor were not sustainable, a third tyrosine kinase inhibitor may be an option for improving patient status until a donor becomes available for transplant. Because the long-term outcome for these patients is poor, the development of new therapies for resistant chronic myeloid leukemia patients is necessary.

OBJECTIVES: In Brazil, imatinib mesylate is supplied as the first-line therapy for chronic myeloid leukemia in the chronic phase through the public universal healthcare program, Sistema Único de Saúde (SUS). We studied the socio-demographic factors that influenced therapy success in a population in the northeast region of Brazil. METHODS: Patients with chronic myeloid leukemia from the state of Piauí were treated in only one reference center. Diagnosis was based on WHO 2008 criteria. Risk was assessed by Sokal, Hasford and EUTOS scores. Patients received 400 mg imatinib daily. We studied the influence of the following factors on the achievement of complete cytogenetic response within one year of treatment: age, clinical risk category, time interval between diagnosis and the start of imatinib treatment, geographic distance from the patient's home to the hospital, years of formal education and monthly income. RESULTS: Among 103 patients studied, the median age was 42 years; 65% of the patients had 2-9 years of formal education, and the median monthly income was approximately 100 US$. Imatinib was started in the first year after diagnosis (early chronic phase) in 69 patients. After 12 months of treatment, 68 patients had a complete cytogenetic response. The Hasford score, delay to start imatinib and years of formal education influenced the attainment of a complete cytogenetic response, whereas income and the distance from the home to the healthcare facility did not. CONCLUSION: Patients require additional healthcare information to better understand the importance of long-term oral anticancer treatment and to improve their compliance with the treatment.

Although myelodysplastic syndromes have a clear definition in theory, the morphologic dysplasia associated with ineffective hematopoiesis may be subtle and difficult to recognize and can commonly be mimicked by systemic conditions, such as infections, autoimmune disorders, nutritional deficiencies, toxic factors and non-hematological malignancies. However, myelodysplastic syndromes may truly coexist with other systemic diseases, which can be masked when the patient's symptoms are attributed exclusively to myelodysplastic syndromes without further investigation. To better illustrate this, we herein describe two cases associated with synchronous gastric cancers.

OBJECTIVES: To evaluate whether risk scores used to classify patients with primary myelofibrosis and JAK-2 V617F mutation status can predict clinical outcome. METHODS: A review of clinical and laboratory data from 74 patients with primary myelofibrosis diagnosed between 1992 and 2011. The IPSS and Lille scores were calculated for risk stratification and correlated with overall survival. RESULTS: A V617F JAK2 mutation was detected in 32 cases (47%), with no significant correlation with overall survival. The patients were classified according to the scores: Lille - low, 53 (73.%); intermediate, 13 (18%); and high, 5 (7%); and IPSS- low, 15 (26%); intermediate-1, 23 (32%); intermediate-2, 19 (26%); and high, 15 (31%). Those patients presenting a higher risk according to the IPSS (high and intermediate-2) had a significantly shorter overall survival relative to the low risk groups (intermediate-1 and low) (p = 0.02). CONCLUSIONS: These results emphasize the importance of the IPSS prognostic score for risk assessment in predicting the clinical outcome of primary myelofibrosis patients.

OBJECTIVE: To evaluate the use of high-dose sequential chemotherapy in a Brazilian population. METHODS: High-dose cyclophosphamide followed by autologous hematopoietic stem cell transplantation is an effective and feasible therapy for refractory/relapsed lymphomas; this regimen has never before been evaluated in a Brazilian population. All patients (106 with high-grade non-Hodgkin lymphoma and 77 with Hodgkin's lymphoma) submitted to this treatment between 1998 and 2006 were analyzed. Chemotherapy consisted of the sequential administration of high-dose cyclophosphamide (4 or 7 g/m²) and granulocyte-colony stimulating factor (300 µg/day), followed by peripheral blood progenitor cell harvesting, administration of etoposide (2g/m²) and methotrexate (8 g/m² only for Hodgkin's lymphoma) and autologous hematopoietic stem cell transplantation. RESULTS: At diagnosis, non-Hodgkin lymphoma patients had a median age of 45 (range: 8-65) years old, 78% had diffuse large B-cell lymphoma and 83% had stage III/IV disease. The Hodgkin's lymphoma patients had a median age of 23 (range: 7-68) years old, 64.9% had the nodular sclerosis subtype and 65% had stage III/IV disease. Nine Hodgkin's lymphoma patients (13%) and 10 (9%) non-Hodgkin lymphoma patients had some kind of cardiac toxicity. The overall survival, disease-free survival and progression-free survival in Hodgkin's lymphoma were 29%, 59% and 26%, respectively. In non-Hodgkin lymphoma, these values were 40%, 49% and 31%, respectively. High-dose cyclophosphamide-related mortality was 10% for Hodgkin's lymphoma and 5% for non-Hodgkin lymphoma patients. High-dose cyclophosphamide dosing had no impact on toxicity or survival for both groups. CONCLUSIONS: Despite a greater prevalence of poor prognostic factors, our results are comparable to the literature. The incidence of secondary neoplasias is noteworthy. Our study suggests that this approach is efficient and feasible, regardless of toxicity-related mortality.

BACKGROUND: Real time PCR has become the most common technique to monitor BCR-ABL transcript levels of patients treated with kinase inhibitors. The aim of this study was to evaluate BCR-ABL levels of chronic myeloid leukemia patients treated with imatinib in the chronic phase and correlate the response to therapy and event-free survival. METHODS: BCR-ABL levels were measured in peripheral blood cell samples using Real time PCR at diagnosis and then every 3 months after starting therapy with imatinib. Major molecular response was defined as a three-log reduction from the standardized baseline value. Major molecular response values were adjusted to international scale using a conversion factor of 1.19. The results are reported as a BCR-ABL/ABL ratio (%). RESULTS: Hematological, major cytogenetic and complete cytogenetic responses were achieved by 57 (95%), 45 (75%) and 38 (63%) patients, respectively. Twenty-four out of sixty patients achieved a major molecular response (40%) in a median time of 8.5 months. Overall survival and event free survival were higher for patients with (100%) versus patients without (77%) a complete cytogenetic response (p-value = 0.01) at 48 months. Patients with complete cytogenetic response and major molecular response had a higher event free survival compared to patients with complete cytogenetic response but without major molecular response (p-value = 0.007). CONCLUSION: In conclusion, the prognostic impact of achieving complete cytogenetic response and a major molecular response and also the importance of molecular monitoring in the follow-up of chronic myeloid leukemia patients were demonstrated.