Lenalidomide Maintenance and Measurable Residual Disease in a Real-World Multiple Myeloma Transplanted Population Receiving Different Treatment Strategies Guided by Access to Novel Drugs in Brazil.

Despite recent advances in multiple myeloma (MM), the incorporation of novel agents and measurable residual disease (MRD) monitoring in low-income countries remains a challenge. Although lenalidomide maintenance (M-Len) after autologous stem cell transplantation (ASCT) has been associated with improved outcomes and MRD has refined the prognosis of complete response (CR) cases, until now, there have been no data on the benefits of these approaches in Latin America. Here, we evaluate the benefits of M-Len and MRD using next-generation flow cytometry (NGF-MRD) at Day + 100 post-ASCT (n = 53). After ASCT, responses were evaluated based on the International Myeloma Working Group criteria and NGF-MRD. MRD was positive in 60% of patients with a median progression-free survival (PFS) of 31 months vs. not reached (NR) for MRD-negative cases (p = 0.05). The patients who received M-Len continuously had a significantly better PFS and overall survival (OS) than those without M-Len (median PFS: NR vs. 29 months, p = 0.007), with progression in 11% vs. 54% of cases after a median follow-up of 34 months, respectively. In a multivariate analysis, MRD status and M-Len therapy emerged as independent predictors of PFS (median PFS of M-Len/MRD- vs. no M-Len/MRD+ of NR vs. 35 months, respectively; p = 0.01). In summary, M-Len was associated with improved survival outcomes in our real-world MM cohort in Brazil, with MRD emerging as a useful reproducible tool to identify patients at an earlier risk of relapse. The inequity in drug access remains a hurdle in countries with financial constraints, with a negative impact on MM survival.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels

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.

Minimal residual disease assessment in acute lymphoblastic leukemia by 4-color flow cytometry: Recommendations from the MRD Working Group of the Brazilian Society of Bone Marrow Transplantation

Abstract Introduction: The minimal residual disease (MRD) status plays a crucial role in the treatment of acute lymphoblastic leukemia (ALL) and is currently used in most therapeutic protocols to guide the appropriate therapeutic decision. Therefore, it is imperative that laboratories offer accurate and reliable results through well standardized technical processes by establishing rigorous operating procedures. Method: Our goal is to propose a monoclonal antibody (MoAb) panel for MRD detection in ALL and provide recommendations intended for flow cytometry laboratories that work on 4-color flow cytometry platforms. Results and conclusion: The document includes pre-analytical and analytical procedures, quality control assurance, technical procedures, as well as the information that needs to be included in the reports for clinicians.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels.

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 eight- to 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.

Minimal residual disease assessment in acute lymphoblastic leukemia by 4-color flow cytometry: Recommendations from the MRD Working Group of the Brazilian Society of Bone Marrow Transplantation.

INTRODUCTION: The minimal residual disease (MRD) status plays a crucial role in the treatment of acute lymphoblastic leukemia (ALL) and is currently used in most therapeutic protocols to guide the appropriate therapeutic decision. Therefore, it is imperative that laboratories offer accurate and reliable results through well standardized technical processes by establishing rigorous operating procedures. METHOD: Our goal is to propose a monoclonal antibody (MoAb) panel for MRD detection in ALL and provide recommendations intended for flow cytometry laboratories that work on 4-color flow cytometry platforms. RESULTS AND CONCLUSION: The document includes pre-analytical and analytical procedures, quality control assurance, technical procedures, as well as the information that needs to be included in the reports for clinicians.

Multidisciplinary diagnostics of chronic lymphocytic leukemia: European Research Initiative on CLL - ERIC recommendations

ABSTRACT Recent advances in chronic lymphocytic leukemia (CLL) includes description of disease genomic landscape, inclusion of prognostic relevant genetic tests in CLL workflow and evaluation of minimal residual disease (MRD)1 in parallel with the increase availability of novel therapy agents.In this review, the theoretical and practical aspects of response assessment have been discussed. These are based on updated recommendations of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) for genetic tests (TP53 mutation and IGHV status) and flow cytometry analysis for CLL. Methodological approaches and interpretation of results were also discussed.2,3

Multidisciplinary diagnostics of chronic lymphocytic leukemia: European Research Initiative on CLL - ERIC recommendations.

Recent advances in chronic lymphocytic leukemia (CLL) includes description of disease genomic landscape, inclusion of prognostic relevant genetic tests in CLL workflow and evaluation of minimal residual disease (MRD)1 in parallel with the increase availability of novel therapy agents. In this review, the theoretical and practical aspects of response assessment have been discussed. These are based on updated recommendations of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) for genetic tests (TP53 mutation and IGHV status) and flow cytometry analysis for CLL. Methodological approaches and interpretation of results were also discussed.2,3.

First proposed panels on acute leukemia for four-color immunophenotyping by flow cytometry from the Brazilian group of flow cytometry-GBCFLUX.

Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2).

"First proposed panels on acute leukemia for four-color immunophenotyping by flow cytometry from the Brazilian Group of Flow Cytometry - GBCFLUX"

Multiparameter flow cytometry (MFC) is a highly sensitive, fast and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an inter-laboratory study to evaluate its effectiveness on the diagnosis and classification of AL. © 2014 Clinical Cytometry Society.

Trisomy 11 as an additional chromosome alteration in a child with acute promyelocytic leukemia with poor prognosis.

The prognostic significance of the additional abnormalities to the t(15; 17) remains controversial. We report a case of promyelocytic leukemia (APL) in a ten-year-old boy. Classical and molecular cytogenetic (FISH) studies of a bone marrow sample obtained at diagnosis revealed the presence of trisomy of chromosome 11 as an additional chromosomal abnormality to the t(15; 17). The presence of the translocation t(15; 17), the cytogenetic marker of APL, is usually associated with good response to treatment with ATRA. In this case, although the patient had risk factors associated with good prognosis, he evolved and died quickly. So it seems that the presence of the trisomy 11 may be associated with disease progression and the poor outcome. To our knowledge, this is the first reported case of t(15; 17) associated with trisomy of chromosome 11 in a child with APL.

JAK2V617F mutation in patients with splanchnic vein thrombosis.

BACKGROUND: Splanchnic vein thrombosis can be the presenting manifestation of myeloproliferative neoplasms. However, the diagnosis of a myeloproliferative neoplasm in these patients is often problematic, and more objective criteria are needed. AIM: To determine the frequency of the mutation JAK2V617F in patients with splanchnic vein thromboses. METHODS: A consecutive series of 108 adult patients with portal vein thrombosis (n = 77) and Budd-Chiari syndrome (n = 31) referred for hemostasis evaluation was retrospectively studied, with a median follow-up of 51 months (1-104). RESULTS: One or more prothrombotic risk factors were present in 63% of the patients. Twenty-four (22%) out of the 108 patients presented the JAK2V617F, including 2 cirrhotic patients. Most had a low mutated allele burden (median 16.5%). JAK2V617F was present in all four patients with a previous diagnosis of a myeloproliferative neoplasm. In nine JAK2V617F-positive patients, the diagnosis of a myeloproliferative neoplasm was made at the thrombosis work-up, during follow-up or after JAK2V617F detection. Among the other 11 patients carrying the mutation, 2 patients have died, 4 had no evidence suggesting a myeloproliferative neoplasm, 1 had a normal bone marrow biopsy, and the other 4 could not be persuaded to undergo a biopsy. Among the patients without an overt myeloproliferative neoplasm, 15 out of 99 (15%) presented the JAK2V617F mutation. None of the JAK2V617F-negative patients have developed signs of a myeloproliferative neoplasm during follow-up. CONCLUSIONS: Our findings suggest that JAK2V617F occurs in a high proportion of patients with splanchnic vein thrombosis, and reinforces the diagnostic utility of JAK2V617F testing in this setting.

HLM/OSBP2 is expressed in chronic myeloid leukemia.

Oxysterols are oxygenated derivatives of cholesterol that have been shown to influence a wide variety of cellular processes including sterol metabolism, lipid trafficking, apoptosis and more recently, cell differentiation. The oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes with high affinity for oxysterols, but their precise function has not been defined yet. One member of this family in humans, HLM/OSBP2 protein, has recently been reported as a potential marker for solid tumor dissemination and worse prognosis in these cases. In this study we focused on the evaluation of HLM/OSBP2 expression in malignant cell lines from different origins (blood and solid tumors) and we also evaluated its expression in chronic myeloid leukemia patients, correlating the molecular findings with clinical outcome. Our results showed that HLM/OSBP2 was expressed in 80% of the analysed CML patients, suggesting that this protein could constitute a helpful tool for disease monitoring and reinforces recent findings that HLM/OSBP2 protein could be involved in the maintenance of the undifferentiated state necessary for leukemogenesis.

Pathological and virological assessment of acute HTLV-I-associated myelopathy complicated with encephalopathy and systemic inflammation.

HTLV-I-associated myelopathy, also known as tropical spastic paraparesis (HAM/TSP), is a chronic inflammatory disease of the spinal cord. Acute cases are uncommon. We report the case of a 41-year-old woman with acute HAM/TSP complicated with encephalitis, an intense inflammatory reaction of the nervous system and lymphocytic infiltration of skeletal muscles, liver, salivary, adrenal and pituitary glands. The immunohistochemical studies of the lymphocytes surrounding blood vessels showed both B- and T-lymphocytes, in similar proportion, with both CD4- and CD8-positive cells. In addition, many perivascular and scattered macrophages were observed. Adult T-cell leukemia/lymphoma (ATL) was ruled out. The marrow aspirate was normal. Serial cerebrospinal fluid (CSF) analysis showed presence of HTLV-I antibodies, but without intrathecal synthesis of specific antibodies. Determination of HTLV-I viral loads demonstrated increased levels in the CSF relative to the peripheral blood and may be associated with widespread inflammation. The pathological and immunological findings may help understand the role of immune-reactive cells in the pathogenesis of HTLV-I-associated myelopathy.

Prevalence of hepatitis C infection in patients with non-Hodgkin's lymphomas.

Several studies have suggested an association between hepatitis C virus (HCV) and low-grade B-cell non-Hodgkin's lymphomas. The results, however, have been controversial. Italian and Japanese studies have reported a 40% prevalence rate, but the data were not confirmed by English and Canadian studies. We evaluated the prevalence of HCV infection in 109 patients with non-Hodgkin's lymphomas, and compared it with a control group composed of 67 patients with Hodgkin's disease and 31 patients with chronic lymphocytic leukemia. The prevalence of HCV infection was also determined in blood donors. HCV infection was detected using second and third generation anti-HCV ELISA. Positive results were additionally confirmed using Inno-LIA AbIII and/or RNA-HCV by PCR. Immunohistochemical stains were used to determine B or T cell lineage when the morphological analysis was not sufficient for lymphoma classification. HCV infection was detected in 9% of patients with non-Hodgkin's lymphomas, in 2% of patients in the control group (p=0.036), and in 1.2% of blood donors. There was no difference in the prevalence of HCV infection between patients with B or T cell lymphomas. Blood transfusions or previous surgeries, both risk factors for HCV infection, were detected in 90% of the patients with a positive anti-HCV test, in average 17 and 36 years before the diagnosis of lymphoma, respectively. Seventy percent of the patients with non-Hodgkin's lymphomas and a positive anti-HCV test presented evidence of chronic liver disease when the lymphoma was diagnosed. This study suggests the presence of an association between HCV infection and non-Hodgkin's lymphomas in Brazil.

Influência da beta-propiolactona em testes laboratoriais hematológicos / Beta-propiolactone influence on haematological laboratory tests

Foram analisados os efeitos da beta-propiolactona em testes hematológicos. A incubaçäo do sangue com este agente resultou numa significante diminuiçäo da VHS e num aumento da concentraçäo da hemoglobina globular média e da contagem dos bastöes. A realizaçäo dos esfregaços logo após a adiçäo da droga evita este artefato. Os demais parâmetros permaneceram inalterados