Diagnosis and treatment of chronic lymphocytic leukemia: recommendations from the Brazilian Group of Chronic Lymphocytic Leukemia

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.

Diversity of breakpoints of variant Philadelphia chromosomes in chronic myeloid leukemia in Brazilian patients

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 regionAbelson 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 fre- quently involved in such rare rearrangements calls attention to possible predisposition that should be further studied. Nevertheless, the pathological implication of these variants remains unclear. .

Identifying the similarities and differences between single nucleotide polymorphism array (SNPa) analysis and karyotyping in acute myeloid leukemia and myelodysplastic syndromes

Objective: To standardize the single nucleotide polymorphism array (SNPa) method in acute myeloid leukemia/myelodysplastic syndromes, and to identify the similarities and differ- ences 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(r) 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 abnor- mal (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. .

Increased angiogenesis in primary myelofibrosis: latent transforming growth factor-β as a possible angiogenic factor

Objective: The aim of this work was to demonstrate a possible relationship between anti-latency-associated peptide human latent transforming growth factor beta 1 (latent TGF-β1) expression in megakaryocytes and microvascular density in bone marrow biopsies from patients with essential thrombocythemia and primary myelofibrosis. Methods: Microvascular density was evaluated by immunohistochemical analysis and the expression of latent TGF-β1 in samples (100 megakaryocytes per bone marrow sample) from 18 essential thrombocythemia and 38 primary myelofibrosis (19 prefibrotic and 19 fibrotic) patients. Six bone marrow donor biopsies were used as controls. Fibrosis in the bone marrow biopsies was evaluated according to the European Consensus. Results: The average fibrosis grade differed between essential thrombocythemia and primary myelofibrosis groups when compared to the control group. Latent TGF-β1 expression differed significantly between the fibrotic primary myelofibrosis (PMF) group and the control group (p-value < 0.01). A high degree of neo-angiogenesis (demonstrated by analysis of CD34 expression) was detected in patients with myelofibrosis. There were correlations between latent TGF-β1 expression and microvascular density (r = 0.45; p-value < 0.0009) and between degree of microvascular density and fibrosis grade (r = 0.80; p-value < 0.0001). Remarkable differences for neo-angiogenesis were not observed between patients with essential thrombocythemia and controls. Conclusion: Angiogenesis participates in the pathogenesis of primary myelofibrosis, in both the prefibrotic and fibrotic stages, while latent TGF-β is differentially expressed only in the prefibrotic stage...

Philadelphia-negative chronic myeloproliferative neoplasms

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.

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia

BACKGROUND: The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. OBJECTIVE: 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 percent) and primary myelofibrosis cases (17.6 percent), 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 percent), primary myelofibrosis (42.8 percent) and essential thrombocythemia (47 percent). 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 ...

Eosinofilia reacional, leucemia eosinofílica crônica e síndrome hipereosinofílica idiopática / Reactive eosinophilia, chronic eosinophilic leukemia and idiopathic hypereosinophilic syndrome

A eosinofilia é freqüente na prática clínica, principalmente quando os valores estão entre 500 e 1000 eosinófilos/uL e indica a presença de doença parasitária, alérgica ou reação a medicamentos. Afora essas situações, a eosinofilia pode ser devida a doenças do tecido conjuntivo, infecções e, mais raramente, a doença hematológica maligna ou a tumores sólidos. Os critérios estabelecidos na década de 70 para a definição para a definição da síndrome hipereosinofílica idiopática se tornaram insuficientes para caracterizar todas as entidades albergadas sob o termo eosinofilia e, hoje, melhor compreendidas graças aos avanços na biologia celular e molecular, que proporcionaram a caracterização de doenças distintas e que envolvem células das linhagens mieloide e linfoide. Nesse contexto, as eosinofilias sanguíneas são categorizadas como reacionais, clonais e idiopáticas (SHE). O advento de terapia antitirosinoquinase (a exemplo do mesilato de imatinibe), eficaz para os casos com o rearranjo gênico FIP1L1/PDGFR, também abriu novas perspectivas para o controle ideal da leucemia eosinofílica crônica. Daí a importância do diagnóstico preciso e rápido para a indicação terapêutica ideal, antes que se instalem as complicações orgânicas, em especial cardíacas, que são irreversíveis. O presente manuscrito objetiva rever as situações de eosinofilia sanguínea e oferecer uma atualização da investigação diagnóstica e terapêutica.

Mild eosinophilia with values of less than 1000 eosinophils/µL is commonly seen in the clinical practice and can be secondary to parasitic, inflammatory or allergic diseases or to drug reactions. Additionally, eosinophilia may be due to connective tissue disorders, infections and occasionally to hematopoietic malignancies or solid tumors. The criteria established in the 1970s, for the definition of idiopathic hypereosinophilic syndrome is today unsatisfactory to characterize all conditions described as eosinophilia. Now these conditions are better understood due to the evolution of cellular and molecular biology. This knowledge has helped to characterize distinct disorders involving myeloid and lymphoid lineages. Hence, eosinophilia is categorized as reactive, clonal or idiopathic. With the introduction of anti-tyrosine kinase (imatinib mesylate) therapy, which is effective for the FIP1L1/PDGFRa rearrangement, there is a possibility to control or cure chronic eosinophilic leukemia. For this reason, precise and fast diagnosis is necessary for ideal therapeutic decisions before organic lesions that are irreversible, such as heart injury, become established. The aim of this manuscript is to review eosinophilia and offer an update on diagnostic and therapeutic investigations.

Leucemia mieloide crônica: tratamento baseado em evidências / Chronic myeloid leukemia: evidence-based treatment

A fisiopatologia da LMC se baseia na presença do cromossomo Philadelphia (Ph), uma anormalidade citogenética adquirida, que resulta na t(9;22)(q34;q11) e origina o rearranjo gênico BCR/ABL.Estudos investigativos conduzidos resultaram na fabricação de droga alvo-específica, o mesilato de imatinibe, que inibe a proteína BCR/ABL anômala.O imatinibe proporcionou resultados terapêuticos impressionantes, tornando-se a primeira opção para o tratamento da doença em fase crônica. A segunda geração de medicamentos antitirosina quinase se apresenta como opção importante para casos de falha, resistência ou intolerância.Assim, uma doença que na década de 60 levava infalivelmente o paciente ao óbito, em média, em quatro anos, apresenta no cenário atual possibilidade de cura ou promissor aumento de sobrevida para a imensa maioria dos casos.

Prevalence of FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD+) in de novo acute myeloid leukemia patients categorized according to cytogenetic risk / Prevalência de duplicação interna in tandem/fms-receptor tirosino-quinase (DIT/FLT3) em pacientes com leucemia mielóide aguda de novo classificados conforme grupos citogenéticos de risco

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 percent) were classified as presenting the favorable karyotype, 11 patients (27.5 percent) as an intermediate group, 7 patients (17 percent) as an undefined group and 9 patients (22.5 percent) as the unfavorable group. FLT3/ITD+ was found in 10 patients (25 percent): 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.

CONTEXTO E OBJETIVO: O mecanismo envolvido na leucemogênese permanece obscuro, e maiores informações a respeito das inadequadas proliferação, diferenciação e apoptose das células neoplásicas é fundamental. TIPO DE ESTUDO E LOCAL: Estudo transversal de prevalência na Disciplina de Hematologia e Hemoterapia, Hospital São Paulo, Universidade Federal de São Paulo. MÉTODOS: Nós pesquisamos a duplicação interna in tandem (DIT) do gene FLT3 (Fms-like tyrosine kinase) em 40 pacientes adultos com leucemia mielóide aguda (LMA) de novo, classificados de acordo com os resultados de cariótipo em banda G, de setembro de2001 a maio de 2005. RESULTADOS: Treze pacientes (32,5 por cento) foram classificados como cariótipo favorável, 11 pacientes (27,5 por cento) como grupo intermediário, 7 pacientes (17 por cento) no grupo de prognóstico indefinido e os restantes 9 pacientes (22,5 por cento) foram alocados como desfavorável. A DIT/FLT3 foi encontrada em 10 pacientes (25 por cento), 3 pacientes com DIT/FLT3 e cariótipo favorável, 4 com DIT/FLT3 e cariótipo intermediário, 2 com DIT/FLT3 e cariótipo de prognóstico indefinido e somente 1 paciente com DIT/FLT3 e cariótipo desfavorável. Entre os pacientes sem DIT/FLT3, 10 apresentaram cariótipo favorável, 8 com cariótipo intermediário, 4 com cariótipo de prognóstico indefinido e 8 com cariótipo desfavorável. Não houve correlação entre a presença de DIT/FLT3 e os grupos de prognóstico conforme resultados de citogenética (P = 0,13). No presente estudo encontramos 2 pacientes vivos por mais de 24 meses. A presença de DIT/FLT3 não influenciou a taxa de sobrevida dos pacientes. CONCLUSÃO: Nós observamos a mesma frequência de LMA com DIT/FLT3 tanto no grupo de cariótipo favorável quanto no grupo intermediário e somente um paciente com LMA e DIT/FLT3 e cariótipo desfavorável, hipoteticamente o pior achado clínico. Desta forma, a vantagem prognóstica do cariótipo favorável em pacientes DIT/FLT3 permanece a ser esclarecida ...

Williams Syndrome: development of a new scoring system for clinical diagnosis

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 themean 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...

OBJETIVOS: Desenvolver um sistema de pontuação (Score) baseado nos achados clínicos para auxiliar os pediatras no diagnóstico clínico da Síndrome de Williams-Beuren e na indicação do teste de hibridização in situ por fluorescência para detectar a microdeleção em 7q11.23. MÉTODOS: O teste de hibridização in situ por fluorescência foi feito em 20 acometidos pela Síndrome de Williams-Beuren, nos quais 42 achados clínicos foram estudados. Para estabelecer quais desses achados estariam associados ao teste de hibridização in situ por fluorescência positivo, realizou-se uma metanálise com 11 trabalhos da literatura em que havia dois grupos, hibridização in situ por fluorescência positivo e negativo. As freqüências dos achados presentes nos indivíduos fluorescência positivo e fluorescência negativo neste estudo foram comparadas em conjunto com os pacientes da literatura através do teste exato de Fisher. Elaboramos um sistema de pontuação (score) baseado nos achados que mostraram correlação significante (p<0,001) para os pacientes hibridização in situ por fluorescência positivo. Determinamos os valores correspondentes aos percentis baseados na média e desvio-padrão, calculados a partir dos 20 pacientes do presente trabalho. RESULTADOS: Dezessete pacientes foram hibridização in situ por fluorescência positivo e três, negativo. Os achados mais discriminativos nos hibridização in situ por fluorescência positivo (3 pontos no score) foram: fáceis típico, baixo peso ao nascimento, dificuldades alimentares, obstipação, estenose aórtica supravalvar, deficiência mental e personalidade amigável.A distribuição dos valores entre os 20 pacientes variou de 19 a 28 pontos com uma média de 23,3 pontos. CONCLUSÕES: O score elaborado permitiu propor o valor de 20 pontos para a indicação do teste de hibridização in situ por fluorescência nos pacientes com suspeita clínica de Síndrome de Williams-Beuren.

Pioderma gangrenoso bolhoso e síndrome mielodisplásica / Bullous pyoderma gangrenosum and myelodysplastic syndrome

O pioderma gangrenoso pode apresentar-se como manifestação paraneoplásica. Relata-se um caso de pioderma gangrenoso, da variante bolhosa, acompanhado de bicitopenia, em que foi evidenciado, por meio de mielograma, biópsia de medula óssea e cariótipo, padrão compatível com síndrome mielodisplásica, subtipo citopenia refratária com displasia de multilinhagens. Foi tratado com dapsona, obtendo cicatrização das lesões. O pioderma gangrenoso pode associar-se a doenças sistêmicas, devendo a síndrome mielodisplásica ser considerada nos casos acompanhados de citopenias. Portanto, o pioderma gangrenoso pode ser um marcador cutâneo de doença sistêmica de prognóstico reservado.

Pyoderma gangrenosum can present as a cutaneous manifestation of paraneoplastic syndromes. A case of bullous pyoderma gangrenosum associated with bicytopenia is described. During the complementary investigation, myelogram, bone marrow biopsy and karyotype were performed, and showed a pattern consistent with myelodysplastic syndrome. The patient was treated with dapsone with improvement. Pyoderma gangrenosum can be a manifestation of systemic diseases. The possibility of myelodysplastic syndrome should always be considered in patients with pyoderma gangrenosum associated with cytopenia. Pyoderma gangrenosum could indicate poorer prognosis in patients with systemic diseases.

Septic arthritis as the first sign of Candida tropicalis fungaemia in an acute lymphoid leukemia patient

Fungal infections caused by Candida species have increased in incidence during the past two decades in England, North America and Europe. Candidal arthritis is rare in patients who are not intravenous drug users or are who not using a prostheses. We report the case of a 24-year-old man with acute lymphoid leukemia, who developed Candida tropicalis arthritis during an aplastic period after chemotherapy. This is the eighth case described in the literature of C. tropicalis causing arthritis without intra-articular inoculation. We call attention to an unusual first sign of fungal infection: septic arthritis without intra-articular inoculation. However, this case differs from the other seven, since despite therapy a fast and lethal evolution was observed. We reviewed reported cases, incidence, risk factors, mortality and treatment of neutropenic patients with fungal infections.