MAPK/ERK and PI3K/AKT signaling pathways are activated in adolescent and adult acute lymphoblastic leukemia.

BACKGROUND: The mitogen-activated protein kinase (MAPK)/ERK signaling cascade and the phosphoinosytol-3 phosphate/Akt (PI3K/Akt) pathways are involved in proliferation and differentiation of hematopoietic cells. The frequency of PI3K/Akt and MAPK pathway activation in adult acute lymphoblastic leukemia (ALL) still need to be elucidated. AIMS: To assess the activity and prognostic implications of MAPK/ERK and PI3K/Akt pathways in adult (ALL). METHODS: We examined 28 precursor-B-cell ALL and 6 T-cell primary ALL samples. Flow cytometry was employed to analyze the expression levels of phosphorylated ERK and phosphorylated Akt. RESULTS: Ten out of 15 (67%) ALL fresh samples (7 B-cell, 3 T-cell) showed constitutive p-ERK expression. The p-ERK mean fluorescent index ratio (MFI (R)) showed a tendency to be higher in ALL than in normal T lymphocytes (1.26 [0.74-3.10] vs. 1.08 [1.02-1.21], respectively [p = .069]) and was significantly lower than in leukemic cell lines (median MFI (R) 3.83 [3.71-5.97] [p < .001]). Expression of p-Akt was found in 35% (12/34) (10 B-cell, 2 T-cell). The median MFI (R) expression for p-Akt in primary blast cell was 1.13 (0.48-9.90) compared to 1.01 (1.00-1.20) in normal T lymphocytes (p = ns) and lower than in leukemic cell lines (median MFI (R) 2.10 [1.77-3.40] [p = .037]). Moreover, expression of p-ERK was negatively associated with the expression of CD34 (1.22 [0.74-1.33] vs. 1.52 [1.15-3.10] for CD34(+) and CD34(-) group, respectively, p = .009). CONCLUSION: Our findings suggest that both MAPK/ERK and PI3K/Akt are constitutively activated in adult ALL, indicating a targeted therapy potential for ALL by using inhibitors of these pathways.

Detection of somatic TP53 mutations and 17p deletions in patients with chronic lymphocytic leukemia: a review of the current methods

ABSTRACT Chronic lymphocytic leukemia is the most common hematologic malignancy among adults in Western countries. Several studies show that somatic mutations in the TP53 gene are present in up to 50% of patients with relapsed or refractory chronic lymphocytic leukemia. This study aims to review and compare the methods used to detect somatic TP53 mutations and/or 17p deletions and analyze their importance in the chronic lymphocytic leukemia diagnosis and follow-up. In chronic lymphocytic leukemia patients with refractory or recurrent disease, the probability of clonal expansion of cells with the TP53 mutation and/or 17p deletion is very high. The studies assessed showed several methodologies able to detect these changes. For the 17p deletion, the chromosome G-banding (karyotype) and interphase fluorescence in situ hybridization are the most sensitive. For somatic mutations involving the TP53 gene, moderate or high-coverage read next-generation sequencing and Sanger sequencing are the most recommended ones. The TP53 gene mutations represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia. Patients carrying low-proportion TP53 mutation (less than 20-25% of all alleles) remain a challenge to these tests. Thus, for any of the methods employed, it is essential that the laboratory conduct its analytical validation, documenting its accuracy, precision and sensitivity/limit of detection.

Detection of somatic TP53 mutations and 17p deletions in patients with chronic lymphocytic leukemia: a review of the current methods.

Chronic lymphocytic leukemia is the most common hematologic malignancy among adults in Western countries. Several studies show that somatic mutations in the TP53 gene are present in up to 50% of patients with relapsed or refractory chronic lymphocytic leukemia. This study aims to review and compare the methods used to detect somatic TP53 mutations and/or 17p deletions and analyze their importance in the chronic lymphocytic leukemia diagnosis and follow-up. In chronic lymphocytic leukemia patients with refractory or recurrent disease, the probability of clonal expansion of cells with the TP53 mutation and/or 17p deletion is very high. The studies assessed showed several methodologies able to detect these changes. For the 17p deletion, the chromosome G-banding (karyotype) and interphase fluorescence in situ hybridization are the most sensitive. For somatic mutations involving the TP53 gene, moderate or high-coverage read next-generation sequencing and Sanger sequencing are the most recommended ones. The TP53 gene mutations represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia. Patients carrying low-proportion TP53 mutation (less than 20-25% of all alleles) remain a challenge to these tests. Thus, for any of the methods employed, it is essential that the laboratory conduct its analytical validation, documenting its accuracy, precision and sensitivity/limit of detection.

Increase of IRF-1 gene expression and impairment of T regulatory cells suppression activity on patients with myelodysplastic syndrome: A longitudinal one-year study.

Studies have demonstrated that abnormalities in interferon regulatory factor-1 (IRF-1) expression might develop myelodysplastic syndromes (MDS). IRF-1 was described as modulator of T regulatory (Treg) cells by suppressing Foxp3 on mice. We aimed to determine the role of Treg and IRF-1 in MDS. Thirty-eight MDS patients fulfilling WHO criteria and classified according to risk scores were evaluated at time 0 (T0) and after 12 months (T12) for: Treg suppression activity in coculture with T effector (Teff) cells; IRF-1 and Foxp3 genetic expression by qRT-PCR; IL-2, -4, -6, -10, -17, TNFα and IFNγ production by Cytometric Bead Array. No differences in Foxp3 expression (T0=0.06±0.06 vs T12=0.06±0.12, p=0.5), Treg number (T0=5.62±2.84×105 vs T12=4.87±2.62×105; p=0.3) and Teff percentage (T0=16.8±9.56% vs T12=13.1±6.3%; p=0.06) were observed on T12. Low risk MDS patients showed a higher number of Treg (5.2±2.6×105) versus high risk group (2.6±1.2×105, p=0.03). Treg suppression activity was impaired on T0 and T12.Cytokine production and IRF-1 expression were increased on T12. The correlation between IRF-1 and FoxP3 was negative (r2=0.317, p=0.045) on T0. These results suggest a hyper activity of the immune system, probably secondary to Treg suppression activity impairment. This state may induce the loss of tolerance culminating in the proliferation of MDS clones.

Single-nucleotide polymorphism-array improves detection rate of genomic alterations in core-binding factor leukemia.

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.

Combined flow cytometric assessment of CD45, HLA-DR, CD34, and CD117 expression is a useful approach for reliable quantification of blast cells in myelodysplastic syndromes.

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.

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes.

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.

Caracterização imunofenotípica das células plasmáticas em pacientes portadores de mieloma múltiplo / Immunophenotypic characterization of plasma cells in patients with multiple myeloma

INTRODUÇÃO E OBJETIVO: O mieloma múltiplo (MM) é uma doença maligna incurável caracterizada pela proliferação de um único clone de plasmócitos na medula óssea. O objetivo deste estudo foi avaliar a frequência e o valor prognóstico da expressão dos fenótipos aberrantes em pacientes com MM por meio de citometria de fluxo multiparamétrica. MÉTODOS: O estudo foi realizado no Hospital São Paulo/Disciplina de Hematologia e Hemoterapia da Universidade Federal de São Paulo (UNIFESP), sendo analisados de maneira prospectiva 30 pacientes portadores de MM ao diagnóstico. Na tentativa de identificar as células mielomatosas por citometria de fluxo (FACScalibur, BD), foram utilizados anticorpos monoclonais anti-CD138, anti-CD38, anti-CD45 específicos para seleção dos plasmócitos. O grupo controle consistiu em quatro doadores saudáveis de medula óssea. RESULTADOS: Todos os plasmócitos mielomatosos expressaram pelo menos um fenótipo aberrante, e CD56+++, CD117++, CD33++, CD13++, CD28++ foram os marcadores mais frequentes, observados em 88 por cento dos pacientes. Os marcadores linfoides foram encontrados nos casos com maior número de fenótipos aberrantes. DISCUSSÃO: Os antígenos CD56+++ e CD28++ apresentaram altos níveis de β2-microglobulina, sendo estes associados a estágios mais agressivos da doença e maior massa tumoral. A ausência da molécula de adesão CD56 foi relacionada com altos níveis de β2M e de cálcio iônico, mostrando que este achado pode ter valor prognóstico. CONCLUSÃO: A partir deste estudo concluiu-se que os fenótipos aberrantes estão presentes na maioria dos casos de MM e que a imunofenotipagem por citometria de fluxo multiparamétrica é uma boa ferramenta para distinguir células plasmáticas normais dos plasmócitos mielomatosos.

INTRODUCTION AND OBJECTIVE: Multiple myeloma is an incurable malignancy characterized by the proliferation of a single clone of plasma cells in bone marrow. The aim of this study was to evaluate the frequency and prognostic value of the expression of aberrant phenotypes in patients with multiple myeloma by multiparametric flow cytometry. METHODS: The study was carried out at Department of Hematology and Hemotherapy of Federal University of São Paulo and 30 patients with MM were analyzed prospectively. In an attempt to identify myeloma cells by flow cytometry (FACSCalibur, BD), specific monoclonal antibodies anti-CD138, anti-CD38 and anti-CD45 were used for the selection of plasma cells. The control group comprised four healthy bone marrow donors. RESULTS: All myeloma plasma cells expressed at least one aberrant phenotype and CD56+++, CD117++, CD33++, CD13++ and CD28++ markers were more frequently observed in 88 percent of patients. Lymphoid markers were found in cases with a higher number of aberrant phenotypes. DISCUSSION: CD56+++ and CD28++ antigens showed high levels of β2-microglobulin, which are associated with more aggressive stages of the disease and larger tumor mass. The absence of adhesion molecule CD56 was associated with high levels of β2M and calcium ion, showing that this finding may have prognostic value. CONCLUSION: From this study it was concluded that the aberrant phenotypes are present in most cases of MM, and immunophenotyping by multiparametric flow cytometry is a useful tool to distinguish normal plasma cells from myeloma plasma cells.

Neoplasias mieloproliferativas: revisão dos critérios diagnósticos e dos aspectos clínicos / Myeloproliferative neoplasms: a review of diagnostic criteria and clinical aspects

As síndromes mieloproliferativas crônicas, atualmente denominadas neoplasias mieloproliferativas (NMP), de acordo com a 4ª. edição da classificação da Organização Mundial da Saúde (OMS), são doenças clonais de célula-tronco hematopoética, nas quais há a proliferação aumentada de uma ou mais das séries mieloides (granulocítica, eritrocítica, megacariocítica ou mastocítica) com maturação eficaz. A progressão de todas é caracterizada por fibrose medular ou transformação leucêmica. Pela classificação da OMS, as NMP incluem: leucemia mieloide crônica (LMC), policitemia vera (PV), mielofibrose idiopática crônica (MF), trombocitemia essencial (TE), leucemia neutrofílica crônica (LNC), leucemia eosinofílica crônica não especificada(LEC), mastocitose (M) e neoplasia mieloproliferativa inclassificável (NMI). É interessante notar que tanto a LMC (BCR/ABL1) como PV, MF e TE (JAK2 V617F e éxon 12, MPLW515L/K) e M (KITD816V) tiveram suas bases moleculares desvendadas e apresentam em comum a ativação constitutiva de tirosino-quinase graças às mutações adquiridas pela célula-tronco hematopoética. A mutação JAK2 V617F é observada em mais de 90 por cento dos casos de PV, mas também em cerca de 50 por cento-60 por cento das MF e TE, levando ao questionamento de como uma única lesão molecular desencadeia três manifestações clínicas diversas. Já há evidências de que eventos genéticos e epigenéticos adicionais contribuem para a patogênese, tais como MPLW515L e MPLW515K. No presente manuscrito são apresentados os aspectos clínicos, a fisiopatologia e os critérios diagnósticos das diferentes NMP.

Chronic myeloproliferative disorders, currently called myeloproliferative neoplasms (MPN), according to the 4th edition of the World Health Organization (WHO) classification are clonal diseases of hematopoietic stem cells, in which there is increased proliferation of the myeloid series (granulocytic, erythrocytic, megakaryocytic series or mast cells) with effective maturation. The progression of all is characterized by marrow fibrosis or leukemic transformation. According to the WHO classification, the MPNs include: chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myelofibrosis (IM), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia not otherwise categorized (CEL-NC), mastocytosis (M) and myeloproliferative neoplasm unclassifiable (MPNU). It is worth noting that the molecular basis of CML (BCR/ABL1), as well as PV,ET, IM (JAK2V617F and exon 12, MPL W515L/K) and M (KITD816V) have been identified and have, in common, constitutive activation of tyrosine kinase due to acquired hematopoietic stem cell defects. The JAK2V617F mutation is observed in around 90 percent of PV cases and in around 50-60 percent of IM and ET leading to the question why a single molecular lesion induces three different clinical manifestations. There is already evidence that additional genetic and epigenetic events contribute to the pathogenesis, including MPL W515L/K mutation. Some clinical aspects, the pathophysiology and diagnostic criteria of MPNs are presented in this paper.

Leucemia eosinofílica crônica com expressão do rearranjo FIPILI-PDGFRa-realto ce caso e revisão da literatura / Chronic easinophilic leukemia with a FIPILI-PDGFRa fuison - case report and literature review

A eosinofilia no sangue e em tecidos está habitualmente associada a condições alérgicas, infecciosas, inflamatórias, neoplásicas, endocrinológicas, uso de medicamentos e exposição a agentes tóxicos. No entanto, pode ocorrer proliferação eosinofílica primária, sem causa aparente ou por expansão clonal da célula-tronco hematopoética. As neoplasias mielo ou linfoproliferativas associadas a rearranjos gênicos como PDGFRα, PDGFRβ e FGFR1 constituem condições raras nas quais ocorre mieloproliferação crônica, alterações no sangue periférico e na medula óssea e lesão tecidual de diferentes órgãos, a partir da liberação de citocinas e fatores humorais pelos grânulos eosinofílicos. A presença do rearranjo PDGFRα relaciona-se comumente à leucemia eosinofílica crônica, com envolvimento de mastócitos e neutrófilos e, mais raramente, à leucemia mielóide aguda ou ao linfoma linfoblástico T, com eosinofilia. O rearranjo PDGFRα mais comum é aquele resultante da deleção intersticial no braço longo do cromossomo 4, que permite a formação de um neogene a partir da fusão dos genes FIP1L1 e PDGFRα. Este codifica uma tirosino-quinase constitutivamente ativa que é inibida pelo mesilato de imatinibe. Em 2002 foi relatado o uso bem sucedido de mesilato de imatinibe em baixas doses em um paciente com síndrome hipereosinofilica e, desde então, vem-se utilizando esta droga com respostas clínicas rápidas, completas e duradouras. Descrevemos um caso de LEC com expressão do rearranjo FIP1L1-PDGFRα.

Chronic eosinophilia is habitually associated with allergic, infectious, inflammatory, neoplastic and endocrine conditions and exposure to certain drugs and toxic agents. However, eosinophilic proliferation may be primary, without identifiable causes, or provoked by clonal hematopoietic stem cell proliferation. Gene fusions involving PDGFR-α, PDGFR-β, and FGFR1 predispose patients to rare conditions with chronic myeloproliferation or lymphoproliferation, alterations in peripheral blood and bone marrow and diffuse tissue injury due to the release of cytokines and humoral factors from eosinophilic granules. The presence of the PDGFR-α rearrangement is commonly related to chronic eosinophilic leukemia, with alterations in peripheral mastocytes and neutrophils, and rarely to acute myeloid leukemia or T lymphoblastic lymphoma with eosinophilia. The most prevalent PDGFR-α rearrangement is one resulting from an interstitial deletion in the long arm of chromosome 4, that allows the formation of a neogene from the fusion of the FIP1L1 and PDGFRα genes. This codes a constitutively active tyrosine kinase, which can be inhibited by imatinib mesylate. In 2002, the successful treatment of a patient using imatinib to treat hypereosinophilic syndrome was reported. Since then, this drug has been utilized with fast, complete and lasting clinical responses. Here we describe a case of chronic eosinophilic syndrome with expression of the FIP1L1-PDGFR-α rearrangement.

Expression of eight genes of nuclear factor-kappa B pathway in multiple myeloma using bone marrow aspirates obtained at diagnosis.

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.

Duas classes de mutação na evolução de policitemia vera para leucemia mielóide aguda / Two-hit model of leukemogenesis in the evolution of polycythemia vera to acute myeloid leukemia

Policitemia vera (PV) é doença mieloproliferativa crônica com risco de transformação para mielofibrose ou para leucemia mielóide aguda (LMA) na evolução da doença. Dez a 25 por cento dos pacientes têm anormalidade citogenética ao diagnóstico, chegando a 50 por cento com a progressão. Relatamos caso de PV com transformação para LMA no qual foi possível demonstrar a teoria de duas classes de mutação: uma indutora de proliferação e outra de bloqueio de maturação. Caso: Paciente feminina, 55 anos, PV diagnosticada em 2002, com mutação JAK2V617F, cariótipo normal, tratada com flebotomias e hidroxiureia. Em 2006 houve progressão para mielofibrose pós-policitêmica e novo cariótipo mostrou 46,XX,del(20)(q13.1) em 4/20 metáfases. Análise por FISH para região 20q13 em amostra estocada confirmou a deleção ao diagnóstico. Após um ano houve transformação para LMA. A mutação JAK2V617F é suficiente para causar proliferação de precursores eritropoéticos, sendo o mecanismo fisiopatogênico primário na PV. Entretanto, a evolução da doença é heterogênea, sugerindo a ocorrência de fenômenos adicionais, levando à instabilidade genômica e contribuindo para a leucemogênese. O caso apresentado sustenta o modelo de dois passos na progressão da PV para LMA, no qual uma classe de mutação gênica confere vantagem proliferativa (JAK2V617F) e outra classe bloqueia a diferenciação hematopoética (deleção 20q). Evidenciaram-se nessa paciente dois eventos contribuindo para a proliferação e para o bloqueio de maturação. Outros mecanismos devem estar implicados e estudos prospectivos devem ser encorajados na tentativa de elucidação dos diferentes passos envolvidos na leucemogênese.

Polycythemia vera (PV) is a chronic myeloproliferative disorder that can evolve to marrow fibrosis or acute leukemia (AML). Cytogenetic alterations can be detected in around 25 percent of patients at diagnosis and in up to 50 percent of those with progression. We report a case of PV with evolution to AML in which it was possible to demonstrate the two-hit model of leukemogenesis: one mutation confers proliferative advantage and another interferes with differentiation. Case: A 55-year-old female patient was diagnosed with PV in 2002 and treated with phlebotomies and hydroxyurea. In 2006, there was progression topost-polycythemic fibrosis with AML one year later. She presented the JAK2V617F mutation. The result of karyotyping performed at diagnosis was normal and at transformation, 46,XX,del(20)(q13.1) was detected in 4/20 metaphases. FISH analysis of a stored sample for 20q13 showed the deletion in 20 percent of interphases confirming the earlier presence of a clonal abnormality that was not detected by karyotyping. The JAK2V617F mutation is sufficient to cause proliferation of hematopoietic cells and has been established as a primary pathogenetic mechanism in PV. However, the evolution of the disease is heterogeneous, suggesting the occurrence of additional phenomena contributing to leukemogenesis. This case demonstrates the two-hit model in the progression of PV to LMA, in which a class of mutation induces proliferative advantage and another blocks differentiation. Two events which contribute to proliferation and to maturation blockade were detected in this patient. Other mechanisms may be implicated and prospective studies should be encouraged in an attempt to elucidate the different steps involved in leukemogenesis.

SAGE analysis highlights the importance of p53csv, ddx5, mapkapk2 and ranbp2 to multiple myeloma tumorigenesis.

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.