Acute leukemias in Piauí: comparison with features observed in other regions of Brazil

Differences in age and sex distribution as well as FAB (French-American-British classification) types have been reported for acute leukemias in several countries. We studied the demographics and response to treatment of patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) between 1989 and 2000 in Teresina, Piauí, and compared these results with reports from Brazil and other countries. Complete data concerning 345 patients (230 ALL, 115 AML) were reviewed. AML occurred predominantly in adults (77%), with a median age of 34 years, similar to that found in the southeast of Brazil but lower than the median age in the United States and Europe (52 years). FAB distribution was similar in children and adults and FAB-M2 was the most common type, as also found in Japan. The high frequency of FAB-M3 described in most Brazilian studies and for Hispanics in the United States was not observed. Overall survival for adults was 40%, similar to other studies in Brazil. A high mortality rate was observed during induction. No clinical or hematological parameter influenced survival in the Cox model. ALL presented the characteristic peak of incidence between 2-8 years. Most of the cases were CD10+ pre-B ALL. In 25%, abnormal expression of myeloid antigens was observed. Only 10% of the patients were older than 30 years. Overall survival was better for children. Age and leukocyte count were independent prognostic factors. These data demonstrate that, although there are regional peculiarities, the application of standardized treatments and good supportive care make it possible to achieve results observed in other countries for the same chemotherapy protocols.33133

Monitoring Of Bcr-abl Levels In Chronic Myeloid Leukemia Patients Treated With Imatinib In The Chronic Phase - The Importance Of A Major Molecular Response

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.333211215Melo, J.V., The molecular biology of chronic myeloid leukaemia (1996) Leukemia, 10 (5), pp. 751-756Wang, L., Pearson, K., Pillitteri, L., Ferguson, J.E., Clark, R.E., Serial monitoring of BCR-ABL by peripheral blood real-time polymerase chain reaction predicts the marrow cytogenetic response to imatinib mesylate in chronic myeloid leukaemia (2002) Br J Haematol, 118 (3), pp. 771-777Muller, M.C., Gattermann, N., Lahaye, T., Deininger, M.W., Berndt, A., Fruehauf, S., Dynamics of BCR-ABL mRNA expression in firstline therapy of chronic myelogenous leukemia patients with imatinib or interferon alpha/ara-C (2003) Leukemia, 17 (12), pp. 2392-2400Branford, S., Hughes, T.P., Rudzki, Z., Monitoring chronic myeloid leukaemia therapy by real-time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics (1999) Br J Haematol, 107 (3), pp. 587-599Radich, J.P., Gooley, T., Bryant, E., Chauncey, T., Clift, R., Beppu, L., The significance of bcr-abl molecular detection in chronic myeloid leukemia patients late, 18 months or more after transplantation (2001) Blood, 98 (6), pp. 1701-1707Hughes, T.P., Kaeda, J., Branford, S., Rudzki, Z., Hochhaus, A., Hensley, M.L., Frequency of major molecular responses to imatinib or interferon-alpha plus cytarabine in newly diagnosed chronic myeloid leukemia (2003) N Engl J Med, 349 (15), pp. 1423-1432Press, R.D., Love, Z., Tronnes, A.A., Yang, R., Tran, T., Mongoue- tchokote, S., BCR-ABL mRNA levels at and after the time of a complete cytogenetic response predict the duration of CCR in imatinib mesylate-treated patients with CML (2006) Blood, 107 (11), pp. 4250-4256Cortes, J., Talpaz, M., O'Brien, S., Jones, D., Luthra, R., Shan, J., Molecular responses in patients with chronic myelogenous leukemia in chronic phase treated with imatinib mesylate (2005) Clin Cancer Res, 11 (9), pp. 3425-3432Iacobucci, I., Saglio, G., Rosti, G., Testoni, N., Pane, F., Amabile, M., Achieving a major molecular response at the time of a complete cytogenetic response (CCgR) predicts a better duration of CCgR in imatinib-treated chronic myeloid leukemia patients (2006) Clin Cancer Res, 12 (10), pp. 3037-3042Baccarani, M., Saglio, G., Goldman, J., Hochhaus, A., Simonsson, B., Appelbaum, F., Evolving concepts in the management of chronic myeloid leukemia: Recommendations from an expert panel on behalf of the European LeukemiaNet (2006) Blood, 108 (6), pp. 1809-1820Hughes, T., Deininger, M., Hochhaus, A., Branford, S., Radich, J., Kaeda, J., Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: Review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results (2006) Blood, 108 (1), pp. 28-37Branford, S., Cross, N.C., Hochhaus, A., Radich, J., Saglio, G., Kaeda, J., Rationale for the recommendations for harmonizing current methodology for detecting BCR-ABL transcripts in patients with chronic myeloid leukaemia (2006) Leukemia, 20 (11), pp. 1925-1930Cortes, J., Baccarani, M., Fea, G., (2008) A Phase III, Randomized, Openlabel Study of 400 Mg Versus 800 Mg of Imatinib Mesylate (IM) in Patients With Newly Diagnosed, Previously Untreated Chronic Myeloid Leukemia in Chronic Phase (CML-CP), Using Molecular Endpoints: One Year Results of TOPS (Tyrosine Kinase Inhibitor Optimization and Selectivity) Study, , 50th ASH Annual Meeting and Exposition Online program and Abstracts. San Francisco, CABranford, S., Fletcher, L., Cross, N.C., Muller, M.C., Hochhaus, A., Kim, D.W., Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials (2008) Blood, 112 (8), pp. 3330-3338O'Brien, S.G., Guilhot, F., Larson, R.A., Gathmann, I., Baccarani, M., Cervantes, F., Imatinib compared with interferon and lowdose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia (2003) N Engl J Med, 348 (11), pp. 994-1004Marin, D., Milojkovic, D., Olavarria, E., Khorashad, J.S., de Lavallade, H., Reid, A.G., European LeukemiaNet criteria for failure or suboptimal response reliably identify patients with CML in early chronic phase treated with imatinib whose eventual outcome is poor (2008) Blood, 112 (12), pp. 4437-444

Molecular characteristics and chromatin texture features in acute promyelocytic leukemia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Background: Acute promyelocytic leukemia is a cytogenetically well defined entity. Nevertheless, some features observed at diagnosis are related to a worse outcome of the patients. Methods: In a prospective study, we analyzed peripheral (PB) leukocyte count, immunophenotype, methylation status of CDKN2B, CDKN2A and TP73; FLT3 and NPM1 mutations besides nuclear chromatin texture characteristics of the leukemic cells. We also examined the relation of these features with patient's outcome. Results: Among 19 cases, 4 had a microgranular morphology, 7 presented PB leukocytes >10x10(9)/l, 2 had FLT3-ITD and 3 had FLT3-TKD (all three presenting a methylated CDKN2B). NPM1 mutation was not observed. PB leukocyte count showed an inverse relation with standard deviation of gray levels, contrast, cluster prominence, and chromatin fractal dimension (FD). Cases with FLT3-ITD presented a microgranular morphology, PB leukocytosis and expression of HLA-DR, CD34 and CD11b. Concerning nuclear chromatin texture variables, these cases had a lower entropy, contrast, cluster prominence and FD, but higher local homogeneity, and R(2)45, in keeping with more homogeneously distributed chromatin. In the univariate Cox analysis, a higher leukocyte count, FLT3-ITD mutation, microgranular morphology, methylation of CDKN2B, besides a higher local homogeneity of nuclear chromatin, a lower chromatin entropy and FD were associated to a worse outcome. All these features lost significance when the cases were stratified for FLT3-ITD mutation. Methylation status of CDNK2A and TP73 showed no relation to patient's survival. Conclusion: in APL, patients with FLT3-ITD mutation show different clinical characteristics and have blasts with a more homogeneous chromatin texture. Texture analysis demonstrated that FLTD-ITD was accompanied not only by different cytoplasmic features, but also by a change in chromatin structure in routine cytologic preparations. Yet we were not able to detect chromatin changes by nuclear texture analysis of patients with the FTLD-TKD or methylation of specific genes.7Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2007/54686-0]CNPq [302277/2009-9, 307270/2010-6, 402022/2010-6

Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes

INTRODUCTION: Myelodysplastic syndromes encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress toward acute myeloid leukemia. The accumulation of genetic alterations is closely associated with the progression of myelodysplastic syndromes toward acute myeloid leukemia. OBJECTIVE: To investigate the presence of mutations in the points most frequent for mutations (hotspot mutations) in phosphatidylinositol-3-kinase (PI3K), Janus kinase 2 (JAK2), FMS-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1), which are involved in leukemia and other cancers, in a population of Brazilian MDS patients. METHODS: Fifty-one myelodysplastic syndromes patients were included in the study. According to French-American-British classification, the patients were distributed as follows: 31 with refractory anemia, 8 with refractory anemia with ringed sideroblasts, 7 with refractory anemia with excess blasts, 3 with refractory anemia with excess blasts in transformation and 2 with chronic myelomonocytic leukemia. Bone marrow samples were obtained and screened for the presence of hotspot mutations using analysis based on amplification with the polymerase chain reaction, sequencing, fragment size polymorphisms or restriction enzyme digestion. All patients were screened for mutations at the time of diagnosis, and 5 patients were also screened at the time of disease progression. RESULTS: In the genes studied, no mutations were detected in the patients at the time of diagnosis. One patient with chronic myelomonocytic leukemia was heterozygous for a Janus kinase 2 mutation after disease progression. CONCLUSIONS: These results show that hotspot mutations in the PI3K, JAK2, FLT3 and NPM1 genes are not common in MDS patients; nevertheless, JAK2 mutations may be present in myelodysplasia during disease progression

Fractal Characteristics of May-Grünwald-Giemsa Stained Chromatin Are Independent Prognostic Factors for Survival in Multiple Myeloma

The use of computerized image analysis for the study of nuclear texture features has provided important prognostic information for several neoplasias. Recently fractal characteristics of the chromatin structure in routinely stained smears have shown to be independent prognostic factors in acute leukemia. In the present study we investigated the influence of the fractal dimension (FD) of chromatin on survival of patients with multiple myeloma.We analyzed 67 newly diagnosed patients from our Institution treated in the Brazilian Multiple Myeloma Study Group. Diagnostic work-up consisted of peripheral blood counts, bone marrow cytology, bone radiograms, serum biochemistry and cytogenetics. The International Staging System (ISS) was used. In every patient, at least 40 digital nuclear images from diagnostic May-Grünwald-Giemsa stained bone marrow smears were acquired and transformed into pseudo-3D images. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R(2) values in the log-log plots. The influence of diagnostic features on overall survival was analyzed in Cox regressions. Patients that underwent autologous bone marrow transplantation were censored at the day of transplantation.Median age was 56 years. According to ISS, 14% of the patients were stage I, 39% were stage II and 47% were stage III. Additional features of a bad prognosis were observed in 46% of the cases. When stratifying for ISS, both FD and its goodness-of-fit were significant prognostic factors in univariate analyses. Patients with higher FD values or lower goodness-of-fit showed a worse outcome. In the multivariate Cox-regression, FD, R(2), and ISS stage entered the final model, which showed to be stable in a bootstrap resampling study.Fractal characteristics of the chromatin texture in routine cytological preparations revealed relevant prognostic information in patients with multiple myeloma

Fractal dimension of chromatin is an independent prognostic factor for survival in melanoma

<p>Abstract</p> <p>Background</p> <p>Prognostic factors in malignant melanoma are currently based on clinical data and morphologic examination. Other prognostic features, however, which are not yet used in daily practice, might add important information and thus improve prognosis, treatment, and survival. Therefore a search for new markers is desirable. Previous studies have demonstrated that fractal characteristics of nuclear chromatin are of prognostic importance in neoplasias. We have therefore investigated whether the fractal dimension of nuclear chromatin measured in routine histological preparations of malignant melanomas could be a prognostic factor for survival.</p> <p>Methods</p> <p>We examined 71 primary superficial spreading cutaneous melanoma specimens (thickness ≥ 1 mm) from patients with a minimum follow up of 5 years. Nuclear area, form factor and fractal dimension of chromatin texture were obtained from digitalized images of hematoxylin-eosin stained tissue micro array sections. Clark's level, tumor thickness and mitotic rate were also determined.</p> <p>Results</p> <p>The median follow-up was 104 months. Tumor thickness, Clark's level, mitotic rate, nuclear area and fractal dimension were significant risk factors in univariate Cox regressions. In the multivariate Cox regression, stratified for the presence or absence of metastases at diagnosis, only the Clark level and fractal dimension of the nuclear chromatin were included as independent prognostic factors in the final regression model.</p> <p>Conclusion</p> <p>In general, a more aggressive behaviour is usually found in genetically unstable neoplasias with a higher number of genetic or epigenetic changes, which on the other hand, provoke a more complex chromatin rearrangement. The increased nuclear fractal dimension found in the more aggressive melanomas is the mathematical equivalent of a higher complexity of the chromatin architecture. So, there is strong evidence that the fractal dimension of the nuclear chromatin texture is a new and promising variable in prognostic models of malignant melanomas.</p

The impact of maternal HIV infection on cord blood lymphocyte subsets and cytokine profile in exposed non-infected newborns

<p>Abstract</p> <p>Background</p> <p>Children born to HIV+ mothers are exposed intra-utero to several drugs and cytokines that can modify the developing immune system, and influence the newborn's immune response to infections and vaccines. We analyzed the relation between the distribution of cord blood lymphocyte subsets and cytokine profile in term newborns of HIV+ mothers using HAART during pregnancy and compared them to normal newborns.</p> <p>Methods</p> <p>In a prospective, controlled study, 36 mother-child pairs from HIV+ mothers and 15 HIV-uninfected mothers were studied. Hematological features and cytokine profiles of mothers at 35 weeks of pregnancy were examined. Maternal and cord lymphocyte subsets as well as B-cell maturation in cord blood were analyzed by flow cytometry. The non-stimulated, as well as BCG- and PHA-stimulated production of IL2, IL4, IL7, IL10, IL12, IFN-γ and TNF-alpha in mononuclear cell cultures from mothers and infants were quantified using ELISA.</p> <p>Results</p> <p>After one year follow-up none of the exposed infants became seropositive for HIV. An increase in B lymphocytes, especially the CD19/CD5+ ones, was observed in cord blood of HIV-exposed newborns. Children of HIV+ hard drug using mothers had also an increase of immature B-cells. Cord blood mononuclear cells of HIV-exposed newborns produced less IL-4 and IL-7 and more IL-10 and IFN-γ in culture than those of uninfected mothers. Cytokine values in supernatants were similar in infants and their mothers except for IFN-γ and TNF-alpha that were higher in HIV+ mothers, especially in drug abusing ones. Cord blood CD19/CD5+ lymphocytes showed a positive correlation with cord IL-7 and IL-10. A higher maternal age and smoking was associated with a decrease of cord blood CD4+ cells.</p> <p>Conclusions</p> <p>in uninfected infants born to HIV+ women, several immunological abnormalities were found, related to the residual maternal immune changes induced by the HIV infection and those associated with antiretroviral treatment. Maternal smoking was associated to changes in cord CD3/CD4 lymphocytes and maternal hard drug abuse was associated with more pronounced changes in the cord B cell line.</p

Lymph vascular invasion in invasive mammary carcinomas identified by the endothelial lymphatic marker D2-40 is associated with other indicators of poor prognosis

<p>Abstract</p> <p>Background</p> <p>Immunohistochemical studies of lymphatic vessels have been limited by a lack of specific markers. Recently, the novel D2-40 antibody, which selectively marks endothelium of lymphatic vessels, was released. The aim of our study is to compare lymphatic and blood vessel invasion detected by hematoxylin and eosin (H&E) versus that detected by immunohistochemistry, relating them with morphologic and molecular prognostic factors.</p> <p>Methods</p> <p>We selected 123 cases of invasive mammary carcinomas stratified into three subgroups according to axillary lymph node status: macrometastases, micrometastases, and lymph node negative. Lymphatic (LVI) and blood (BVI) vessel invasion were evaluated by H&E and immunohistochemistry using the D2-40 and CD31 antibodies, and related to histologic tumor type and grade, estrogen and progesterone receptors, E-cadherin, Ki67, p53, and Her2/<it>neu </it>expression.</p> <p>Results</p> <p>LVI was detected in H&E-stained sections in 17/123 cases (13.8%), and in D2-40 sections in 35/123 cases (28.5%) (Kappa = 0.433). BVI was detected in H&E-stained sections in 5/123 cases (4.1%), and in CD31 stained sections in 19/123 cases (15.4%) (Kappa = 0.198). LVI is positively related to higher histologic grade (p = 0.013), higher Ki67 expression (p = 0.00013), and to the presence of macrometastases (p = 0.002), and inversely related to estrogen (p = 0.0016) and progesterone (p = 0.00017) receptors expression.</p> <p>Conclusion</p> <p>D2-40 is a reliable marker of lymphatic vessels and is a useful tool for lymphatic emboli identification in immunostained sections of breast carcinomas with higher identification rates than H&E. Lymphatic vessel invasion was related to other features (high combined histologic grade, high Ki67 score, negative hormone receptors expression) associated with worse prognosis, probable reflecting a potential for lymphatic metastatic spread and aggressive behavior.</p

Imbalances in serum angiopoietin concentrations are early predictors of septic shock development in patients with post chemotherapy febrile neutropenia

Background: Febrile neutropenia carries a high risk of sepsis complications, and the identification of biomarkers capable to identify high risk patients is a great challenge. Angiopoietins (Ang -) are cytokines involved in the control microvascular permeability. It is accepted that Ang-1 expression maintains endothelial barrier integrity, and that Ang-2 acts as an antagonizing cytokine with barrier-disrupting functions in inflammatory situations. Ang-2 levels have been recently correlated with sepsis mortality in intensive care units. Methods: We prospectively evaluated concentrations of Ang-1 and Ang-2 at different time-points during febrile neutropenia, and explored the diagnostic accuracy of these mediators as potential predictors of poor outcome in this clinical setting before the development of sepsis complications. Results: Patients that evolved with septic shock (n = 10) presented higher levels of Ang-2 measured 48 hours after fever onset, and of the Ang-2/Ang-1 ratio at the time of fever onset compared to patients with non-complicated sepsis (n = 31). These levels correlated with sepsis severity scores. Conclusions: Our data suggest that imbalances in the concentrations of Ang-1 and Ang-2 are independent and early markers of the risk of developing septic shock and of sepsis mortality in febrile neutropenia, and larger studies are warranted to validate their clinical usefulness. Therapeutic strategies that manipulate this Ang-2/Ang-1 imbalance can potentially offer new and promising treatments for sepsis in febrile neutropenia