The expression of p53, p21/WAF-1, Mdm2, c-Myc, and proliferating cell nuclear antigen (PCNA) proteins was examined by the immunohistochemistry of paraffin-embedded tissues of 62 patients with aggressive non-Hodgkin's lymphomas (NHL) and correlated to clinical data. Expression of p53, p21/WAF-1, Mdm2, and c-Myc protein was observed in 17 out of 62 cases (30%), 25 out of 60 (42%), 13 out of 44 (30%), and 39 out of 51 (76.5%), respectively. The p53 + /p21WAF-1 phenotype, which is more frequently found in p53 mutations, was associated with a worse overall survival (P = 0.04) and with a lower rate of complete response (CR) (PF = 0.01). p53 and c-Myc negative expression was related to a better response to chemotherapy (PF = 0.005 and 0.035, respectively). The expression of p53, c-Myc, and Mdm2 was related to a shortened overall survival (P < 0.001, 0.05, and 0.037, respectively), suggesting that the expression of these proteins could be associated with a poor outcome in these patients. Am. J. Hematol. 67:84-92, 2001.

4477 Tyrosine kinases Inhibitors (TKI) of first and second generation are presently the choice of treatment for chronic myeloid leukemia (CML) and adherence to the treatment is an important factor to obtain good therapeutic results. We used the World Health Organization adherence definition that enrolls a global person's behavior including how the medication is used, diet, and/or lifestyle change. Objective: To evaluate the adherence to TKI in patients with CML and to identify factors that can affect adherence to TKI treatment. Methods: A prospective and observational analysis was performed with 122 patients taking TKI, in a public institution. The median of observation time was 169 days (131-240). We used the Hasford scale to risk assessment of diagnosis. The adherence was calculated using the mean medication possession ratio (MPR), calculated as total days’ dose of TKI divided by the number of the days in the observation time. Statistical analysis began with descriptive analysis and after that we applied Pearson or Spearman's correlation and t-Test, what it is adequate, considering significant p-value £ 0.05. Results: A total of 122 patients were evaluated: 92 (75.5%) were taking Imatinib, 16 (13%) Nilotinib, 09 (7.5%) Dasatinib and 5 (4%) Bosutinib. The mean age was 48 (20-82) years. The majority of patients were male (61%) and the median of TKI treatment time was 45 months (7-114). There were 103 (84.5%) patients in first line treatment, 17 (14%) in second line and 2 (1.5%) in third line. Considering all patients, median and mean of adherence was 96% and 90% whereas 23% of patients had 100% of MPR. There was a significant difference between adherence distribution and cytogenetic results, best adherent patients had most complete and major cytogenetic response (p= 0.01) in CML Hasford lower risk group. In all patients, the MPR decreased with longer use of TKI treatment (p= 0.02) and with longer time of diagnosis (P= 0.002). The adherence was superior in patients participating in clinical trials (p= 0.01) and using second generation TKI (p= 0.001). Conclusion: The best adherence was correlated to a better cytogenetic response in Hasford lower risk group. Longer time of treatment was related to a poorer adherence, suggesting a continuous approach by health team can make a difference. Disclosures: No relevant conflicts of interest to declare.

1681 Recently the European LeukemiaNet has developed a new scoring system (European Treatment and Outcome Study [EUTOS] score) for newly diagnosed chronic myeloid leukemia (CML) in chronic phase (CP) treated with imatinib. The EUTOS score classifies patients in high or low risk on the basis of the percentage of basophils in the peripheral blood and the spleen size at diagnosis, with significant correlations with the achievement of an 18-month complete cytogenetic response (CCyR) and progression-free survival (PFS). The aim of this work was to evaluate EUTOS score in CML-CP treated in our center with imatinib as a predictive factor for overall survival (OS), event-free survival (EFS) and PFS. Patients and methods: Between February 2003 and May 2012 consecutive patients with newly diagnosed CML-CP were treated with imatinib 400 mg daily (n= 144) or imatinib 600–800 mg daily (n= 14) were included in the analysis. The criteria recommended by European LeukemiaNet (ELN) were used for the definitions of CCyR and the progression to accelerated phase (AP) or blast phase (BP). The EUTOS score was defined by (7×basophils) plus (4×spleen size) at diagnostic. A EUTOS score of more than 87 indicates high risk, and less than or equal to 87 low risk. EFS was measured from the start of imatinib treatment to the date of any of the following events: death from any cause at any time, loss of complete hematologic response, loss of CCyR, or progression to AP or BP. PFS was measured from the start of treatment to the date progression to AP or BP, last follow-up, or death from any cause. Survival probabilities were estimated by the Kaplan-Meier method and compared by the log-rank test whereas it was applied cumulative incidence for the probability to achieve CCyR. Results: A total of 158 patients were treated, 94 (59.5%) male. The median age at Imatinib was 47 years (17–86 years). The median time from diagnosis to TKI therapy was 2 (0–6) months, with 153 (96.8%) receiving previous treatment with Hydrea. The median follow-up was 29 (1–110) months. The median splenomegaly size was 4 (0–29) cm and the median basophil percentage was 2.5% (0–18%). According to the Sokal score, 43 (34,4%) patients, 46 (36,8%), and 36 (28,8%) were in low, intermediate, and high Sokal score category, respectively (33 not available). Concerning the Hasford score, 60 (48,3%), 50 (40,4%) and 14 (11,3%) were in low, intermediate and high risk categories (34 NA). A total of 137 (86,8%) patients were in the low EUTOS score category and 21 (13,2%) in the high risk category. The cumulative probability of achieving a CCyR and MMR at 36 months for all patients was 78% and 64%, respectively. Patients who had not achieved CCyR after 6 months (51/153 – 33%) had a 2% risk of subsequent progression, which increased to 12% after 12 months, 14% in 18 months and 19% after 24 months. EFS, PFS, and OS rates for the whole group were 60%, 89%, and 92%, respectively. Patients with a low EUTOS score had higher rates of cumulative CCyR compared with patients with high EUTOS score (82% vs. 53%, p= 0.06) (figure 1). There were no differences in the cumulative CCyR rates in patients stratified by Sokal or Hasford scores (and 0.21 and P=0.82, respectively). Patients with CCyR at 18 months had a higher EFS (81% vs. 18%, p< 0.0001) and PFS rates (96% vs. 82%, p= 0.03). There was no difference in PFS (figure 2) and OS rates between patients with low and high EUTOS score. However, patients with high and intermediate Sokal score had an inferior PFS rates in comparison with low risk group (77%, 84% and 100%, respectively, p= 0.02). There was a superior EFS rates in low risk in comparison with high EUTOS score (63% vs. 36%, p= 0.01) (figure 3), whereas the overall survival there was no difference (91% vs. 100%). Sokal scores EFS rates were 68%, 60% and 40% for low, intermediate and high risk groups respectively (p= 0.03). In conclusion, similarly to the original report, EUTOS score seems to predict CCyR, but not PFS in our population. However, EFS was significantly better in the low than high risk group. The score can discriminate patients with poor outcome, with lower probability of achieving responses to first line imatinib therapy. Disclosures: No relevant conflicts of interest to declare.

Introduction : Treatment with tyrosine kinase inhibitors (TKIs) has dramatically increased the overall survival of patients with chronic myeloid leukemia (CML) but second generation TKI has been associated with an increased risk of cardiovascular events. Objectives: The aim of this study was to evaluate the incidence of cardiovascular adverse events (CVE) in CML patients treated with TKIs and to correlate with the cardiovascular (CV) risk of the patients. Methods: this is a retrospective analysis of consecutive CML patients treated with TKIs between 2005 and 2013at our Institution. Baseline risk factors for CV diseases were collected at baseline and included age, arterial hypertension (AH), dyslipidemia, obesity, hypothireoidism, smoking, diabetes mellitus (DM), coronary artery disease and chronic renal failure. Cardiovascular events during TKI treatment were collected and included: myocardial infarction, unstable angina, peripheral arterial disease, stroke, arrythmia,hypertension and cardiac failure. Cardiovascular risk was calculated using the SCORE chart of the European Society of Cardiology and patients were classified in low, moderate, high and very high risk. Results: We analyzed CML patients treated with imatinib (n=117), dasatinib (n=91) and nilotinib (n=60). The median time of follow-up was 748, 519 and 851 days, respectively. Baseline risk factors: 90 patients (38,5%) had hypertension, 34 (14,5%) DM, 67 (28,6%) dyslipidemia, 51 (21,8%) obesity, 22 (9,4%) hypothyroidism, 14 (6%) coronary arterial disease, 21 (9%) systolic cardiac dysfunction, 4 (1,7%) stroke, 20 (8,5%) chronic kidney failure and 36 (15,4%) were smokers. SCORE chart classification: 106 patients (39,5%) were in the low-risk category, 70 (26%) in the moderate risk, 46 (17,2%) in the high risk, 46 (17,2%) in the very high risk group. Overall, the cumulative incidence of CVE was 4.1%. Five (5.5%) events occurred during dasatinib treatment (P=0.015), 6 (10%) events during nilotinib and no events during imatinib treatment (P=0.001). The incidence of CVE was 10.8% in the high and very high-risk groups and 0.52% in moderate and low risk group (P≤0.001). The incidence of arterial ischemic events (AIE) was 10% (n=6) in patients treated with nilotinib, 2.2% (n=2) with dasatinib and 0% with imatinib (P≤0.001). Arterial events were exclusively observed in high and very high-risk groups (8 events, 8.7%) (P≤0.001). The risk factors associated with a higher risk of CVE were hypertension (P≤0.001), dyslipidemia (P≤0.001), coronary arterial disease (P=0.003), congestive heart failure (P=0.002) and chronic renal failure (P=0.011). Disease progression was the main cause of death in all groups. Conclusions: CVE were more frequent in patients treated with second generation TKIs. AIE were more frequent in patients treated with nilotinib, in those having a high or very high risk SCORE. The CV risk stratification of CML patients before and during TKI therapy can help in TKI selection and to identify patients at high risk, in order to reduce the morbidity and mortality associated with CVE. Disclosures Pagnano: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Miers-Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Besides the known factors such as the presence of oncogenes, the macro-environment (pollution, infections) or organic microenvironment (dysregulation of the immune system) can be the triggering factor of the process of leukemogenesis. It is known that the amount of rainfall can affect the distribution (dilution) of pollutants in the air and water reserves. There is no description of the climate influence in the incidence of Acute Promyelocytic Leukemia (APL), which has its own clinical laboratory characteristics, and is defined by the presence of the PML-RARA rearrangement. The aim of this study was to investigate the impact of seasonality in the incidence of Promyelocytic Leukemia in Brazil, and its characteristics. Patients and methods we analyzed the clinical laboratory data and origin of participant cases of the International Consortium on Acute Promyelocytic Leukemia (IC-APL), a group multicenter treatment of APL with standardized diagnosis and treatment. We included all patients diagnosed with APL of Brazilian centers between 2006 and 2011. We excluded patients without demographics. Patients were divided into macro-climate (Northeast, South and Southeast). Northeast: 49 cases of Pernambuco, Southeast: 16 cases of Minas Gerais, São Paulo 88 cases; South: 27 cases of Rio Grande do Sul and 19 cases of Paraná. Meteorological data were extracted from the database Meteorological Research and Education (BDMEP) of the National Institute of Meteorology (INMET), and grouped by quarter. We studied the mean maximum temperature, mean minimum temperature and rainfall. The relationship between the number of cases and meteorological data were analyzed by the Spearman test. Results We included 149 patients with APL. In the South, there were 46 patients, 50% female and 50% male, mean age: 37 years, 16 cases occurred on the first quarter (January-March), 12 on the second quarter (April-June), 8 cases on third quarter (July-September) and 10 on the fourth quarter (October to December). In the Northeast, there were 49 cases, 25 female and 24 male, mean age 34 years with 11 cases on the first and second quarters, 12 cases on the third quarter and 15 cases on the fourth quarter. Southeast: 54 cases with 29 female cases and 25 male cases, mean age 25 years, with 12 cases on the first and second quarter, 11 cases on the third quarter and 19 cases on the fourth quarter. In the South, there was no statistically significant correlation between the weather and the number of registered cases of APL. In the Northeast, there was a negative correlation between the number of cases of APL and rainfall (r = -0.57, p = 0.004) and a trend with the maximum temperature (r = 0.34, p = .07). In the southeast, there was positive correlation between rainfall (r = 0.42, p = 0.02) but not with temperature. In the northeast, the smallest amount of rainfall is associated with higher temperatures (r = -0.49, p <0.0002), whereas in the Southeast, the greater amount of rainfall is associated with warmer temperatures. Conclusion There is no known etiology of APL, but the correlations found between rainfall and number of cases could be related to the dispersion of pollutants into the environment. Disclosures: No relevant conflicts of interest to declare.

Using the overall survival (OS), disease free survival (DFS) and progression free survival (PFS), as well as associated toxicity, the purpose of this work was to CR, 1 partial response (PR), 2 relapsed disease (RD) and 28 disease progression (DP)], 11 (31%) had not performed ASCT. OS was 37%; DFS was 49% and PFS 28%. OS by diagnosis was 42% for DLBCL, 40% for T-cell (8 y) and 20% for Mantle Cell (6 y) (P=NS). OS by B symptom patients was 22% vs. 58% (P=0.002) and PFS was 23% vs. 37% (P=0.03). Patients who achieved CR after HDCY (38) had significantly better OS and PFS (38% and 17%) than patients who remained in DP (P<0.0001). Cox Regression demonstrated therapeutic lines before HDCY P= 0.02) and PD both before (RR = 2.70; P<0.001) and after HDCY (RR = 5.38; P<0.0001). Conclusions: Our study suggests HDS is an efficient treatment to improve status and to reduce tumoral burden. Regardless of toxicity-related mortality it is feasible, especially considering the poor prognosis of patients. Rev. Bras. Hematol. Hemoter. 2009;31(Supl. 2):9-14.

Background: Primary Myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) characterized by increased myeloid proliferation and associated with mutations that induce tyrosine-kinase activation mainly via JAK-STAT pathway, culminating in extensive bone marrow (BM) fibrosis in the course of disease progression. In contrast to the monoclonal origin of hematopoietic cells, fibroblasts proliferation is polyclonal, and mediators involved in fibrosis, neoangiogenesis and osteosclerosis seem to be involved in disease progression. Metformin (MTF) is a biguanide that exerts selective antineoplastic activity in a variety of malignancies, through its action on nutrients privation and hypoxia, leading to apoptosis. In JAK2-mutated cell lines, MTF reduced cell viability, proliferation and clonogenicity, while in Jak2V617F knock-in-induced mice, MTF reduced Ba/F3 JAK2V617F tumor burden and splenomegaly. These data suggest that MTF could have a therapeutic effect in PMF patients. Aims: To conduct an open label phase II study to evaluate MTF effects on BM fibrosis, inflammation mediators, JAK-STAT pathway activation and disease progression in PMF patients. Methods: PMF non-diabetic adults were eligible. Subjects with severe renal function impairment were not included. Patients received MTF (Glifage XR®) in rising doses until a maximum of 2500mg PO daily, according to tolerance. Primary endpoint was BM fibrosis reversion. Secondary endpoints included reduction of inflammation and downregulation of the JAK-STAT pathway. Clinical data was systematically compiled. Blood and BM samples were collected at the time points: pretreatment (0), 3 mo and 6 mo. Collagen was evaluated in BM biopsy specimens by Masson's trichrome stain: three representative areas from each slide were analyzed and the collagen/sample area was quantified using Image J software; the mean percentage of each slide was used for statistics. IL-6, IL-8 and TNF-α levels were analyzed in BM samples using multiplex assay. Phosphorylation status of intracellular proteins STAT3 and STAT5 was analyzed by flow cytometry and the percentage of cells was recorded using FlowJo software. In order to evaluate gene modulation following MTF exposure, samples at time points 0 and 6 mo were analyzed by PCR array for insulin signaling genes (PAHS-030Z, Qiagen). Genes with ±1.5 fold-change in both directions were selected for validation. For each experiment, statistical analysis was performed and a p value &lt;0.05 was considered statistically significant. Results were expressed as medians (min-max). This trial was approved by the Institutional and National Review Board; written informed consent was obtained from all subjects. Results: 11 patients (aged 40-84y) were included. Two subjects had early treatment discontinuation due to non-related causes. The median exposure to MTF was 10 mo (5-11) and the median dose was 2000mg/day (1500-2500mg). The most frequent adverse event was diarrhea (n=3). No life threatening event occurred. A reduction in BM collagen area percentage was observed comparing pretreatment biopsies (26.9% (14.8-53.1%)) versus 3 months (3.8% (2.3-4.0%), p=0.062) and versus 6 months of MTF use (0.84% (0.12-17.1%), p=0.125), however, this result was not statistically significant probably due to the low number of patients analyzed (n=5). IL-6, IL-8 and TNF-α levels did not differ between time points. Flow cytometry analysis demonstrated a trend in STAT3 phosphorylation decrease when comparing pretreatment samples versus 6 months of MTF use, though this result was not statistically significant (p=0.06). Mean fluorescence intensity for pSTAT3 was: pretreatment 10.53 ± 5.75, 3 mo 7.34 ± 2.4, 6 mo 5.41 ± 1.14; and for pSTAT5: pretreatment 14.03 ± 7.41; 3 mo 10.71 ± 7.74; 6 mo 6.03 ± 1.41. PCR array for insulin signaling genes showed 21 genes downregulated after 6 months of MTF treatment, including genes previously associated with MPN phenotype: INS (0 and 6 mo treatment fold-decrease: 0.18), NOS2 (0.24), VEGFA (0.34), LEP (0.34), IGFBP1 (0.38) and IRS2 (0.62). Conclusions: In this study, metformin showed to be a safe and well-tolerated drug. Our preliminary results demonstrated a trend in BM collagen reduction in PMF patients following metformin treatment. Downregulation of important genes associated with MPN phenotype was also noted. The trial is ongoing and these results will be validated at other time points for all subjects. Disclosures Pagnano: Abbvie: Consultancy; Pint Pharma: Consultancy; Sandoz: Consultancy.

4850 Introduction: The WHO 2008 classification of acute myeloid leukemia (AML) is based on morphology, cytogenetics and molecular features. Among them, mutations and internal tandem duplications of FLT3 in AML with a normal karyotype have been associated to a poor prognosis. Mutated NPM1 in the absence of FLT3-ITD is associated to a favorable. On the other hand, variables of nuclear chromatin texture have been described as independent risk factors in several malignancies (ALL, melanoma and multiple myeloma). Aim: To compare the influence on overall survival of the chromatin fractal dimension and molecular features in adult patients with AML. Patients and Methods: We analyzed 106 consecutive cases diagnosed at our Institution between 2007 and 2009. Diagnosis was made by bone marrow (BM) cytology and karyotype, and cases were classified by WHO criteria. Genomic DNA was extracted by phenol-chloroform. Genotyping was made with the MegaBACE 1000 equipment and analyzed in the Fragment Profiler v1.2. For detection of the FLT3-TKD mutation, genomic DNA was amplified by PCR followed by restriction analysis. Blasts from the diagnostic BM cytology were digitalized, segmented and nuclear morphometric variables were examined. Their influence on overall survival was analyzed in the Cox model. Results: Median age: 52 years; peripheral blood (PB) leukocytes: 24.0×109/l (0.7-281.3). In the univariate analysis were significant: PB leukocyte count (p=0.005), low-risk karyotype (p=0.002), FLT3 ITD+ (p=0.002), FLT3+NPM1- (p=0.029) and “goodness of fit” (R245) of the chromatin fractal dimension (Minkowski) (p=0.03). Age, fractal dimension and methylation status of p15, p16, p57, p73, ER and MDR1 were not significant. In the multivariate analyses including age, PB leukocytes, R245 and mutations, high leukocyte counts (p=0.03) and low R245 (p=0.01) were independent unfavorable and FLT3-NPM1- (p=0.04) and FLT3-NPM1+ (p=0.02) were favorable prognostic variables. Conclusions: The blast chromatin texture measured by R245 was an independent prognostic factor together with known risk variables in AML. Supported by: FAPESP and CNPq Disclosures: No relevant conflicts of interest to declare.