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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.

Background The MLL5 gene encodes a histone methyltransferase implicated in positive control of several genes related to hematopoiesis. Its close relationship with retinoic acid–induced granulopoiesis suggests that the deregulated expression of MLL5 might lead acute promyelocytic leukemia (APL) blasts to become less susceptible to differentiation-inducing ATRA effects. Here, we retrospectively determined the MLL5 transcript levels in samples from APL patients enrolled in the International Consortium on Acute Promyelocytic Leukemia (IC-APL) trial and analyzed its relationship with clinical and laboratory features, hematologic recovery, relapse, and survival. The results of the IC-APL have been previously reported (Blood 2013, 121(11) pp: 1935). In brief, complete hematological remission (CR) was achieved by 153/180 patients enrolled in Brazil, Mexico, Chile and Uruguay and after a median follow up of 28 months, the 2-year cumulative incidence of relapse (CIR), overall survival (OS) and disease-free survival (DFS) were 4.5%, 80% and 91%, respectively. Design and Methods One hundred and twenty-one APL patients (age, 15-73y) from seven different Brazilian institutions and treated according to the IC-APL protocol were included. The treatment schedule was identical to that adopted in the PETHEMA/HOVON LPA2005 trial, except for the replacement of idarubicin by daunorubicin. ATRA treatment was initiated immediately in all cases in which the diagnosis of APL was suspected based on morphology. As normal controls, total bone marrow (BM, n=8) and peripheral blood (PB, n=101) cells from healthy donors (age, 18-60y) were collected and mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. Additionally, 28 CBF-leukemia samples were included. Gene expression profile was analyzed by real-time quantitative PCR using the ABL FusionQuant Standard Kit as an endogenous control. Based on the continuous distribution of MLL5/ABL expression on APL samples (Figure 1), we adopted the median value of MLL5/ABL expression as cut-off to dichotomize APL patients in “low” and “high” MLL5 transcript levels. Results MLL5 expression was not different between APL, CBF-leukemia and healthy donors samples (Figure 1; P=0.19). There was no relevant difference between APL patients with low (n=62) and high (n=59) MLL5 transcript levels with respect to clinical and laboratory features, although high MLL5 transcript levels were more likely to be present in female gender (P=0.029). Overall, 102 (84%) achieved CR. Patients with low MLL5 transcript levels had significantly lower CR rate than patients with high MLL5 transcript levels (74% vs 95%; P=0.002). Twelve patients (10%) experienced early mortality (i.e., death during induction therapy) due to hemorrhage (n=6; 50%), disease progression (n=1; 8.3%), thrombosis (n=1; 8.3%), therapy-related infection (n=3; 25%) and differentiation syndrome (n=1; 8.3%); MLL5 transcript levels had no impact on early mortality (P=0.155). With a follow-up of 33 months among survivors (range, 1-72 months), patients with low MLL5 transcript levels had significantly lower 2-year OS (P=0.005) and 2-year DFS rate (P=0.037) than patients with high MLL5 transcript levels. Up to January 2013, a total of six relapses (5%) had been recorded. The 2-year CIR among patients with low and high MLL5 transcript levels was 14% (95%CI: 5% to 27%) and 2% (95%CI: 0.1% to 11%), respectively (P=0.04). We have further evaluated the prognostic impact of MLL5 transcript levels in those patients who remain alive after induction therapy (107 patients). Low MLL5 transcript levels were predictive of lower CR rate (P=0.042) and 2-year OS rate (P=0.009), but had no impact on DFS (P=0.106). Conclusion Our results show that MLL5 transcript levels may predict lower remission rate, short survival and higher risk of relapse in APL patients treated with ATRA and anthracycline-based chemotherapy. This is the first report describing the MLL5 expression as a prognostic factor in APL; nevertheless, our results should be confirmed in an independent cohort. Disclosures: No relevant conflicts of interest to declare.

Second-generation tyrosine kinase inhibitors (2G-TKI), nilotinib or dasatinib used after imatinib failure can induce complete cytogenetic response (CCR) in 50% of chronic myeloid leukemia (CML) patients. BCR-ABL transcript levels reduction in the initial months of treatment has been associated improved outcome. Aims to evaluate the predictive value of early molecular responses, at 3 and 6 months after treatment with 2G-TKI in CML patients with imatinib failure or intolerance; to correlate these responses with CCR, overall survival (OS), progression-free survival (PFS) and event free survival (EFS). Methods Between September 2007 and August 2012, 71 consecutive patients with CML resistant or intolerant to imatinib were treated with dasatinib (n= 50) or nilotinib (n=21). BCR-ABL transcripts were measured in peripheral blood using real-time quantitative PCR (RQ-PCR) at 3 months intervals. Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1% (IS). Cytogenetic analysis was performed at baseline, 3, 6, 12 and 18 months after starting therapy with 2G-TKI. BCR-ABL mutation analysis by direct sequencing was investigated in resistant patients. Probabilities of OS, PFS and EFS were calculated using Kaplan-Meier method. An event was defined as the loss of CHR, CCR, progression to advanced phases, death or 2G-TKI discontinuation. The CCR probabilities according to molecular responses were calculated by c2 method and cumulative incidence, considering as competitive event death or progression. Results 71 patients were analyzed, median age of 47 years (15-81); Disease status before 2G-TKI was: 50 (71%) CP; 13 (18%) AP and 8 (11%) BC. Responses: 59/71 (83%) obtained CHR; 38/55 (69%) CCR and 37/60 (62%) MMR. At 3 months of therapy, 81.5% (44/54) had a BCR-ABL ratio ≤10% and at 6 months 66% (33/50) had ≤ 1%. At 3 months, CCR was obtained 65% (19/29) pts with ≤10% RQ-PCR and 16% (1/6) with >10% RQ-PCR (p= 0.06). At 6 months, CCR was 100% (12/12) in pts with RQ-PCR ≤ 1% and 14% (1/7) in those with >1% (p< 0.0001). The probability to achieve RQ-PCR < 10% at 3 month was 43% (95% CI 32-54%). During treatment 3 (4%) progressed to AP and 5 (7%) to BC. The 5-year probability of OS was 78% (95% CI: 68-88%) albeit by disease status was 86% in CP, 92% in AP and 12% in BC (p< 0.0001). OS was inferior in pts with RQ-PCR > 10% at 3 months (60 vs 84%, p= 0.03) and >1% at 6 months (68 vs100%, p= 0.006). PFS was 68% in 5-year, and was lower in BC pts (p< 0.0001) and pts with RQ-PCR >1% at 6 months (p= 0.004). EFS was 53% and lower in BC pts (p< 0.0001), in pts with RQ-PCR > 10% at 3 months (p= 0.005) and > 1% at 6 months (p< 0.0001). RQ-PCR at 3 and 6 months were also predictive of a worse survival when patients in CP were analyzed separately. 2G-TKI was discontinued in 44% (31/71) due to: resistance (n=18), intolerance (n=5), death (n= 3), HSCT (n=3) and loss of follow-up (n=2). Eleven BCR-ABL mutations were detected in 36 pts; 3 previously 2G-TKI (L387M-1, E255K-1, M351T-1) and 9 after therapy (T315I-5, M244V-2, E255V-1, Y253H-1). OS by mutation was 45% with any mutation and 88% with no mutation (p= 0.05). Conclusion BCR-ABL transcript levels at 3 and 6 months can identify patients with worse prognosis and less chance to obtain CCR with 2G-TKI after imatinib treatment. Disclosures: No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) patients with imatinib failure are usually treated with second generation tyrosine kinase inhibitors (TKI). In case of dasatinib or nilotinib intolerance or resistance patients with HLA matched donor are submitted to allogeneic hematopoietic stem cell transplantation (HSCT) or switch treatment to a different TKI. Aims to evaluate complete hematological response (CHR), cytogenetic and molecular responses, overall, progression free and event free survival (OS, FPS and EFS) in CML patients in third line therapy that previously used 2 TKI. BCR-ABL mutations was also evaluated in this population Patients and methods between July 2008 and August 2012 26 CML patients were evaluated: 10 patients (pts) treated with dasatinib (38,5%) and 16 with nilotinib (61,5%) in third line therapy. OS, PFS and EFS were calculated with Kaplan-Meier method and Log-Rank was used for comparison, from the date of the third TKI start. The events for PFS were progression to accelerated phase (AP) or blast crisis (BC) and death. For EFS, were considered loss of CHR, loss of complete cytogenetic response (CCR), TKI discontinuation for failure or intolerance, progression or death. Patients were censored in HSCT date or TKI discontinuation for cytogenetic and molecular responses evaluation, but not for OS. Mutational analysis was done with Sanger sequencing. Results 13 male (50%) and 13 female patients with median age 54 years (22-75) and median follow-up of 32 months were analyzed (1-59). Previous response to imatinib: only one patient has achieved CCR. Status before third TKI start: 19 (73%) less than partial cytogenetic response (PCyR); 2 (7,7%) PCyR; 4 (15,5%) CCR;and 1 (3,8%) clonal evolution. 8/18 (42%) patients presented the following mutations: F317L (2), E255V (1), Y253H (1), M351T (1), M244V (1) e F359V (2). The median time of the third TKI treatment was 171 days (15-1140). Responses to third line TKI: CHR 17/19 (89%) in CP; 3/3 AF; 0/4 BC; CCR: 3/19 in CP (2 pts at 3 months and one in the 12º month); 1/3 AP; 0/4 BC; MMR: 4/19 in CP; 1/3 AP; 0/4 in BC. Nine lost CHR (45%) (8 in CP and one in AP). At 3 months, 1/21 pts (4.5%) achieved MMR; 2/13 (15%) at 6 months, while at 12 months 1/11 (9%) had MMR. At 3 months 11/21 had a BCR-ABL/ABL(IS) >10% and at 6 months 10/13 >1%. Three patients progressed to advanced phases (11,5%) ; 6 (23%) died (4 due to LMC, 1 acute myocardial infarction and 1 from graft vs host disease after HSCT; one lost follow-up. Third line treatment was discontinued in 12 (46%) pts: 6 for resistance, 2 death; 2 intolerance and resistance; 1 intolerance and one loss of follow-up. OS, PFS and EFS were 69%, 68% e 30% respectively(figure 1). OS was inferior in AP and BC in comparison with CP (0% vs 50% vs 90% - p< 0,0001)(figure 2). In summary responses may be obtained with dasatinib or nilotinib after 2 TKI failure, but they are not durable in advanced phases. Besides the results, that is an alternative for disease stabilization until the identification of a suitable donor or for patients with no performance status for HSCT. Disclosures: No relevant conflicts of interest to declare.

Introduction The majority of chronic myeloid leukemia (CML) patients (pts) in chronic phase (CP), present satisfactory response to imatinib treatment. However, 25-30% of these pts exhibit suboptimal response or treatment failure. The probability of achieving optimal response may be related with several factors. The human organic cation transporter 1 (hOCT1, SLC22A1), an influx transporter, is responsible for the uptake of imatinib into chronic myeloid leukemia (CML) cells The aim of this study was to analyze hOCT-1 levels at diagnosis of CML patients and correlate with cytogenetics and molecular responses. Methods hOCT-1 expression was evaluated in 58 newly diagnosed CML pts. Pts were treated with imatinib 400-600mg in first line. Samples were collected from peripheral blood at diagnosis and RNA was obtained from total leucocytes. For cDNA synthesis, 3 ug of RNA was used. hOCT-1 expression was evaluated by real-time PCR with TaqMan probe SLC22A1 (Applied Biosystems) and endogenous GAPDH control. The results were analyzed using 2-ΔΔCT. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter if CCR was achieved. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RT-PCR (RQ-PCR). Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1%. Results 58 CML pts, 60% male, median age of 46 years (19-87) were evaluated, 71% in chronic phase (CP), 21% in accelerated phase (AP) and 5% in blast crisis (BC). The mean and median of hOCT-1 transcript levels in the total group was 2.03 and 0.961 respectively (0.008–19.039) and CP pts was 1.86 and 1.00 (0.008-10.34).The median duration of imatinib treatment was 27 months (1-109) and 96.6% achieved complete hematological response, 79.3% complete cytogenetic response and 69% major or complete molecular response. The regression analysis showed correlation between higher transcript levels of hOCT-1 and BCR-ABL transcripts Conclusions In this report, we found that high hOCT-1 expression was predictive of BCR-ABL transcripts Disclosures: No relevant conflicts of interest to declare.

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 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.

The incidence of Grade 3 (hemoglobin [Hb] level 6.5–8 g/dL) or Grade 4 (Hb level < 6.5 g/dL) anemia was reported to be 3% with imatinib therapy for newly diagnosed CML in the IRIS trial, with a median follow-up of 19 months and 7% for chronic-phase (CP) chronic myeloid leukemia (CML) after IFN-α failure. However, there is few data regarding development of anemia after long-term follow-up and its causes. The aim of this work was to evaluate the incidence of anemia after at least two years of treatment of CML patients in CP treated with imatinib and to identify other contributing causes of anemia in this population. Patients and Methods Between 2006 and 2010 121 patients with CML were treated with imatinib 400-800 mg in our center. Forty-six patients were excluded from the analysis for accelerated phase at diagnosis (n=20), discontinuation of imatinib before 2 years of treatment (n=21) and 5 for other causes. A total of 65 patients were evaluated. Hb levels were analyzed at diagnosis, at imatinib start, and at two years of imatinib. Anemia developed after the second year of imatinib therapy was classified as grade 1 (Hb level < 10.0 g/dL), grade 2 (Hb level of 8.0 to < 10 g/dL), or grade 3 (Hb level of < 8.0 g/dL) for the purpose of treatment evaluation according to National Cancer Institute (NCI) Common Toxicity Criteria - Version 4.0. Other comorbidities were analyzed. Results The median age was 53 years (24-86 years), 34 male (52%). 38% of the patients did not present other medical conditions. Comorbidities found in 62% of the patients: systemic arterial hypertension (31%), diabetes mellitus (6.7%), cardiopathy (5%), hypothyroidism (6.1%) and renal failure (only one patient with renal dysfunction in dialysis). Two patients received interferon: one prior imatinib and another during pregnancy, requiring interruption of imatinib in this period. Patient’s responses: 54 (83%) achieved complete cytogenetic response (CCR) and 10 lost response; eight of them developed anemia; 7 had (10.7%) partial response, 1 minor response (1,5%), 1 no response and 1 was not available. Fifty-three patients (81.5%) achieved major molecular response, with loss of response during follow-up in 8 cases (12.5%), seven of these developed anemia.The median Hb (g/dL) level was 12.1, 13 and 13.2 at diagnosis, at imatinib start and at 2 years of treatment respectively. Most of the patients developed Hb bellow the reference level (73.8%; n = 48) during the follow-up, after the second year of treatment. Anemia classification according to NCI criteria: 41 cases presented grade 1, 6 (12.5%) grade 2 and one grade 3. Of these, 68.7% (n = 33) had no etiological causes for the anemia, five patients (10.4%) were resistant, two (4.2%) were not adherent to treatment and in 8 cases (16.7%) the etiology of anemia was identified: hypothyroidism (n=1), erosive gastritis (Helicobacter Pylori positive)(n=1), B12 vitamin deficiency (n=2), HIV/AIDS (n=1), pulmonary tuberculosis (n=1) and renal failure (n=2). Fourteen (29.2%) patients had normalization of Hb levels without medical intervention and the majority (56.2%) still persists with the anemic status until the last follow-up. Four (8.3%) patients had resolution of the anemia after switching to a second generation inhibitor and 3 (6.2%) showed clinical improvement after treatment of the cause. In summary, several degrees of anemia may occur in patients with CML on long-term imatinib therapy, most of them not severe. Regular hematological follow-up is required to identify causes not CML related that can be corrected or if related to imatinib toxicity, dose modification or change of therapy. It should be emphasized the importance of investigating secondary causes for anemia, especially in patients with good adherence to treatment and satisfactory therapeutic response. Disclosures: No relevant conflicts of interest to declare.

Several studies demonstrated the prognostic significance of an early molecular response in chronic myeloid leukemia (CML) patients (pts) treated with imatinib in first line or other tyrosine kinase inhibitors. Aims: The aim of this study was to evaluate the impact of early molecular responses, at 3 and 6 months after treatment with imatinib in CML pts and correlate these responses with CCR, MMR, overall survival (OS) and event free survival (EFS). Patients and Methods Between February 2006 and June 2012, 95 adult pts with newly diagnosed CML in chronic phase (CP) received imatinib 400mg/daily. CP was defined using WHO 2008 criteria. All pts received a short course of hydroxiurea until imatinib was available. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RT-PCR (RQ-PCR). Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1% (IS). Statistical analysis: OS was measured from imatinib start until date of death or last visit. An event was defined as death from any cause. EFS was measured from imatinib start until the first event (loss of complete hematological response (CHR); complete cytogenetic response (CCR), progression to advanced phase, death or imatinib discontinuation) or last visit. OS and EFS rates were calculated using Kaplan-Meier method and log-rank test to compare its curves. The MMR probabilities according to molecular responses at 3 and 6 months were calculated by c2 method and cumulative incidence, considering as competitive event death or progression, before the event. Results 95 pts were analyzed, 57 (60%) male, with a median age of 47 years (17-79); Sokal score: high, intermediate and low was 30, 38.6 and 31.4% respectively; EUTOS scores was 81.5% low and 18.5% high. The median time from diagnosis until imatinib therapy was 1 month (0-5) and the follow-up was 39 month (3-89). Responses: 88% achieved CHR; 50% CCR and 53% MMR. One patient progressed to advanced phase during follow-up, while on imatinib treatment. 21 (22%) pts discontinued imatinib due to intolerance (47.6%); resistance (42.9%), death (4.8%) and Allo-HSCT (4.8%). At 3 months from the start of therapy, 30/64 (46.8%) achieved CCR, 15/64 (23.4%) partial cytogenetic response and 20/64 (31.2%) less than partial; by RQ-PCR, 72.3% (68/94) achieved at 3 months BCR-ABL transcripts ≤10% and 27.7% (26/94) > 10%. At 6 months 55.2% (48/87) had BCR-ABL transcripts ≤ 1% and 44.8% (39/87) >1%. The OS was 97% (95%CI: 95-99%) and EFS 63% (95%CI: 52-75%).There was no significant difference in OS and EFS in pts with RQ-PCR > 10% vs ≤ 10% at 3 months (figure 1), but pts with BCR-ABL transcripts > 10 and >1-10% at 6 months had an inferior EFS in comparison with pts with BCR-ABL transcripts ≤ 1% (41%,50%,89% respectively - p= 0.005), (figure 2). The CI showed that CCR pts at 3 months reached MMR earlier at 24 month (54% vs 18%, p=0.03), as well as CCR pts at 6 months, albeit no significance statistically (52% vs 37%, p= 0.16). For RQ-PCR at 3 months, pts with BCR-ABL transcripts 0-1% had a probability of 88% to achieve MMR, 1-10% had 52% and >10% 42%, p< 0.0001 (figure 3). In conclusion, our results show that early molecular responses are predictive of achieving MMR and BCR-ABL transcripts Disclosures: No relevant conflicts of interest to declare.

Rationale for Study Primary Myelofibrosis (PMF) mainly affects individuals in their sixth decade of life or older. It is the most difficult myeloproliferative neoplasm to be defined, as the phenotype mimics a variety of hematological and non-hematological diseases. No symptom or sign is specific for PMF. Many patients are asymptomatic at presentation and the disease is usually detected by the discovery of a splenomegaly or abnormal blood counts during a routine examination. The only treatment with curative potential for PMF is allogeneic stem cell transplantation. This should be reserved only for high-risk patients, after careful consideration, taking into account the option for participation in clinical trials of new drugs. It is therefore extremely important the early identification of poor prognosis patients, to offer the best therapy. In Brazil, we do not have sufficient data to assess a patient's journey between the first symptoms and diagnosis of PMF. Moreover, the real incidence/ prevalence of this disease is unknown and a sub-diagnosis rate is speculated in our country. Objectives Understand and identify the journey of patients with PMF (clinical characteristics, diagnosis process and therapeutics interventions) and the problems between the first symptoms and initiation of treatment. Methods Given the observational nature of the study and the lack of a specific hypothesis to be tested, the statistical analysis is descriptive. This partial analysis includes descriptive results of contents of the database. Categorical data are presented as frequencies and percentages. For continuous data, mean, standard deviation, median, minimum, and maximum values are presented. Results 103 patient disposition (2008-2012); all of them met WHO criteria 2008: median age: 69; 58% male and 13.6% had died. Based on medical history, professional or environmental exposure was related in 4%, but this information was lack in 25% of the medical charts. In almost 42% of the medical records, the family history was not available. Two patients had a positive family history (brother of one and sister from another one with the same disease). Most frequent reason (63,1%) for medical evaluation seeking was symptoms and fatigue happened in 67% of them. Time from symptoms onset date until the confirmed diagnosis took a median of 9.7 months. General physician was the most frequent specialist to do the first evaluation of the patient until confirmed or suspected diagnosis. Bone marrow aspiration and bone marrow biopsy were performed in all patients; mutation status for JAK V617F in 82.5%, PCR for BCR-ABL in 71.8% and bone marrow cytogenetics in 64.1%. According to IPSS (78 patients), 8.5 % were low risk, 63.4% intermediate-1 63.4%, intermediate-2 28.1% and none high risk. Anemia was the most frequent laboratorial finding (67%) and most of the patients were treated with hydroxiurea (75.7%). Conclusions Although most of the patients have apparently the diagnosis of PMF, it took almost 10 months to be done. An expressive number of medical reports do not show a complete history, while 20-30% of them don’t have all the laboratory tools for a precise diagnosis. It is important to know Brazilian needs to improve diagnostic and provide a better treatment, since we are facing new drugs for PMF. Disclosures: No relevant conflicts of interest to declare.

Summary Hypogamaglobulinemia is frequently associatedwith B-CLL rendering the patients prone to bacterialinfections. The use of purine analogs that causedepletion of T lymphocytes in the treatment of thisdisease, increases the spectrum of susceptibility ofthese patients to oportunistic agents. We report acase of a young B-CLL patient treated with 2-CdA,who presented multiple infectious complicationsduring the course of his disease associated with asevere cellular immune deficiency.Rev.bras.hematol.hemoter.,2000, 22 (3):420-423 Key words : Chronic lymphocityc leukemia,cryptococcal meningitis, cladribine Referencias Bibliograficas 1. Lorand-Metze I. Leucemias linfoidescronicas. Diagnostico, estadiamento, fatoresprognosticos e tratamento. Ser. Monogr.Esc. Bras. Hematol. 1999; 6; 18-22.2. Keating M.J. Translational research inchronic lymphocytic leukemia. AmericanSociety of Hematology 1999; 249-54.3. Cheson B.D. Infectious and immunossupressivecomplications of purine analog therapy. J ClinOncol

Abstract 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.

Abstract 3787 Dasatinib is effective at inducing complete cytogenetic response (CCyR) in approximately half of chronic myeloid leukemia (CML) patients treated in the chronic phase (CP) after failing imatinib. The aim of this work was to evaluate the role of molecular monitoring in predicting the outcome of patients treated with dasatinib after imatinib therapy. Between 2008 and 2012, it was analyzed 55 consecutive patients with CML with imatinib intolerance or resistance treated in a single center. Patients received dasatinib (50–140 mg) as second or third line therapy. Cytogenetic analysis was performed at 3, 6, 12 and 18 months after dasatinib introduction. BCR-ABL1 transcripts were measured in the blood at 3 months intervals using real-time quantitative PCR (RQ-PCR). Results were expressed as percent ratios relative to an ABL1 internal control. Major molecular response (MMR) was defined as a transcript level ≤ 0.1% on the international scale. Kinase domain (KD) mutations were performed before starting therapy and/or after dasatinib resistance. The probabilities of overall survival (OS), progression-free survival (PFS), and event-free survival (EFS) were calculated using the Kaplan-Meier method. An event was defined as the loss of a CCyR or complete hematologic response, progression to AP and BP, death, or discontinuation of dasatinib. The probabilities of cytogenetic and molecular responses were calculated using cumulative incidence (CI) and x2method. Results: 33 patients were male (60%) and 22 female (40%), with median age of 48 years (15–81). At diagnosis Sokal scores were low for 11/34 (32.4%), intermediate for 6 (17.6%) and high for 17 (50%) (21 NA). Thirteen patients had a previous CCyR with imatinib. The median time between diagnosis and dasatinib treatment was 25 (2–223) months. The median follow-up was 12 months. Disease phase at beginning of dasatinib treatment: 32 (58%) CP, 13 (24%) accelerated phase (AP) e 10 (18%) blast phase (BP). Eight-seven percent achieved RHC, 55% CCyR and 38% MMR. At 3 months 67% (25/37) had BCR-ABL1/ABL1 transcript ratio '10%, at 6 months 48% (14/29) ≤ 1% and at 12 months 27% (6/22) RQ-PCR ≤ 0.1%. After introduction of dasatinib, patients with the 3-month BCR-ABL1/ABL1 transcript ratio of >10% had a lower chance of achieving CCyR (12.5% vs 81.5%, p= 0.001) and MMR (8.3% vs. 58.3%, p= 0.005). Patients with the 6-month BCR-ABL1/ABL1 transcript ratio of >1% had a lower chance of achieving CCyR (8% vs. 75%, p= 0.01) and MMR (26.6% vs. 64.2%, p=0.06) compared with patients with ratio ≤ 1%. The probability of OS, PFS and EFS in 48 months while on treatment was 83%, 70% and 29%, respectively. PFS was 88%, 76% e 11% in CP, AP and BP respectively (p< 0.0001). EFS was 36%, 32% e 10% in CP, AP and BP (P< 0.0001). Dasatinib was discontinued in 26/55 because of resistance (12), intolerance (5) or transplant (1). BCR-ABL KD mutations were detected in 13/38 cases, two before (L387M e M351T) and 11 during dasatinib treatment (T315I-6, M244V-2, E255V-1, E499E-1, M351T-1). Patients with mutations had an inferior EFS (p=0,05). In conclusion, this study indicates that evaluation of molecular response at 3 and 6 months can identify patients with less chance of response to dasatinib in patients with imatinib failure. The early identification of patients with poor outcome is important for planning future treatments. Disclosures: No relevant conflicts of interest to declare.

Abstract 5125 The TOB1 gene is a transcription factor responsible for the transduction of the gene ERBB2. It is a member of a family of cell suppressor proliferation proteins called TOB/BTG1 family; also, this gene operates on the inhibition of neoplastic transformation. The TOB1 gene presents a decreased expression in several types of cancer such as lung, breast, thyroid and stomach cancer. However, the function of this gene in chronic myeloid leukemia (CML) remains unknown. Aiming to evaluate the inhibition of gene TOB1 into BCR-ABL positive cells and trying to elucidate the molecular mechanisms associated with the inhibition of this gene in the CML we proceed to a more detailed study of this gene. The inhibition of this gene in K562 cells was performed using specific lentivirus. The effect of silencing TOB1 in the proliferation of K562 cells was assessed by the MTT assay after 48 hours of culture; in shTOB1 the proliferation was increased in comparison with shControl cells. To evaluate the synergistic effect between the inhibition of kinase tyrosine activity of BCR-ABL and the inhibition of TOB1 we performed a treatment with different concentrations of imatinib (0. 1, 0. 5 and 1μM), but we observed the decrease in cell proliferation of shTOB1 cells to similar levels of shControl cells only at the 1μM concentration. Therefore, the TOB1 silencing increased the proliferation of K562 cells without an additional effect of a treatment with Imatinib. To analyze the clonogenicity, we performed a formation of colonies assay, in methylcellulose, to determine whether silencing TOB1 could cause a change in the clonal growth of positive BCR-ABL cells. There was no significant change in the number of colonies that grew in cell culture shTOB1 compared to shControl cells. These results suggest that silencing TOB1 in K562 cells may not change the clonogenicity. In the assessment of cell cycle, the flow cytometry analysis revealed a significant accumulation of K562 cells in S phase, with consequent reduction of cells in the G2 phase of the cell cycle in cells shTOB1 compared to cells shControl. The TOB1 gene silencing in K562 cells kept the cells in the S phase and prevented the entry of cells in the G2 phase showing that the inhibition of gene TOB1 induced an increase in proliferation of K562 BCR-ABL cells. The level of apoptosis was assessed by flow cytometry after labeling the cells with anexin-V/PI. The Imatinib treatment presented dose-response in the induction of apoptosis as expected. However, a cumulative effect with TOB1 silencing was not observed. Furthermore, the apoptosis was also assessed by assays of caspases 3, 8 and 9, which showed an increase of the caspase activity of shControl cells in relation of the shTOB1 cells, showing that inhibition of this gene also changes the level of apoptosis. These results corroborate the literature data that report the relationship of this tumour suppressor gene in signalling pathways related to angiogenesis, carcinogenesis, apoptosis and metastasis. When we relate the results obtained with the LMC, we can consider the possibility of TOB1 regulation changes be related to modification of important signalling pathways such as AKT, PI3K, STAT3 and STAT5, among others. Furthermore, the inhibition of TOB1 may be related with an increase on the number of BCR-ABL positive cells and subsequent disease progression. In conclusion, this study confirmed literature data showing that TOB1 gene works as a tumour suppressor protein in cells of many types of cancer. From this work we can infer that in CML the expression of this gene is transformed, resulting in changing of the capacity of induction of apoptosis, decrease tumour necrosis and increase cell proliferation. This work was supported by FAPESP and INCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 4415 The CML is a clonal disease of stem cells and its main feature is the unregulated production of a tyrosine kinase protein called BCR-ABL, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. Because of this, the use of tools for the study of gene expression could bring new insights in the understanding of these mechanisms in the CML. In a recent study using SSH libraries, we compared the gene expression pattern between granulocytes of health control and CML patients, and we identified the gene SEPT5 expressed only in CML patients. Although the studies in the literature, there is not a clear relationship between the expression of this gene and the development or progression of CML. SEPT5 is a member of nucleotide binding proteins called septins that were firstly described in yeast as cell division cycle regulatory proteins. This gene was reported in patients with AML translocated with MLL gene, in adult human brain and heart; it is also associated with alpha granules of human blood platelets. The aims of this study are to carry a functional analysis of SEPT5 in differents cells line and to study the relationship of this gene and the development and/or progression of CML. The gene expression evaluation was made in granulocytes, mononuclear cells and total leukocytes of CML patients and healthy blood donors in peripheral blood. It was also evaluated in bone marrow donors, in human cell lines (K562, HL60 and NB4) and in mice cell lines (BaF3/BCR-ABLp210 and BaF3T315I), performed by real-time PCR for the following genes: SEPT5, β-actin and GAPDH. Experiments were also performed to verify the difference between the chemotaxis of granulocytic cells from controls and patients by ELISA. Data were analysed statistically using the ANOVA followed by Dunnett’s test – P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethic Committee of the Faculty of Medical Sciences of University of Campinas. The gene expression of SEPT5 was evaluated by real time PCR using the same samples used in the library construction to validate the results found in the SSH library. The data confirmed our previous results, showing that the SEPT5 expression is increased in all cells of patients compared to controls. The same results were observed when we studied the expression comparing individually patients and health blood donors, suggesting that this protein could be increased in all human cells that present the translocation BCR-ABL. The level of expression of this gene in HL60 and NB4 was significantly lower than in K562 cell line. The experiments with mice cell lines showed a higher expression of this gene in BaF3T315I when compared to BaF3BCR-ABLp210. We obtained a significant expression difference in all experiments (p Disclosures: No relevant conflicts of interest to declare.

Abstract 1701 Mutations within the BCR-ABL domain are the most frequent mechanism of imatinib (IM) resistance. The second generation inhibitors (SGI) are indicated for imatinib intolerance or resistance and the initials trials showed similar response rates in IM resistant patients after IM failure, independent of mutation status, with exception of T315I. The aim of this work was to report the frequency of BCR-ABL mutations in chronic myeloid leukemia (CML) patients of a Latin American population and to evaluate the clinical impact of the presence and type of mutations in overall survival (OS), progression free survival (PFS) and in the response to second generation inhibitors (SGI). Methods: retrospective analysis of 17 centers from Latin America. A total of 529 CML patients with mutation analysis performed in samples collected between 2002 and 2011 were included. Mutations were detected by direct sequencing from bone marrow or peripheral blood samples, collected from CML patients. After imatinib resistance, patients were treated with SGI (69%) or other treatments. Overall survival (OS) was calculated from date of mutation detection until last follow-up or death, and progression-free survival (PFS) from date of mutation detection until progression to accelerated phase or blast crisis, last follow-up or death. Survival curves were calculated using the log-rang test (SPSS 14.0 software).Results: the median age of patients at diagnosis was 45 years (5–87). 81% were in chronic phase (CP), 13% in accelerated phase (AP), 6% in blast crisis (BC). According to Sokal score, patients were stratified in low (36%), intermediate (30%) and high risk (34%); 36% had previously used Interferon. The median time from diagnosis until Imatinib treatment was 8 months (0–310) and from Imatinib start until mutation detection was 31 months (1–104). Mutations were found in 188 patients, in the following sites: P-loop (75/40%), nucleotide contact site (34/18%), catalytic domain (44/23%), A-loop (11/6%) and others (24/13%). The most frequent mutations detected were: T315I (30/16%), F359V/C/I (27/14%), M244V (18/9.6%), E255K/V (17/9%), G250E (17/9%), Y253H/F/Y (15/8%), M351T/L (12/6%); Ten patients presented concomitant mutations. On dasatinib treatment 29 mutations (27% T315I) were detected whereas 18 during nilotinib (16.5% T315I). Overall survival in the total group was 61% (95%CI: 51–71%) with a median time of 12 months. There was a significant difference in OS and PFS between non-mutated and mutated patients (76% vs 44% and 64% vs 44% respectively (P= 0.008 and P= 0.001). There was no difference in survival comparing P-loop mutations and others, excluding T315I. Patients with T315I mutations had a poorer outcome in comparison with other mutations (OS 21% vs 62%; PFS 35% vs 55%) (P= 0.04 and 0.06, respectively). In the group with BCR-AL mutations, OS was superior in patients that received a SGI in comparison with other treatments after resistance (50% vs 36% P= 0.007). One hundred patients (19%) died due to: disease progression (72), infections (8), graft versus host disease (2), central nervous system bleeding (2), cardiac failure (1), second neoplasia (1). 14 causes were not reported. Conclusions: Patients with BCR-ABL mutations had an inferior OS and PFS. T315I mutations were associated to a poor outcome. Treatment with SGI prolonged survival of patients after imatinib failure. Disclosures: Pagnano: Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Boquimpani:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Pasquini:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Spector:Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract 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.

Abstract 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.

Abstract 2215 Poster Board II-192 The exact action mechanism of the protein BCR-ABL is unknown, studies in vitro and in animal models showed that the activity of protein tyrosine kinase (TK), by itself, is sufficient to cause the development of CML. Thus, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. The use of methods to detect the difference in gene expression between CML and normal granulocytes could bring new insights in the understanding of these complex mechanisms, highlighting new therapeutic targets for the CML treatment. Evaluation of the differential gene expression between CML and normal granulocytes using the Subtractive Suppression Hybridization (SSH) and investigation of the involvement of a set of genes in modulating the development of CML. The SSH libraries were constructed using a pool of granulocytes RNA's extracted from CML patients and from healthy blood donors. Real-Time (RT-PCR) was used to validate the results and evaluation of gene expression in granulocytes and in mononuclear cells in the pool of patients and controls used for the construction of libraries. The expression of the RUNX1 gene, whose expression was the most differential between the libraries, was evaluated in mononuclear cells of patients with CML using RT-PCR. The comparison between granulocytes of CML patients and control granulocytes showed 39 genes exclusively expressed in CML and 169 genes in controls. For validation, we evaluated the expression of genes RUNX1, KPNA6, TOB1, SEPT5, CDC42SE, ITCH, MIER and GP1BA by RT-PCR in the pool of patients and controls used for the construction of libraries. Among these genes, the RUNX1 gene showed substantial difference, and was also studied in mononuclear cells from 20 patients with CML in different stages of the disease and using different types of treatment with TK inhibitors. The expression of this gene was highly altered in patients in advanced phase of disease when compared with patients who are responding to treatment. The genes identified in this study may be related to the development and progression of CML. Among them, the RUNX1 gene was shown to be one of the most promising, since the occurrence of chromosomal translocations in this gene has been associated with different types of acute leukemia – however its association with CML is not yet clear. The results showed a relationship between the expression of this gene and progression of the disease, beyond the identification of several genes that may lead to a better understanding of CML and help in identifying new therapeutic targets. This work was supported by FAPESP. Disclosures: Off Label Use: Development of TKI in the treatment of CML.

Abstract 3260 Poster Board III-1 Background: CML treatment with tyrosine kinase inhibitors induces high and durable rates of complete cytogenetic response. Despite treatment efficacy, a significant proportion of patients develop resistance to these drugs. We measured gene expression profiles in an attempt to identify gene pathways that may be associated with dasatinib resistance. Patients and Methods: Mononuclear cells were separated from peripheral blood samples from seven CML patients resistant to imatinib, collected prior and after dasatinib treatment. Three patients who achieved partial cytogenetic response (Ph-positive cells: 1% - 35%) within twelve months were considered responders (R), whereas four patients who failed to achieve PCyR within 12 months of treatment were classified as non-responders. RNA samples prepared from peripheral mononuclear cells were hybridized to Agilent Technologies 4×44K Whole Human Genome Microarrays (WHGM) and 4×44K intronic-exonic custom oligoarrays. The latter was developed by Verjovski-Almeida's group (Nakaya et al, Genome Biology 2007, 8:R43) and contains sense and antisense probes that map to intronic regions in the human genome representing totally (TIN) and partially (PIN) intronic non-coding RNAs (ncRNAs), in addition to probes for the corresponding protein-coding genes of the same loci. Raw microarray data were normalized by the Affy package in statistical R language implemented in the Bioconductor platform. Each sample was labeled in replicate with Cy3 or Cy5 and the two were considered technical replicates. Two independent statistical approaches SAM (Significance Analysis of Microarrays) and Golub's discrimination score (SNR, Signal to Noise Ratio, with permutations) were performed to identify differentially expressed transcripts between responder and non-responder patients. For the intronic-exonic platform, the analysis parameters were FDR 10%, SNR>1.5 and p1.5 and p Results: We identified 34 ncRNAs with altered expression (26 over and 8 underexpressed in responders) in pre-treatment samples and 33 ncRNAs (20 over and 13 underexpressed in responders) in post-treatment samples. Functions associated with protein-coding genes from the same genomic loci as those of the intronic differentially expressed ncRNAs were: regulation of transcription (PRMT5, SOD2, SSBP3, BCL7A, MLL), signal transduction (PRKCB1, RASGRP2, NF1, PXN) and apoptosis (BCL2, PCSK6, TNFAIP8, EIF4G2). WHGM platform data analysis showed 63 and 250 protein-coding genes differentially expressed in pre and post-treatment samples, respectively. We observed a higher number of protein-coding genes with altered expression after treatment in the following functions: cell communication, immune response and metabolic process (p Conclusions: Overall, these findings indicate that protein-coding genes and intronic ncRNAs may be related to dasatinib resistance and response to treatment. In particular, altered expression of ncRNAs transcribed from the introns of ‘regulation of transcription' genes could be part of an important alternative mechanism of gene expression control during emergence of resistance.Support: FAPESP (2005/60266-8) Disclosures: No relevant conflicts of interest to declare.

Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.

From May 1998 to November 2006, 77 HD patients who used the regimen of high dose cyclophosphamide HD (CY) 7 or 4g/m2, methotretaxe 8g/m2 and etoposide 2g/m2 followed by AHSCT were analyzed. Their median age was 25.8 (8.8–71.5) years, 46 males and 31 females. At diagnosis 50 (65%) were stage III or IV disease, 10 (13%) had bone marrow involvement, 29 (37.7%) bulky disease and 55 (71.4%) B symptoms. Besides that, all patients were submitted to a mean of 2 (1–4) chemotherapy lives and their status were 3 (3.9%) complete remission (CR), 17 (22.1%) partial remission (PR), 57 (74%) progression disease (PD) or in non-specified sensitivity relapse. Concerning CY dose 30 (39%) received 4 g/m2 and 47 (61%) received 7 g/m2. After the HDCY 16 (20.8%) were in CR, 22 (28.6%) PR, 28 (36.3%) remained in DP and 11 (14.3%) died most in PD. After a median follow-up of 3.97 (1.13–55.9) months, 53 (68.8%) patients were submitted to AHSCT and their present status is 30 alive [18 CR, 2PR and 10 DP]; 23 dead [2 CR, 14PD and 7 related to the procedure]. Overall survival for transplanted patients was 55% in 8 years. Currently we have 33/77 (43%) alive patients, 19 CR, 3 PR, 11 PD. Overall Survival (OS) for whole group was 32%, Disease Free Survival (DFS) 64% and Progression Free Survival (PFS) 40%. Patients who were in DP or relapse prior to the HDCY (57) compared to their status after that had a significant improvement (P=0,001), their OS was 40% to CR-PR group (24) versus 16% PD group (33). In general, mortality was 44 (57%), their cause was 19 PD (43.2%), 8 (18.2%) related to HDCY, 2 (4.5%) related to HDMTX, 7 (16%) related to AHSCT, 6 (13.6%) infections, 1 (2.3%) chronic GVHD after a reduced intensity conditioning regimen transplant and 1 (2.2%) AML. Besides that, 3 patients developed MDS and 1 developed AML. In conclusion, although it had happened a significant number of toxicity related deaths, the sequence is feasible, mainly for sensitive patients and presents an acceptable response. The authors emphasize the high frequency of poor prognosis patients and occurrence of MDS/AML in 4 (5.2%) patients.

The purpose of this study was to evoluate the use of high dose cyclophosphamide HD (CY) 7 or 4g/m2 and HD etoposide (VP-16) 2g/m2 followed by AHSCT in patients affected by aggressive NHL. From January 1998 to November 2006, 106 patients, 66 males, 40 females, median age 47 (8–66) years underwent into this regimen. The diagnosis was 83 (78.3%) DLBCL, 13 (12.3%) with T-cell Lymphoma and 10 (9.4%) Mantle Cell. At diagnosis, 88 (83%) were stage III or IV, 34 (32.1%) presented bone marrow involvement, 65 (61.3%) bulky disease, 67 (63.2%) B symptoms and 45 (42.5%) were high or high intermediate risk according to IPI. Prior to HDCY patients had been submitted to a mean of 2 (1–3) therapy lines and 6 (5.7%) were in complete remission (CR), 38 (35.8%) partial remission (PR), 62 (58.5%) disease progression (PD) or relapse. Concerning CY dose 42 (39.6%) patients received 4 g/m2 and 64 (60.4%) 7 g/m2, respectively. After HDS, 13 (31%) patients who had used 4 g/m2 and 30 (47%) used 7 g/m2 were in CR (P= 0.03). After the HDCY 43 (40.6%) were in CR, 33 (31.1%) PR, 21 (19.8%) remained in PD and 9 (8.5%) died. After a median follow-up of 4.1 (1.5–57) months, 80 patients (75.5%) were submitted to AHSCT and their present status is 44 alive [33 CR, 4 PR, 7 PD]; 36 dead [4 CR, 1 PR, 2 relapse, 17 PD and 12 had death related to procedure]. Their overall survival was 45% in 8 years. Currently we have 50/106 (47%) alive patients, 35 CR, 6 PR and 9 PD. Overall Survival (OS) was 37%, Disease Free Survival (DFS) 49%, Progression Free Survival (PFS) 42%, Event Free Survival (EFS) 28%. OS by diagnosis was 42% DLBCL, 40% T-cell in 8 years whereas 20% Mantle Cell in 6 years (P=NS). OS by B symptoms patients was 22% versus 58% (P= 0.002) and EFS was 23% versus 37% (P= 0.03). Patients who were in PD prior to the HDCY compared to their status after that had a significant improvement (P< 0.001), their OS was 38% to CR-PR group (38) versus 0% PD group (24). In general, mortality was 56 (53%), their cause was 23 (41.1%) PD, 7 (12.5%) related to HDCY, 12 (21.4%) related to AHSCT, 13 (23.2%) infections and 1 (1.8%) GVHD a RIC transplant. Besides that 1 (0.9%) patient developed MDS and is alive. B symptoms at diagnosis have appeared as a predictor factor for survival. Our study suggests HDS is an efficient treatment to improve status and to reduce tumor burden. Although it presented high toxicity related mortality, we consider the treatment feasible, especially considering the patients’ poor prognosis.

Background: Imatinib is now the standard therapy for newly diagnosed CML patients. However, some patients develop resistance, mainly those treated in advanced stages. Mutations in the kinase domain of BCR/ABL are the most frequent mechanisms associated to resistance in vivo and some of them do not respond even to other kinase inhibitors (nilotinib and dasatinib). Aims: detect mutations in the kinase domain of BCR/ABL in CML patients with primary or secondary resistance to Imatinib and describe their clinical outcome after mutation detection. Patients and Methods: we evaluated 17 CML patients with primary or secondary resistance to Imatinib. After RNA extraction from peripheral blood samples, amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR, to cover amino acids 244–486. PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank). Results: We found seven mutations in ten patients resistant to imatinib: 5 in blast crisis (BC), 3 in accelerated phase (AP) and 2 in chronic phase (CP). Mutations identified: T315I (3), L248V(1), G250E(1), F359V(1), M244V(1), E255K (2) and E279K (1). Five patients were treated with dasatinib (trials CA-180–005, 006, 034 and 035). Patients with T315I mutation (3) did not respond to treatment. One is dead due disease progression and two are currently been treated with hidroxiurea in CP and AP. Patient with mutation M244V is still using dasatinib, presenting major cytogenetic response. Patient with F359V mutation was submitted to a non-myeloablative stem cell transplantation from an identical sibling donor, in AP. The transplantation was uneventful, and no acute or chronic GVHD was observed. On day +57 the chimerism analysis showed a mixed chimerism followed by a hematological relapse. Two lymphocytes infusions were done, on days +77 and +114, with no response. The F359V mutation was still present after the SCT and the patient was included in a phase II trial of dasatinib (CA180-005), which is on going and is in partial hematological response. Patients with other mutations died in blast crisis. In seven patients mutations were not found, but two patients had clonal evolution. Summary/Conclusions: BCR-ABL mutations are frequent in patients resistant to imatinib. Patients with P-loop mutations presented a worse prognosis with rapid evolution to blast crisis and patients with T315I mutation had no response to dasatinib, as recently described. We identified two patients with non-P-loop mutations with good response to dasatinib. P-loop mutations have been associated with poor prognosis, but its prognostic impact depends on patient selection and treatment received after detection of mutation. Mutations detection is helpful in deciding strategies to overcome resistance to imatinib.

High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT) is considered standard of care for patients with Hodgkin’s disease (HD) who relapse or progress after first-line chemotherapy. Recently, high-dose sequential chemotherapy (HDS) before ASCT has been evaluated, and phase II data suggest that it may improve the outcome without increasing significantly the toxicity. However, the lack of comparison between HDS and a more conservative strategy hampers any conclusion about the effectiveness of HDS. In this study, we compared in a non-randomized fashion, HDS (n=52) with a less-intensive chemotherapeutic regimen (non-HDS, n=60) followed by ASCT in patients with relapsed or refractory HD. Patients were treated at 2 Brazilian centers (HDS - UNICAMP; non-HDS - UFRJ). HDS consisted of cyclophosphamide (7 g/m2) followed by stem cell collection, methotrexate (8 g/m2) plus vincristine (1.4 mg/m2) and etoposide (2 g/m2), followed by BEAM and peripheral blood ASCT. Non-HDS patients received 2 cycles of DHAP, followed by cyclophosphamide (1,5 g/m2) followed by stem cell collection, followed by CBV and peripheral blood ASCT. There were more HDS patients with advanced disease (stage IV 40% vs. 13%, p=0.001), bulky disease (62% vs. 39%, p=0.02), and less patients in complete remission (CR) (6% vs. 18%, p=0.04). Toxic death before HSCT occurred in 5 patients in the HDS group and in none in the non-HDS group (p=0.02). Among 31 patients with progressive disease (PD) in the HDS group, 8 (25% remained in PD before ASCT, compared to 100% of 28 patients in the non-HDS group (p

Minimal Residual Disease (MRD) monitoring is recognized as a clinically useful tool for the management of Acute Promyelocytic Leukemia (APL) and has been performed by reverse transcriptase Polymerase Chain Reaction (RT-PCR) and Real-Time Quantitative Polymerase Chain Reaction (RQ-PCR). The vast majority of the published studies were conducted in developed countries within well established clinical trials, but the feasibility of MRD monitoring in developing countries is controversial. Here we describe the experience of Brazilian centers that participated in the International Consortium on Acute Promyelocytic Leukemia (IC-APL). In the present study, the participating centers were located at distances up to 2.500 km of central national laboratory and delivery sample time is less than two days. The aim of this study was to determine the feasibility and compare the effectiveness of RQ-PCR and RT-PCR in the MRD research in the context of IC-APL. We analyzed 398 bone marrow (BM) samples from 74 Brazilians patients with de novo APL; mean follow-up of 18 months. Samples were collected at diagnosis (n=74), at post-induction (n=48) after the third consolidation (n=41), and at maintenance (n=235). Standardized assays developed by Europe Against Cancer (EAC) program were used. Clinical characteristics were similar among the full cohort (patients from Brazil [n=122], Mexico [n=30], Chile [n=23], Uruguay [n=8]). Thirty-nine (52%) patients were classified as intermediate risk. PML breakpoint was bcr1 (n=45), bcr2 (n=1), and bcr3 (n=27). The median NCN of transcripts at diagnosis was 0.5151 (n=41) and 0.4690 (n=27) for the bcr1 and bcr3 subtypes, respectively. At the end of induction, there was a reduction of about 3 logs (0.0004 for bcr1 and 0.0005 for bcr3). In this stage, six discrepant cases were observed, all positive by RQ-PCR. There were no relapsed cases. 66/74 (89%) patients completed induction phase and achieved complete remission, and 8/74 (10%) died of hemorrhagic causes. The rate of molecular remission in our study was 37.5% (18/48) by RQ-PCR and 50% (24/48) by RT-PCR. Considering samples obtained at the end of consolidation phase, one discrepant result was detected as negative by RT; however it was not confirmed by RQ-PCR. Both cases did not relapse. The analysis for RQ-PCR was performed in 64% (41/64) of samples. Two patients have died of infectious diseases during the consolidation phase. All patients achieved molecular remission based on the results of RT-PCR. One patient was positive by RQ-PCR (NCN: 0.00006), but all subsequent samples were negative for both techniques, and the patient is alive, in remission. The median NCN of all samples after the third consolidation phase was 0.00001 PML-RARA/104 copies of ABL copies, regardless of the breakpoint. Within 53 patients who have completed the third cycle of consolidation, only 48 started the maintenance. 235 samples were evaluated during maintenance; 87.6% (206/235) were negative by RQ-PCR technique and 94.4% (222/235) by RT-PCR. The median NCN of all samples in the maintenance was 0.00001 PML-RARA/104 copies of ABL copies. The RQ-PCR technique proved to be predictive of relapse in three out of four cases of molecular relapse. All three cases showed positive results for RT-PCR during the early stages of the maintenance cycle and were confirmed by a second sample taken within 15 days. These results are confirmed in literature, observing that most patients who had negative PCR after consolidation relapsed after few months. In one case of molecular relapse, the RQ-PCR analysis provided much earlier warning of recurring disease, testing positive 5 and 6 months, respectively, before documentation of molecular relapse by conventional RT-PCR assay. In two cases, the negative result was performed by RT-PCR post induction, and was not confirmed by RQ-PCR, which remained positive on subsequent samples, and was confirmed once by RT-PCR after long time. According to transcripts numbers at maintenance, there was a decrease of approximately 5 logs when compared to samples after third consolidation. RQ-PCR technique was more sensitive than RT-PCR, providing earlier warning of relapse, thereby allowing greater opportunity for successful delivery of pre-emptive therapy. In sum, the implementation of the IC-APL allowed the improvement of laboratory standards in parallel to advances in clinical management. Disclosures No relevant conflicts of interest to declare.

Background: Myeloproliferative disorders are a risk factor for deep venous thrombosis. The most common venous event is deep vein thrombosis of the lower limbs, cerebrovascular thrombosis is one of the major causes of morbidity and mortality among patients with MPD and splanchnic vein thrombosis are frequent in patients with latent MPD. The recently identified Janus kinase (JAK2) V617F somatic mutation is an acquired mutation frequently found in MPD patients and has been related to a higher risk of vascular events among those patients. Objective: Determine the presence of JAK 2 V617F mutation among patients with deep venous thrombosis of the lower limbs, splanchnic veins and central nervous systems without overt MPD. Patients and methods: We accessed the medical histories of 240 adult patients with the diagnosis of deep vein thrombosis who have been treated in the Haematology Department of the State University of Campinas, 63 patients with splanchnic vein thrombosis, 126 patients with deep vein thrombosis of the lower limbs and 51 patients with central nervous system vein thrombosis. The DNAs obtained at the diagnosis of all those 240 patients were than tested for the JAK 2 V617F mutation by polimerase chain reaction followed by restriction enzyme analysis. None of them fulfilled criteria for diagnosis of MPD at the time of the thrombosis. Results: The three groups were similar with regard to age of the thrombosis, platelets and hemoglobin values at the diagnosis. There was also no diference between the three groups regarding the presence of hereditary thrombofilia and antiphospholipid antibodies. Females accounted for 47% of the patients with splanchnic vein thrombosis, 72.2% in thrombosis of the lower limbs and 74% in central nervous system (p values of 0.0013 and 0.042 respectively). The median neutrophils values at diagnosis were significantly lower among patients with splanchnic vein thrombosis compared with patients with thrombosis of the lower limbs or central nervous system (p= 0.007 and p= 0.004 respectively). The thrombotic event was provoked in 23.8% of patients with splanchnic vein thrombosis versus 57.8% in thrombosis of the lower limbs (95% CI 0.12–0.45, p Conclusion: The JAK2 V167F mutation in frequently present in cases of splanchnic vein thrombosis without overt MPD but not in case of deep vein thrombosis of the lower limbs and probability not in cases of central nervous system vein thrombosis. The further development of MPD in the positive JAK2 V167F mutation cases is yet to be determined.

Abstract 1407 Background: Aberrant expression of MLL5, BAALC, ID1, and WT1 genes is frequently associated with inferior outcome in cytogenetically normal acute myeloid leukemia patients (Damm et al. Blood 2011; 117(17):4561–8). The expression levels of these genes vary in patients with acute promyelocytic leukemia (APL), but the clinical significance of these findings remains unclear. Objective: (1) to determine if the gene expression levels of MLL5, BAALC, ID1, and WT1 are associated with clinical outcome of APL patients treated with ATRA and anthracycline-based chemotherapy, (2) to generate an integrative score (IS) based on these potential prognostic factors and clinical parameters and (3) to use this score for outcome prediction in APL. Design and Methods: One hundred and fifty APL patients (age, 15–73y) from seven different Brazilian institutions and treated according to the IC-APL protocol were included. The treatment schedule was identical to the PETHEMA-LPA 2005, except for the replacement of idarubicin by daunorubicin; ATRA treatment was initiated immediately in all cases in which the diagnosis of APL was suspected based on morphology. Gene expression profile was analyzed by Real-time PCR. Integer weights for the IS were derived from Cox proportional hazard model, using overall survival (OS) as outcome parameter. Hazard ratios (HR) for OS were calculated for each variable separately (Table 1). Variables with P 9 (high-IS). Results: The integrative weights of variables analyzed are summarized in Table 1. The IS was modeled in 137 patients (median score: 6; range, 1–17). According to PETHEMA-GIMEMA relapse risk criteria, 22%, 23% and 70% of patients assigned in the low-IS (n=46), intermediate-IS (n=57) and high-IS (n=34) groups were deemed high-risk of relapse (P Conclusions: Our results suggest that MLL5, BAALC, ID1, and WT1 expression levels are associated with clinical outcome and that the IS may become a useful tool for outcome prediction in APL. Disclosures: Lo-Coco: Cephalon: Speakers Bureau; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees. Löwenberg:Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees.

Abstract 3552 The International Consortium on Acute Promyelocytic Leukemia (IC-APL) is an initiative of the International Members Committee of the ASH that aims to improve the treatment outcome of acute promyelocytic leukemia (APL) patients in developing countries, which was launched in Mexico, Brazil, Chile and Uruguay. The protocol is identical to the PETHEMA-LPA2005, except for the replacement of idarubicin by daunorubicin. In our interim analysis, estimated 2-year overall and disease-free survivals are 80% and 90%, respectively. The 2-year cumulative incidence of relapse was 5.6%. The median follow-up among survivors was 23 months (range: 1 – 56 months). A secondary aim of IC-APL was to establish molecular monitoring for minimal residual disease (MRD) as a standard practice for APL patients in these countries and to use the results obtained to guide therapy. According to the IC-APL protocol, testing for the PML-RARA fusion transcript was to be performed at diagnosis, end of induction (optional), after the third cycle of consolidation, and every 3 months during maintenance. Considering that real-time quantitative polymerase chain reaction (RQ-PCR) provides a number of advantages compared to conventional non-quantitative reverse transcriptase PCR (RT-PCR), we retrospectively compared the results obtained by both techniques. We analyzed 400 bone marrow (BM) samples from 97 patients with de novo APL enrolled in the IC-APL protocol in Brazil. Of the 97 patients, 78 were considered eligible. The mean age was of 35.8 years with 46 males. Among eligible patients, 49 corresponded to bcr1 ; one to bcr2 and 28 to bcr3 subtype of PML breakpoint. To quantify the fusion transcript PML-RARA we used standardized assays developed in the Europe Against Cancer (EAC) program, normalized to the expression of the ABL gene. The results were compared to plasmid standards (Ipsogen, Marseille) and expressed as Normalized Copy Numbers (NCN). Follow-up samples were considered PCR positive when PML-RARA transcripts amplified with Cycle Threshold (Ct) values of ≤40 in at least 2 out of 3 replicates, according to EAC criteria. A total of 71 samples at diagnosis, 50 at the end of induction, 47 after the third consolidation, 202 during maintenance phase and 30 samples after completion of treatment were analyzed. The median NCN of PML-RARA transcripts at diagnosis was 0.5151 and 0.5092 for the bcr1 and bcr3 subtypes, respectively. At the end of induction there was a reduction of about 3 logs (0.0004 for bcr1 and 0.0005 for bcr3). In this phase, six discrepant cases were observed, all presenting positivity by RQ-PCR. None of these cases relapsed and presented consecutive negative results. Considering samples obtained at the end of consolidation, we detected one case of molecular persistence detected by both methods, and two discrepant results, one positive by RT and another by RQ-PCR. Both cases did not relapse. Among samples collected during maintenance and after the end of treatment, two patients (2.5%) relapsed. Both of them were molecular relapses, defined as the detection (in the context of morphological remission) of the PML-RARA transcript by RT-PCR in two consecutive samples collected 15 days apart. RQ-PCR analysis provided much earlier warning of recurring disease, testing positive 5 and 6 months, respectively, before documentation of molecular relapse by conventional RT-PCR assay. Figure 1 show the kinetics of NCN in these two cases. Our results reinforce that the PML-RARA transcript may be detected after induction but this finding was not of prognostic value. However, our study underlines the importance of sequential monitoring to distinguish patients likely to be cured following front-line therapy from those destined to relapse. The RQ-PCR technique was shown to be more sensitive than RT-PCR, providing earlier warning of impending relapse, thereby allowing greater opportunity for successful delivery of pre-emptive therapy. Finally, our results demonstrate that the implementation of the IC-APL allowed the improvement of laboratory standards in parallel to advances in clinical management. Disclosures: Pasquini: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Pagnano:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau.