ALBERT

Beschreibung: Background Despite the high efficacy of IM treatment in chronic myeloid leukemia (CML), some patients fail to achieve optimal response. Several studies demonstrated that IM is a substrate of membrane transporters, such as ABCB1 (P-gp, MDR1) and variations in protein expression or activity could affect the pharmacokinetics of IM by reducing or increasing its bioavailability. These alterations could be related with single nucleotide polymorphisms in ABCB1 gene. Previous data confirms that haplotypes containing the mutated alleles for ABCB1 c.1236C〉T, c.3435C〉T and c.2677G〉T showed major structural modifications that result in changes in the conformation of the binding sites of P-gp. These modifications could affect the pharmacokinetics of IM. Aim The aim of this study was to evaluate the influence of the different haplotypes for ABCB1 c.1236C〉T, c.3435C〉T and c.2677TG〉T polymorphisms in IM plasma concentration, P-gp activity and IM response from CML patients treated with standard dose of IM (400 mg/day). Methods Twenty eight patients in chronic phase of CML were selected according to the haplotypes for ABCB1 c.1236C〉T, c.3435C〉T and c.2677G〉T polymorphisms at two health centers in São Paulo, Brazil. Ten patients with ABCB1 1236CC/3435CC/2677GG haplotype comprised the wild-type group and 18 carriers of haplotypes with at least one mutated allele in each genotype for three ABCB1 polymorphisms (10 patients with 1236CT/3435CT/2677GT and 8 with 1236TT/3435TT/2677TT) comprised the mutated group. Patients were matched for IM time of use. All patients were in chronic phase of CML, treated with a standard dose of IM (400 mg/day) for a median time of 63.5±12.6 months and with complete cytogenetic response (CCyR). Major molecular response (MMR) was defined as a reduction of BCR-ABL1 transcripts levels to ≤ 0.1% in the peripheral blood standardized on the International scale. Complete molecular response (CMR) was defined as a reduction ≤0.0032% of BCR-ABL1 transcripts levels. Real-Time PCR was performed to evaluate ABCB1 mRNA expression to control gene GAPDH. P-gp functional activity was determinated by rhodamine123 efflux assay. Analysis of P-gp expression and functional activity were performed by flow cytometry. The determination of plasma concentration of IM was performed by capillary electrophoresis. Results Patients without MMR had lower plasma concentration of IM when compared to those that achieved this response (0.51 µg/mL vs. 1.42 µg/mL, P=0.001) but no association was found between the different haplotypes and IM plasma levels or ABCB1 mRNA/P-gp expression. The median of Rh123 efflux in wild-type and mutated groups was 59.1 (54.8 - 69.5) and 38.3 (27.4 - 47.9) (P

Beschreibung: 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

Beschreibung: TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors (QIN Q et al., 2012) and has been described as an emerging risk factor in hematological neoplasms (MERINDOL et al., 2014). . Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to disease biology and therapeutic targets for patients with AML. Objectives: 1) to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients. Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak¨ 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan¨ Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of β-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V-phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information . Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test. Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1) showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04) (fig 1). We found increased TWIST1 mRNA levels in bone marrow samples of 23 AML patients (3.88 ± 1.59) compared with 5 healthy controls (0.54 ±0.25) (p= 0.02) (fig 2). Patients in the highest tertile of TWIST1 expression did not show differences in percentage of blasts in bone marrow and complete remission after treatment compared with patients in low and middle tertile. Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Background: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in tumor neoangiogenesis which plays an important role in hematological neoplasias progression and prognosis. Myelofibrosis (MF) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPNs) resulting from acquired hematopoietic stem cell mutations. Most patients share the JAK2V617F mutation and may exhibit substantial overlap. The molecular mechanisms involved in MPNs pathogenesis are not completely elucidated. In this context, evaluation of genes and proteins related with angiogenesis could be useful to better understand MPN pathobiology. Aims: To investigate the mRNA and protein expressions of MMPs and TIMPs and their relationship with plasma markers of angiogenesis (VEGFA and FGF2) and JAK2V617F mutation status in MF and ET patients. Methods: Forty-three MPNs patients (MF=29 and ET=14) and 36 control subjects were studied. The controls were matched with patients according to age and gender. MPN diagnosis was defined according to the 2008 WHO criteria. The MMP2, MMP9, TIMP1 and TIMP2 mRNA expression from peripheral leukocytes and serum proteins levels were measured by real time PCR and Luminex technology. VEGFA and FGF2 concentration were measured in plasma from MPN patients and controls. JAK2V617F mutation status and allele burden and MPL W515K/L analysis were performed in total leukocytes DNA by real time PCR, using Taqman MGB probes. Results: Higher MMP2, MMP9 and TIMP1 mRNA expression were observed in MF patients compared to controls (P

Beschreibung: 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

Beschreibung: Abstract 4262 AML is a heterogeneous group of genetically diverse hematopoietic malignancies arising from blood cell progenitors and is considered an oncologic emergency. Patients present prognostic characteristics (specifically karyotype and age) that influence OS. Substantial progress achieved in AML therapy includes early chemotherapy initiation, aggressive large broad antibiotic usage, early antifungal prescription, RBC and platelet transfusions, HSCT, and development of new drugs. Most published AML data are from developed countries and only a few are from emergent ones in which the overall context is distinct. We aimed to study the effect of elapsed time from AML diagnosis to treatment (TDT) on OS in a group of patients from public Hospital Sao Paulo, Brazil. 41 consecutively AML patients were selected (23 males and 18 females, median age: 41 yrs, range 18 – 84 yrs) from Jan 1st, 2001 to Mar 31st, 2004 and last contact was on Jul 24th, 2011. AML diagnosis was set according to WHO classification and by G-banding karyotype (ISCN, 2009). Cytogenetic risk classification categories were adapted from MRC (Grimwade et al). Patients were treated with 3+7 daunorubicin (45mg/m2/d/3d), APL patients received ATRA-based regimen (Ruutu et al). Kaplan-Meier and student t-test were used. Results: 38 were de novo AML and 3 secondary, median pretreatment WBC was 9.1/μ L (range 0.5–240/ μ L), median TDT was 6 days (range 1–82 d); 26 patients were young (60 yrs). Cytogenetic risk distribution was: favorable 20.7%; intermediate 27.6%; unfavorable 27.6% and no metaphases 27.6%. 20 patients achieved CR (48.7%): 61.5% in the young group and 26.6% in the old. Median survival was 26.8 weeks (figure 1), 33 weeks for the young group and 10.7 weeks for the old (p =0.034). No statistical differences were found in Hb, WBC and platelet count among groups. Young patients tended to be treated earlier than old ones (TDT 4 days vs 11, p=0.07). An inverse correlation was seen in TDT with age, leukocyte, CR and OS. Longer TDT (〉 10 d) was associated with worse CR rates (p=0.02) and OS (p=0.04). When patients were categorized into TDT from 1–4 d (I) vs 〉 5 (II), those from I presented better OS than II (p=0.004). When TDT was longer than 7 days OS decreased even more. Hb was higher in patients with TDT I vs II (8.3 vs 7.5 g/dL, p=0.03) but WBC (p=0.34) and platelet count (p=0.75) were not different. Patients with TDT of 10 d were younger than TDT 〉10 d (median age 41 vs 70 yrs, p=0.001). For the entire AML cohort OS was 15.1% in 2 yrs and 8.6% in 7 yrs. Discussion: The results showed that less than half of the patients reached CR. Younger patients presented better CR than elderly and this is similar to other series (Appelbaum et al; Juliusson et al). Despite chemotherapy regimen younger patients presented poorer OS compared to Northern Hemisphere series (Juliusson G et al). Several reasons account for such results: poorer PS, delayed diagnosis and bleeding and infectious complications. Elderly patients who were treated presented dismal OS and notably a median TDT almost three times longer than younger. Reasons for a longer TDT in AML were: significant comorbidities, severe infectious complications, and delay to start chemotherapy due to logistics reasons and the need to involve caregivers and family members to aid in decision making process. With more comorbidity in elderly population, reluctance toward intensive therapy is common and probable medical decision is critical with this.Therefore measures to shorten the TDT should be taken: address rapid AML diagnosis, evaluate and treat comorbidities, and develop a supportive and continuous system to promote early AML diagnosis and prompt treatment. Our data suggest longer TDT, when analyzed continuously, predicted for lower CR rates and OS rates therefore we need to address identify factors and start therapeutic measures to aid decision making and improve OS in AML patients in our country. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 4903 The Philadelphia chromosome (Ph), t(9;22), is detected in around 90% of the chronic myeloid leukemia (CML) patients, but in the remaining 5–10% cases a variant type may be present. Variant Ph-chromosome is characterized by the involvement of another chromosome in addition to the 9 and 22. It can be a simple type of variant when one more chromosome is involved, or complex, in which two or more chromosomes take part in the translocation. The aim of this report is to relate the diversity of variants Ph found in CML patients at the Cytogenetic service of Fleury Lab (São Paulo, Brazil) during the past 12 years. All Ph-positive CML patients (ALL&AML were excluded) were surveyed and the variant Ph ones were listed. All the karyotype analysis were from non-stimulated short term cultures of bone marrow or peripheral blood and classified according to ISCN (1999-2009). Of the 1,071 Ph-positive cases, 967 were classic Ph, while 50 (5.17%) were variants. Regarding the variant form, 44 (88%) were simple type and 6 (12%) complex type (table 1), similar to Marzocchi et al (2011), who found 93% and 7% of each type, respectively. Some of the variant Ph translocations we detected are listed in table 1. In agreement with what Marzocchi et al reported, the most involved chromosome was the 17 (eleven times in the present report and four cases in theirs), followed in frequency by the chromosome 1 (six times), 20 (five times), 6, 11 (four times each), and 2, 10, 12 and 15 (three times each). The chromosomes not involved in the Ph translocation were: 8, 9, 18, 21, 22, X and Y (Figure 1). Among all breakpoints seen in this survey, six were repeated: 11p15, 14q32, 15q11.2, 16p13.1, 17p13 e 17q21, differing from Marzocchi et al who denied finding any recurrent breakpoint. Reid et al. (2003) listed nine recurring breakpoints in variant Ph translocations most reported by other authors, who also assert that most of breakpoints related to variant Ph occur in regions of known oncogenes or typical secondary breakpoints in other cancers. Indeed we endorse this statement, since we also found some breakpoints related to other cancers. Of the list Reid et al reported, three were found in this study as well: 12p13, 17p13 and 17q21. Some of the genes located in these regions are related to other cancers (i.e. ETV6, CD9, RARA, GAS7). However, the gene TP53, located in the region 17p13 was the only one previously reported as related to CML. Oliver M. (2007) states that this gene is involved in 20–30% of cases of blast crisis CML. Regarding the prognosis, evolution of the disease and response to the treatment, it seems that variant type of Ph translocation has no implication for survival (Marzocchi et al 2011 and Maha et al 2004. In conclusion, as we presented a large variety of variant Ph translocations, it becomes clear that there are lots of possible interactions to form variant type and most of them occur in regions already reported as oncogenes or related to cancers, indicating that there must be local phenomena favoring the predisposition to these breakpoints involvement.Table 1–Examples of variant Ph karyotypes found in this survey, showing a little of the diversity of interaction.Karyotype46,XY,t(1;9;22;16)(q32;q34.1;q11.2;p13)[2]/46,XY46,XX,t(9;22;1;13)(q34.1;q11.2;p12;q34)[17]/46,XX[3]46,XY,t(9;22;1;?3)(q34.1;q11.2;q36.3;?q26)[20]46,XX,t(1;9;22;14)(p21;q34.1;q11.2;q32)[20]46,XY,t(9;22;2)(q34.1;q11.2;q24)[8]/46,XY[12]46,XY,t(9;22;4)(q34.1;q11.2;p16)[20]46,XY,t(9;22;5)(q34.1;q11.2;p13)[20]46,XY,t(9;22;6)(q34.1;q11.2;q22)46,XX,t(9;22;7)(q34.1;q11.2;p22)[20]46,XY,t?(9;22;10;11)(q34.1;q11.2;q22;q25)[20]46,XY,t(9;22;12)(q34.1;q11.2;p13)[20]46,XY,t(9;22;14)(q34.1;q11.2;q32)[19]/46,XY[1]46,XY,t(9;22;15)(q34.1;q11.2;q11.2)[20]46,XX,t?(9;22;16)(q34.1;q11.2;p13.3)[20]46,XY,t(9;22;17)(q34.1;q11.2;p13)[20]46,XY,t(9;22;19)(q34.1;q11.2;q11.2)[20]46,XY,t(1;7)(p22;q22),t(9;22;20)(q34.1;q11;q11.2)[2]/46,XY[18]Figure 1– Distribution of chromosomes involved in variant Ph translocation, according to the chromosome region (p arm or q arm).Figure 1. – Distribution of chromosomes involved in variant Ph translocation, according to the chromosome region (p arm or q arm). Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Background: Myelofibrosis (MF) and essential thrombocythemia (ET) are clonal hematopoietic stem cell malignancies classified as myeloproliferative neoplasms (MPNs). FGF2 and TGF-β were associated with pathophysiology and progression of these diseases. Although FGF2 and TGF-β roles in angiogenesis and fibrosis are known, their mRNA expression in leukocytes of MPNs patients was not yet evaluated. Furthermore, the detailed mechanism of TGF-β signaling in the pathophysiology of these diseases remains unclear. In regular signaling process, Smads are a group of proteins critical for transmitting signals from TGF-β superfamily of the cell surface to the nucleus. Thus, Smads signaling plays an important role in the regulation of hematopoiesis and it could modulate TGF-β action in MPNs. Aims: The aim of this study was to investigate if there are differences in mRNA expression of FGF2, TGFB1 and SMADS 1 to 7 in MPNs patients and healthy controls and if it is related to JAK2 mutation status and allele burden. Methods: Fifteen primary myelofibrosis (PMF), 15 myelofibrosis post essential thrombocytemia (MPET), and 22 essential trombocythemia (ET) patients diagnosed according to the World Health Organization criteria (2008) were selected from the Hematology Department of the Federal University of de Sao Paulo and from the Hematology Division of the Catholic University of Sao Paulo. Other group with healthy subjects was also included. The control group was matched by age and gender with MPNs patients: 30 for PMF, 17 for MPET, and 34 for ET. Expressions of FGF2, TGFB1 and SMADs 1 to 7 mRNA were evaluated in total leukocytes by Real Time PCR, using Taqman assays. JAK2V617F mutation status and allele burden and MPL W515K/L analysis were performed in total leukocytes DNA by Real Time PCR, using Taqman MGB probes. Results: No difference was found between frequencies of JAK2V617F mutated subjects (P=0.520) and in allele burden percentage (P=0.415) in MPNs groups. Allele burden was also not associated with mRNA levels of studied genes in each group of MPNs, and none of the patients presented MPL W515K/L mutations. MF and ET patients had higher TGFB1 and FGF2 mRNA levels than its controls (P

Beschreibung: 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.

Beschreibung: Abstract 773 Mantle cell lymphoma (MCL) remains an incurable disease and has the worst outcome among B-cell lymphomas. Patients generally have a good response to first line treatment but most relapse and tend to have shorter responses or resistant disease. Thus, novel treatment strategies capable of providing and sustaining durable responses are clearly needed. The translocation t(11;14), a hallmark of MCL, leads to cyclin D1 overexpression and is invariably accompanied by different secondary genetic lesions that collaborate for lymphomagenesis. In a previous study, we found that several genes related to the AKT, WNT and TGFβ signaling pathways were aberrantly expressed in MCL. The role of the AKT and WNT pathways in MCL pathogenesis has been well established by other groups, but little is known about the role of the TGFβ pathway. To address this issue, we tested whether halofuginone, a small molecule with recognized anti-TGFβ and antifibrotic activity, would have cytotoxic effect against a panel of MCL cell lines. We found that halofuginone at nanomolar levels had significant cytotoxic activity against MCL cell lines as measured by the MTT assay. The IC50's for Mino and HBL-2 cell lines were 30 and 61 ng/mL at 48h, respectively, with IC50's for Jeko-1, JVM-2 and Granta-519 falling in between. Halofuginone induced apoptosis in Mino and HBL-2 cells in a time- and concentration-dependent fashion, as evidenced by annexin V/7-AAD staining by flow cytometry and electron microscopy studies. However, halofuginone failed to inhibit SMAD2 phosphorylation induced by recombinant TGFβ1 in Mino and HBL-2 cells, as shown by Western blot analysis, and co-treatment experiments with TGFβ1 failed to show antagonism, suggesting that the effect of halofuginone in MCL is not mediated by TGFβ inhibition. Cell cycle analysis of Mino and HBL-2 cells exposed to halofuginone revealed time- and concentration-dependent accumulation in G1 (83% of Mino cells at G1 upon exposure to 50 ng/mL for 24h vs. 48% in untreated Mino cells), and immunocytochemical analysis showed that this effect was accompanied by striking down-regulation of cyclin D1 protein levels starting as early as 3h after exposure to halofuginone, a finding that was reproduced in primary MCL cells. Real-time RT-PCR experiments, however, revealed up-regulation of cyclin D1 mRNA levels by halofuginone over time, suggesting a post-transcriptional mechanism for the observed down-regulation of cyclin D1 protein levels. Western blot analysis of Mino and HBL-2 cells exposed to halofuginone for 24h showed a concentration-dependent phosphorylation of GCN2, PERK and EIF2α, and up-regulation of ATF4. These findings point to an activation of integrated stress response pathways (amino acid starvation response and endoplasmic reticulum stress response) that causes a general shutdown in protein synthesis and explain, at least partially, the down-regulation in cyclin D1 levels. To further characterize the proteins targeted by halofuginone in MCL we employed a proteomic profiling approach in which differentially expressed proteins were revealed by label-free liquid chromatography tandem mass spectrometry (LC-MSE) analysis on a nanoAcquity system coupled to a Synapt MS Q-Tof mass spectrometer. A comprehensive catalogue representing 147 proteins was generated from this analysis and we found that several members of the heat shock protein family are up-regulated in Mino cells exposed to 100 ng/mL of halofuginone for 14h, the relevance of which is currently under investigation. Together, our data demonstrate that halofuginone at nanomolar levels has significant antiproliferative and cytotoxic effects in MCL cells that are induced by the activation of integrated stress response pathways. More importantly, our study provides a rationale for exploring the clinical activity of this oral agent in patients with MCL. Disclosures: No relevant conflicts of interest to declare.