Abstract: Luceno-Araujo et al use assays of mutations associated with myeloid malignancy to propose an integrative prognostic score for acute promyelocytic leukemia (ISAPL) in patients treated with all-trans retinoic acid and anthracycline-based therapy. They demonstrate that the ISAPL is superior for predicting outcomes and identifying patients who may benefit from alternative therapies to maximize their chance of a cure.

Abstract: Background: The Slit-Robo pathway has been shown to participate in the pathogenesis of several malignant diseases in addition to its physiologic role during the development of the central nervous system (CNS). However, the relevance of the Slit-Robo pathway in acute promyelocytic leukemia (APL) is presently unknown. We investigated the status of the Slit-Robo pathway in APL following the recent demonstration by Amodeo et al (CellRep. 2017) that the PML protein induces neural stem/progenitor cells migration by inhibiting the SLIT2 gene, which was associated with an increased presence of H3K27me3 in the SLIT2 promotor region. Moreover, sensitivity towards the PML-targeting drug arsenic trioxide in primary glioblastoma cells was shown to be regulated by the PML/SLIT axis. Aims: Here, we quantified SLIT2 transcript levels in bone marrow (BM) samples from APL patients and healthy subjects and determined whether they are associated with clinical and laboratory features at diagnosis and treatment outcomes. In addition, we evaluated the effect of increased SLIT2 protein on leukemic cell survival, proliferation and clonogenecity. Methods: Cell cycle distribution, proliferation index (Ki-67/proliferation curve), colony formation and rate of apoptotic cells (annexin V/PI) were evaluated in both APL cell lines NB4 (ATRA-sensitive), NB4R2 (ATRA- resistant) and four primary APL samples following the treatment with the human recombinant SLIT2 protein (50 ng/ml) for 24 to 72 hours. In addition, 88 patients (age, 18-73y) with newly diagnosed APL enrolled in the International Consortium on Acute Leukemia (ICAL) study - ICAPL2006 were included. For comparison, BM mononuclear cells from five healthy volunteers (age, 18-60y) were also included. SLIT2 transcripts was determined by qPCR and expressed as comparative Ct method. Patients were dichotomized into "low" and "high" SLIT2 expression groups using a cut off value of 1.53 based on survival receiver operating characteristic (ROC) curve and the C index analyses. The following parameters were used to evaluate treatment outcome: complete hematological remission (CHR); 5-year Disease-Free Survival (DFS) and 5-year Overall Survival (OS) rates. Results: At the end of the sixth day, in vitro treatment with SLIT2 significantly reduced the proliferation rate (P=.01) and clonogenicity (P=.01) in primary APL samples, while significantly increasing the apoptotic rate after 72 hours treatment with SLIT2 (P=.03). In accordance, SLIT2 treatment decreased cell proliferation and clonogenicity (all P 〈 .01) and increased the drug-induced apoptosis in a time-dependent manner in NB4 and NB4R2 cells (P 〈 .01). Cell cycle analysis revealed that the cell cycle was arrested in the S-phase (P=.001) with a reduction of the G2/M phase (P=.001) in NB4 after 72 hours of treatment but no such impact was observed on NB4R2. In the clinical setting, patients with APL had a higher-than-normal expression of SLIT2 (6.7-fold higher than BMMC; P=.01). Baseline characteristics were similar between patients with low and high SLIT2 levels, except for a higher white blood cell count in patients with low SLIT2 expression (P=.024). Overall, 80/94 (85%) patients achieved complete hematological remission (CR). SLIT2 transcript levels had no impact on CR rate (P=.099). The estimated 5y DFS and the CIR rates were 87% (95% CI: 80-92%) and 12% (95% CI: 7-17%), respectively. Patients with low SLIT2 expression presented a lower 5y DFS rate (79%, 95% CI: 48-92%) than those with high SLIT2 expression (96%, 95% CI: 64-97%) (P=.026). In addition, patients with low SLIT2 expression exhibited an enhanced CIR rate compared to high SLIT2 expression (21% vs 11%) (P=.03). With a median follow up of 32 months (1-101 months), the estimated 5y OS rate was 79% (95% CI: 72-84%). Patients with lower SLIT2 expression had a lower 5y OS rate (68%, 95% CI: 45-83%) compared to those with higher SLIT2 expression (84%, 95% CI: 73-91%) (P=.008). This result was consistent with the multivariable proportional hazards analysis (HR: 1.02; 95% CI: 1.01-1.03; P=.001). Conclusion: Our results suggest that SLIT2 transcript levels may predict outcomes in APL patients treated with ATRA and anthracycline-based chemotherapy. Our data show that SLIT2 treatment represses APL cell growth, colony formation and induces apoptosis in vitro and we are currently assessing the role of the Slit-Robo pathway in the hCG-PML/RARA transgenic mouse model. Disclosures Pagnano: EMS: Other: Financial support for participation in congress; Novartis: Consultancy; Shire: Other: Lecture; Abbvie: Consultancy. Tallman:Daiichi-Sankyo: Other: Advisory board; Cellerant: Research Funding; ADC Therapeutics: Research Funding; AROG: Research Funding; AbbVie: Research Funding; Orsenix: Other: Advisory board; BioSight: Other: Advisory board. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Lowenberg:Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees.

Abstract: Introduction: FOXO3A is a transcription factor shown to be involved in all-trans retinoic acid (ATRA)-induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL). Its biological activity may be significantly enhanced upon metformin, raising the possibility that ATRA and Metformin may act synergistically; which could be useful to overcome ATRA resistance. Despite progress in APL treatment, approximately 10-15% of patients will relapse after treatment with ATRA and anthracyclines and frequently present with ATRA resistance. Relapsed patients respond well to arsenic trioxide (ATO) treatment, but the cost of ATO is a significant barrier in many countries. Aims: Here, we investigated the effects of in vitro treatment with ATRA plus metformin in APL cell lines and primary blasts, and quantified FOXO3A expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Finally, we evaluated the effect of induced overexpression of FOXO3A gene in regards to cell viability and proliferation. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=4) and APL patients (age, 25-47y; n=4) were treated with metformin alone (5mM/ 10mM) or in combination with ATRA (1µM) and evaluated for cell viability. In addition, 106 patients (age, 18-82y) with newly diagnosed APL enrolled in the ICAPL2006 study were included. As controls, eight samples of bone marrow mononuclear cells (BMMC) from healthy volunteers (age, 18-60y) were analyzed. To validate our data, FOXO3A transcript levels from an independent cohort was used (31 patients from Amazonia!, Probe n. 204131_s_at, and five normal BMMC samples included in the same databank). NB4/NB4R2 (ATRA-resistant) cell lines were transduced with FOXO3A or empty vector (control). After synchronization using double thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For dose-response curves, cells were treated with graded concentrations of ATRA, ATO and metformin. For the apoptosis analysis, cells were treated with ATRA (1µM), metformin (5mM) and combination for 24, 48 and 72 hours. The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b surface levels. Results: In primary cells (from TM/APL patients) a 2-fold reduction in viable cells was observed after metformin and metformin plus ATRA treatment (P 〈 .05), compared to only 0.5-fold reduction rate using ATRA alone. In the clinical setting, APL patients expressed a lower absolute level of FOXO3A than normal BMMC (P 〈 .001). These results were corroborated in an external validation cohort (P=.004). The retrospective analysis of patients enrolled in the ICAPL2006 study revealed that the baseline characteristics were similar between patients with low and high FOXO3A levels. Low FOXO3A expression was associated with lower DFS (84%; 95% CI: 63-94%)(HR: 1.25, 95% CI: 0.64-10.4) and OS (76%; 95% CI: 63-84%) (HR: 1.43, 95% CI: 0.12-1.48). The overexpression of FOXO3A in APL cell lines was associated with reduced basal cell viability (P=.02), clonogenicity (P=.001), and proliferation (P=.001) in both APL cell lines. Subsequent cell cycle analysis showed no significant difference between FOXO3A-cells and controls in normal conditions and when treated with ATRA. Using a nonlinear regression analysis, IC50 for ATO was significantly lower for NB4-FOXO3A cells (0.27μM) than control (2.27µM), with no corresponding response for NB4R2 cell line (2µM for empty vector vs 1.46μM for NB4R2 FOXO3A). NB4 (IC50: 14mM) and NB4R2 (IC50: 4.2mM) FOXO3A cells presented higher sensibility to metformin treatment versus control group (IC50: 23mM for NB4; IC50: 22mM for NB4R2). For ATRA treatment alone, only NB4-FOXO3A presented increased sensitivity to ATRA (P=.001). In accordance, ATRA treatment (alone or in combination with metformin) increased the drug-induced apoptosis in a time-dependent manner (P 〈 .05) in NB4-FOXO3A cells, but had no effect on NB4R2-FOXO3A, which presented increased apoptosis rate only with metformin treatment. The differentiation rate was higher in FOXO3A-expressing cells (P 〈 .05). Conclusion: Based on clinical and functional data, the FOXO3A pathway could be an interesting target to overcome ATRA resistance in APL in offsetting where ATO is unavailable in low- and middle-income countries. Disclosures Tallman: BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; Orsenix: Other: Advisory board. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Lowenberg:Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract: High ΔNp73/TAp73 expression ratio is associated with lower overall survival and higher cumulative incidence of relapse in APL. ΔNp73/TAp73 expression ratio is an independent prognostic marker in APL.

Abstract: 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 〈 0.05 in univariate analyses were included in the model. Variables considered for the model inclusion consisted in 2 clinical (WBC counts, albumin levels) and 5 molecular markers (FLT3-ITD status and gene expression levels of MLL5, BAALC, ID1, and WT1). Other candidates, such as age, platelet count, gender, ECOG performance status, PML breakpoint and FAB subtype were not significant and not included in the score. The HR were converted to integer weights according to the following: variables with HR 〈 1 were excluded from analyses; variables with HR 3 1 and 〈 1.5 were assigned a weight of 1; variables with HR 3 1.5 and 〈 2.5 were assigned a weight of 2; variables with HR 3 2.5 were assigned a weight of 3. The final score was the sum of these integer weights. Based on maximally selected rank statistics, the scores were grouped into 3 risk-groups: 0–5 (low-IS), 6–9 (intermediate-IS), and 〉 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 〈 0.001). Overall, 118 (86%) patients achieved CR; the remaining 19 patients (14%) experienced early death due to hemorrhage (n=12), therapy-related infection (n=6) and differentiation syndrome (n=1). Induction mortality was significantly higher in the high-IS group (low: 2%; intermediate: 15%; high: 26%) (P=0.001). CR was achieved in the low-, intermediate-, and high-IS group in 98%, 84%, and 73% of the patients, respectively (P=0.007). With a follow-up of 24 months among survivors, patients assigned in the high-IS group had a lower 2-y OS rate (63%) compared with those in the intermediate- (80%) and low-IS groups (97%; P 〈 0.001). Eight relapses were recorded. The IS was not predictive of relapses (P=0.351). 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: Introduction: Described as a well know marker of worse prognosis in acute myeloid leukemia (AML), MN1 overexpression has been associated with inv(16) or EVI1 overexpression (Heuser et al., Blood 2007). The promoter region of the MN1 gene has Retinoic Acid Response Elements (RAREs), and higher levels of MN1 expression have been associated with decreased response to retinoic acid (RA) in vitro. Nevertheless, in the context of acute promyelocytic leukemia, little is known about MN1 gene expression and functionality in vivo. Aims: Here, we investigated the effects of in vitro treatment with RA plus arsenic trioxide (ATO) in APL cell lines and primary blasts overexpressing MN1. Additionally, we quantified MN1 expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=2) and APL patients (age, 36-45y; n=2) were transduced with MN1 or empty vector (EV, control) to evaluate cell proliferation, differentiation and apoptosis. Confirmatory assays were performed using transduced NB4 and NB4R2 (RA-resistant) cell lines. After synchronization using double thymidine block, transduced cells were submitted to proliferation and clonogenic (treated with RA and ATO, as well) assays. To evaluate the apoptotic rate, cells were treated with ATO (1 µM) alone or in combination with RA (1 µM each), for 24, 48 and 72 hours. The granulocytic differentiation in response to RA treatment alone (1 µM) or in combination with ATO (1 µM) was evaluated based on the CD11b and CD11c surface levels. In addition, 116 patients (age, 18-82y; 51% males) with newly diagnosed APL enrolled in the ICAPL2006 study were included. To validate our data, Bootstrap resampling procedure with 1000 repetitions from the original database was performed to assess the model bias. Results: Primary APL cells transduced with MN1 (from TM/APL patients) presented higher proliferation rates compared to controls (P 〈 .05). Similarly, the overexpression of MN1 in APL cell lines was associated with increased proliferation (P=.001) and clonogenicity (P 〈 .05). Furthermore, NB4-MN1 cells are able to form colonies in the presence of RA (1 µM) (P 〈 .01) but not under ATO (1 µM) treatment (P 〉 .05), while NB4R2-MN1 cells were able to form colonies in the presence of ATO (P 〈 .05). To investigate whether MN1 promotes resistance to drug-induced apoptosis, we treated lentivirally transduced cells with RA plus ATO for 72 hours. No differences were founded between the MN1-transduced and the EV cells (P 〉 .05). In accordance with our results using primary APL samples, ATO treatment (alone or in combination with RA) does not modulate the drug-induced apoptosis in a time-dependent manner in NB4 and NB4R2-MN1 cells (P 〈 .05). For NB4 cells, the differentiation rate was lower in MN1-expressing cells under RA alone or in combination with ATO for 48 hours (P 〈 .05), although these effects were abrogated after 72 hours of RA and ATO treatment. In contrast, NB4R2-MN1 cells exhibited decreased differentiation rate at 48 and 72 hours in presence of RA alone or in combination with ATO, in comparison with EV cells (P 〈 .05). In the clinical setting, the retrospective analysis of patients enrolled in the ICAPL2006 study revealed that the baseline characteristics were similar between patients with low and high MN1 levels. According to PETHEMA/GIMEMA criteria for predicting relapse, 34% and 46% of patients assigned to the low- and high-MN1 groups were deemed high-risk patients, respectively (P=.045). High MN1 expression was associated with lower 5-y Disease-Free Survival rates (74%; 95% CI: 69-91%)(HR: 2.61, 95% CI: 0.86-8.36) and Cumulative Incidence of Relapse (25%; 95% CI: 13-34%) (HR: 2.27, 95% CI: 0.75-6. 8). Bootstrap analysis for internal validation resulted in an AUC (0.63, 95% CI: 0.57-0.809) very similar to the original described data. Conclusion: We show that MN1 is relevant for RA-induced differentiation both in vitro and in vivo and may be involved in RA-resistance. Additionally, treatment with ATO circumvented the differentiation blockage in MN1 cells. Disclosures Pagnano: Sandoz: Consultancy; Pint Pharma: Consultancy; Abbvie: Consultancy. Tallman:International Conference in Leukemia: Honoraria; 14th Annual Miami Cancer Meeting: Honoraria; New Orleans Summer Cancer Conference: Honoraria; Indy Hematology Review: Honoraria; ADC Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding; Cellerant Therapeutics: Research Funding; Orsenix: Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Mayo Clinic: Honoraria; Rigel: Membership on an entity's Board of Directors or advisory committees; Nohla: Membership on an entity's Board of Directors or advisory committees, Research Funding; Salzberg Weill Cornall MSKCC Seminar in Hematologic Malignancies: Honoraria; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Oklahoma Medical Center: Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy; Danbury Hospital Tumor Board: Honoraria; Hematology Oncology of Indiana: Honoraria. Dillon:Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria. Heuser:Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding. Löwenberg:Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees.

Abstract: The KMT 2E ( MLL 5 ) gene encodes a histone methyltransferase implicated in the positive control of genes related to haematopoiesis. Its close relationship with retinoic acid–induced granulopoiesis suggests that the deregulated expression of KMT 2E might lead acute promyelocytic leukaemia ( APL ) blasts to become less susceptible to the conventional treatment protocols. Here, we assessed the impact of KMT 2E expression on the prognosis of 121 APL patients treated with ATRA and anthracycline‐based chemotherapy. Univariate analysis showed that complete remission ( P  =   0·006), 2‐year overall survival ( OS ) ( P  =   0·005) and 2‐year disease‐free survival ( DFS ) rates ( P  =   0·037) were significantly lower in patients with low KMT 2E expression; additionally, the 2‐year cumulative incidence of relapse was higher in patients with low KMT 2E expression ( P  =   0·04). Multivariate analysis revealed that low KMT 2E expression was independently associated with lower remission rate (odds ratio [ OR ]: 7·18, 95% confidence interval [ CI ]: 1·71–30·1; P  =   0·007) and shorter OS (hazard ratio [ HR ]: 0·27, 95% CI : 0·08–0·87; P  =   0·029). Evaluated as a continuous variable, KMT 2E expression retained association with poor remission rate ( OR : 10·3, 95% CI : 2·49–43·2; P  =   0·001) and shorter survival ( HR : 0·17, 95% IC : 0·05–0·53; P  =   0·002), while the association with DFS was of marginal significance ( HR : 1·01; 95% CI : 0·99–1·02; P  =   0·06). In summary, low KMT 2E expression may predict poor outcome in APL patients.

Abstract: Abstract 3536 ΔNp73 is an alternative TP73 gene transcript lacking the transactivation (TA) domain that is generated via alternative splicing and/or P2 promoter. The encoded protein acts as a potent transdominant inhibitor of wild type TP53 and full-length TAp73. In several human malignancies the unbalanced expression of transcriptionally active (TAp73) and inactive (ΔNp73) variants correlates with treatment outcome. We have previously reported that higher ΔN/TA isoform expression ratio was associated with poorer prognosis and resistance to cytarabine induced apoptosis in patients with acute myeloid leukemia (AML) (Lucena-Araujo et al., 2008). In acute promyelocytic leukemia (APL), both isoforms are expressed, but the clinical significance remains unknown. The aim of this study was to determine whether the ΔN/TA expression ratio was associated with treatment outcome of APL patients and to investigate the mechanisms by which ΔNp73 may contribute to PML-RARa+ cell survival. Using isoform-specific probes for ΔNp73 and TAp73, their expression was analyzed in 166 APL patients by Real-time quantitative polymerase chain reaction (RQ-PCR). Patients were divided into tertiles for ΔN/TA expression ratio (median value=23.62; 33rd/66th percentiles=12.8/42.3) and their clinical and laboratory characteristics were compared. Patients in the highest tertile presented higher white blood cells (WBC) counts than those in intermediate/lower tertiles (p 〈 0.001), but no significant differences were observed for age, gender, PML breakpoint, or platelets count. Higher ΔN/TA expression ratio values were significantly associated with the presence of FLT3-ITD (p =0.001). Treatment outcome was obtained for 131 APL patients enrolled in the APL99 (n=41) and IC-APL (n=90) trials. The mean follow-up was 29.1 months, ranging from 1 to 85.5 months. The mean overall survival (OS) of all patients was of 66.8 months [95%CI; 60.8 to 72.8], whereas it was of 67.1 months [56.5 to 77.7] for patients in the lower and 41.7 months [32 to 51.4] for those in the higher tertile for ΔN/TA expression ratio (p=0.014, Figure 1A). Univariate analysis identified WBC counts above 10,000/μl (p =0.003), FLT3-ITD mutation (p =0.011) and ΔN/TA expression ratio (p =0.014) as predictive factors for OS. However, in multivariate Cox analysis, these three prognostic factors were not independent. Until April 2011, a total of eight relapses (6.1%) were recorded. The disease free survival (DFS) rate at five-years for all patients was of 88.3% ± 4.2% and the mean DFS was of 76.1 months [71.2 to 80.9] . DFS was significantly shorter in patients at the higher tertile ΔN/TA expression ratio compared with patients at the lower tertile (72.1 ± 11.2% vs 97.1 ± 2.8%, respectively; p 〈 0.001; Figure 1B) and was the only variable found to be significant in the univariate analysis. To test the functional significance of the association of PML-RARa with high ΔNp73 gene expression, primary murine bone marrow cells from hCG-PML-RARa transgenic mice were transfected with MSCV-based retroviral vector carrying the ΔNp73 cDNA upstream of IRES-GFP cassette (PML-pMIG-ΔN). Expression of ΔNp73 in PML-RARa+ cells increased cell proliferation rate by 2.5-fold compared to PML-RARa+ transfected with the empty vector (p =0.03). This increase resulted from accumulation of cells at the G2/M phase (5.79 ± 0.08% for PML-pMIG vs 9.8 ± 0.35% for PML-pMIG-ΔN, p 〈 0.001), as well as at S phase of the cell cycle (27.74 ± 0.89% for PML-pMIG vs. 36.78 ± 0.81% for PML-pMIG-ΔN, p =0.001). In addition, transfection of ΔNp73 resulted in resistance to cytarabine-induced apoptosis. After 24h of culture with 50μg/ml of cytarabine (ED-50%), the fractional effect for the drug (% Anexin V-positive in (treated – untreated) cells /100 - % Anexin V-positive in non-treated cells) was 32.1% for PML-pMIG-ΔN and 54.8% for PML-pMIG (p 〈 0.001). In conclusion, ΔN/TA expression ratio was associated with shorter OS and DFS in APL, which may reflect increased cell proliferation and apoptosis resistance due to ΔNp73 activity.Figure 1.Overall (A) and disease-free survival (B) in acute promyelocytic leukemia patients according to ΔN/TA expression ratio.Figure 1. Overall (A) and disease-free survival (B) in acute promyelocytic leukemia patients according to ΔN/TA expression ratio. Disclosures: No relevant conflicts of interest to declare.