Search

Supplemental digital content is available in the text., Imatinib is the treatment of choice for FIP1L1/PDGFRA (F/P)-associated chronic eosinophilic leukemia (F/P+ CEL), but its optimal dosing, duration, and possibility of discontinuation are still a matter of debate. A retrospective multicenter study was conducted with 44 F/P+ CEL patients identified in the French Eosinophil Network and treated with imatinib. The most frequently involved systems were skin (57%), spleen (52%), and lung (45%), and eosinophilic heart disease was observed in 15 patients (34%). Complete hematologic response (CHR) was obtained in all patients, and complete molecular response (CMR) in 95% of patients (average initial imatinib dose, 165 mg/d). For 29 patients the imatinib dose was tapered with a maintenance dose of 58 mg/d (±34 mg/d), allowing sustained CHR and CMR. None of the patients developed resistance during a median follow-up of 52.3 months (range, 1.4–97.4 mo). Imatinib was stopped in 11 patients; 6 of the patients subsequently relapsed, but 5 remained in persistent CHR or CMR (range, 9–88 mo). These results confirm that an initial low-dose regimen of imatinib (100 mg/d) followed by a lower maintenance dose can be efficient for obtaining long-term CHR and CMR. Our data also suggest that imatinib can be stopped in some patients without molecular relapse.

Allogeneic hematopoietic stem cell (HSC) transplantation is a curative treatment for many hematologic malignancies for which umbilical cord blood (UCB) represents an alternative source of HSCs. To overcome the low cellularity of one UCB unit, double UCB transplantation (dUCBT) has been developed in adults. We have analyzed the outcome of 136 patients who underwent dUCBT reported to the SFGM-TC registry between 2005 and 2007. Forty-six patients received myeloablative regimens, and 90 patients received reduced-intensity conditioning regimens. There were 84 cases of leukemia, 17 cases of non-Hodgkin lymphoma, 11 cases of myeloma, and 24 other hematologic malignancies. At transplantation, 40 (29%) patients were in complete remission. At day 60 after transplantation, the cumulative incidence of neutrophil recovery was 91%. We observed one UCB unit domination in 88% of cases. The cumulative incidence of day 100 acute graft-versus-host disease, chronic graft-versus-host disease, transplant-related mortality, and relapse at 2 years were 36%, 23%, 27%, and 28% respectively. After a median follow-up of 49.5 months, the 3-year probabilities of overall and progression-free survival were 41% and 35%, respectively, with a significant overall survival advantage when male cord engrafted male recipients. We obtained a long-term plateau among patients in complete remission, which makes dUCBT a promising treatment strategy for these patients.

Background Gene expression profiling has shown its ability to identify with high accuracy low cytogenetic risk acute myeloid leukemia such as acute promyelocytic leukemia and leukemias with t(8;21) or inv(16). The aim of this gene expression profiling study was to evaluate to what extent suboptimal samples with low leukemic blast load (range, 2-59%) and/or poor quality control criteria could also be correctly identified. Methods Specific signatures were first defined so that all 71 acute promyelocytic leukemia, leukemia with t(8;21) or inv(16)-AML as well as cytogenetically normal acute myeloid leukemia samples with at least 60% blasts and good quality control criteria were correctly classified (training set). The classifiers were then evaluated for their ability to assign to the expected class 111 samples considered as suboptimal because of a low leukemic blast load (n = 101) and/or poor quality control criteria (n = 10) (test set). Results With 10-marker classifiers, all training set samples as well as 97 of the 101 test samples with a low blast load, and all 10 samples with poor quality control criteria were correctly classified. Regarding test set samples, the overall error rate of the class prediction was below 4 percent, even though the leukemic blast load was as low as 2%. Sensitivity, specificity, negative and positive predictive values of the class assignments ranged from 91% to 100%. Of note, for acute promyelocytic leukemia and leukemias with t(8;21) or inv(16), the confidence level of the class assignment was influenced by the leukemic blast load. Conclusion Gene expression profiling and a supervised method requiring 10-marker classifiers enable the identification of favorable cytogenetic risk acute myeloid leukemia even when samples contain low leukemic blast loads or display poor quality control criterion.

Purpose To investigate the impact of prior-to-transplantation azacitidine (AZA) on patient outcome after allogeneic stem-cell transplantation (alloSCT) for myelodysplastic syndrome (MDS). Patients and Methods Of the 265 consecutive patients who underwent alloSCT for MDS between October 2005 and December 2009, 163 had received cytoreductive treatment prior to transplantation, including induction chemotherapy (ICT) alone (ICT group; n = 98), AZA alone (AZA group; n = 48), or AZA preceded or followed by ICT (AZA-ICT group; n = 17). At diagnosis, 126 patients (77%) had an excess of marrow blasts, and 95 patients (58%) had intermediate-2 or high-risk MDS according to the International Prognostic Scoring System (IPSS). Progression to more advanced disease before alloSCT was recorded in 67 patients. Donors were sibling (n = 75) or HLA-matched unrelated (10/10; n = 88). They received blood (n = 142) or marrow (n = 21) grafts following either myeloablative (n = 33) or reduced intensity (n = 130) conditioning. Results With a median follow-up of 38.7 months, 3-year outcomes in the AZA, ICT, and AZA-ICT groups were 55%, 48%, and 32% (P = .07) for overall survival (OS); 42%, 44%, and 29% (P = .14) for event-free survival (EFS); 40%, 37%, and 36% (P = .86) for relapse; and 19%, 20%, and 35% (P = .24) for nonrelapse mortality (NRM), respectively. Multivariate analysis confirmed the absence of statistical differences between the AZA and the ICT groups in terms of OS, EFS, relapse, and NRM. Conclusion With the goal of downstaging underlying disease before alloSCT, AZA alone led to outcomes similar to those for standard ICT.

El objetivo fue evaluar los efectos de la alimentación y el esquileo, durante el último tercio de la gestación, sobre el metabolismo energético de las ovejas y su repercusión sobre las reservas energéticas de sus corderos. A los 90 días de gestación 38 ovejas Corriedale adultas, gestando un solo feto, fueron divididas en dos grupos, R (n = 12, suplementado con concentrado) y CN (n = 26, alimentado a campo natural), evaluándose su estado metabólico mediante determinaciones de la glucemia y del ß-hidroxibutirato. A los 110 días de gestación se esquiló a la mitad de las ovejas del grupo CN, quedando dividido en dos nuevos grupos (E, esquiladas, n = 13 y campo natural, CN, n = 13), estudiándose el efecto de la esquileo sobre los mismos parámetros. En los corderos se determinó la glucemia (al parto y a las 24 horas), el peso (al parto, a las 24, 48 y 72 horas) y el peso y valor energético de grasa parda perirrenal. En el día 140 de gestación las ovejas alimentadas con concentrado y las esquiladas presentaron valores superiores de glucemia que las alimentadas exclusivamente con pasto natural; los valores de ß-hidroxibutirato sérico fueron más elevados en las ovejas de pasto natural y las esquiladas que las suplementadas con concentrado. Al nacimiento el peso de los corderos no presentó diferencias entre los tres grupos. La glucemia de los corderos nacidos de ovejas suplementadas con concentrado fue mayor que los de ovejas de pasto natural, al parto y a las 24 horas y en las esquiladas mayor en que se alimentaban exclusivamente de pasto a las 24 horas. Los corderos de las ovejas esquiladas presentaron grasa parda con mayor valor energético que los provenientes de ovejas que pastaban exclusivamente hierba, demostrándose así relación entre el estado energético de las ovejas al parto y las reservas energéticas con las que nacen sus corderos.

Monoclonal gammopathies of undetermined significance (MGUS) have been shown to be associated with an increased risk of fractures. This study describes prospectively the bone status of MGUS patients and determines the factors associated with vertebral fracture. We included prospectively 201 patients with MGUS, incidentally discovered, and with no known history of osteoporosis: mean age 66.6±12.5 years, 48.3% women, 51.7% immunoglobulin G (IgG), 33.3% IgM and 10.4% IgA. Light chain was kappa in 64.2% patients. All patients had spinal radiographs and bone mineral density measurement in addition to gammopathy assessment. At least one prevalent non-traumatic vertebral fracture was discovered in 18.4% patients and equally distributed between men and women. Fractured patients were older, had a lower bone density and had also more frequently a lambda light chain isotype. Compared with patients with κ light chain, the odds ratio of being fractured for patients with λ light chain was 4.32 (95% confidence interval 1.80–11.16; P=0.002). These results suggest a high prevalence of non-traumatic vertebral fractures in MGUS associated with lambda light chain isotype and not only explained by low bone density.

We performed a prospective, randomized, open, blinded end point (PROBE) study to assess the efficiency of transfusing high doses of platelets in patients with thrombocytopenia, either acute leukemia (AL) or those undergoing autologous hematopoietic stem cell transplantation (AT). Patients were randomly assigned to receive transfusions with a target dose of 0.5 × 1011/10 kg (arm A) or 1 × 1011/10 kg (arm B). A total of 101 patients were included, of whom 96 were given at least one transfusion. The median time between the first transfusion and when the platelet count reached at least 20 × 109/L increased from 63 hours to 95 hours in the arm B group (P = .001), and the median number of transfusions was lower in this group (2; P = .037). The total number of transfused platelets did not differ between groups (14.9 × 1011 for arm A versus 18.5 × 1011 for arm B; P = .156). In such patients, a prophylactic strategy of high doses of platelets could improve platelet transfusion efficiency.

The prognosis of poor risk AML in elderly pts justifies either supportive care only or investigational studies without obvious benefit of post-remission chemotherapies. A phase I study has demonstrated that the combination of azacitidine and lenalidomide is well-tolerated in AML with efficacy superior to that of single-agent therapy in higher-risk MDS (Sekeres JCO2010). The GOELAMS group investigated the efficacy of 12 maintenance cycles alternating monthly azacitidine (sc 75 mg/m²/d1-7) and lenalidomide (10mg/d1-21) every 28 days for pts in CR and compared the results to previous SA2002 and BGMT95 prospective trials which shared the same LIA induction followed by chemotherapy-based maintenance. Between 3/2011 and 2/2013, 117 pts from 27 centers (median age 69 yrs; 60-80, 9 pts ≥ 75) with previously untreated poor prognosis AML were included. Risk factors allowing inclusion were either: i) centrally reviewed (IL) poor risk cytogenetics (n=83) defined as complex karyotype (≥3 abnormalities), monosomal karyotype, t(6;9), del or – 5 or 7, and EVI1 or MLL rearrangement except for t(9;11); ii) preceding MDS (n=52) or iii) secondary leukemia occurring after previous cancer (n=37). Median WBC was 2.9 G/L (0.5-160), 19 pts had WBC > 30 G/L. Induction chemotherapy included lomustine 200 mg/m² po d1, Idarabucine 8 mg/m²/d (d1-5), cytarabine 100mg/m²/d CI (d1-7) and G-CSF (d15 to recovery). At the end of induction, CR (excluding CRp) was achieved in 56% (65 pts), 9% died from infection (n=6), cerebral hemorrhage (n=1) or multiorgan failure (n=4) and 35% (41pts) failed to achieve CR. Despite a trend towards a higher CR rate in pts without previous MDS (59% vs 48% CR), CR was not related to any of the risk factors studied. Median follow-up for survivors is 16 months (3-25). Monthly alternating azacitidine-lenalidomide maintenance therapy was started in 65 pts. Except for 1 pt who died during cycle1 of lenalidomide without any obvious explanation, tolerance was good. Median nadir of neutrophils and platelets was 1 and 96 G/L respectively. Grade 3/4 adverse events occurred in 6% of cycles and were due to infection, haemorrhage, hepatitis or fatigue (3%, 1%, 0.5%, 0.5% of cycles respectively). Median CR duration was 4.5 mo (1-23) and 40 pts relapsed. Allogeneic SCT with non myelo-ablative preparative regimen was performed in 4 pts less than 70 years in CR1: 1 is alive in relapse, 2 died of GVH and 1 is in continuous CR. Pts achieving CR had a median OS of 13 mo (95% CI 7.3-18.6), 54% being alive at 1 year and 15% at 2y. In refractory pts, the median OS was 7 mo (95% CI 2.8-11.2) with 32% alive at 1 year (P = 0.0001). OS and DFS reported in the current trial and compared to historical controls are described below: induction results are similar to those observed in the 128 poor risk cytogenetic pts of the previous SA2002/BGMT trials treated with the same LIA induction (CR 58%, early death 15%, failure 27%) but who received maintenance therapy with 6 mini-reinductions (Ida 10mg/m² d1, cytarabine 50 mg/m²x2/d d1-5) and 6MP-MTX for 2years. No significant survival benefit was observed compared to pts with poor risk cytogenetics treated with chemotherapy maintenance in our previous trials. However, pts included in this trial have probably an even worse prognostic than those included in the GOELAMSSA2/BGMT95 trial which excluded AML secondary to MDS and previous cancers. This type of alternating azacitidine/lenalidomide maintenance improves OS and DFS of pts without poor risk cytogenetics (median DFS not reached and 19 mo) and could be randomly compared with conventional chemotherapy maintenance in AML secondary to MDS or cancer but also in older pts with intermediate risk cytogenetics.Median (95% CI)6mo1y2yOS maintenance aza-rev (117)9 mo (7.5-10.5)70%41%17%OS 2002/BGMT95 (128)6 mo (4.1-7.8)54%32%21%DFS maintenance aza-Rev (65)10 mo (2.5-17.4)61%49%8%DFS 2002/BGMT95 (74)9 mo (6.0-11.9)63%43%26%OS poor risk cytogenetic (83) vs other (33)8 mo vs NR P=0.000162% vs 83%27% vs 70%10% vs 60%OS previous cancer (37) vs no cancer (80)9 mo vs 9 mo68% vs 73%44% vs 40%24% vs 17%OS previous MDS (52) vs no MDS(64)12 mo vs 7 mo P=0.00177% vs 65%58% vs 26%27% vs 12%DFS poor risk cytogenetic (44) vs other (20)6 mo vs 19 mo P=0.00748% vs 84%33% vs 67%11% vs 28%DFS previous cancer (20) vs no K(45)8 mo vs 8 mo80% vs 61%46% vs 47%0% vs 13%DFS previous MDS (27) vs no MDS(38)15 mo vs 6 mo, P=0.0874% vs 48%59% vs 32%12% vs 0% Disclosures: No relevant conflicts of interest to declare.

Introduction The treatment of Adult Acute Lymphoblastic Leukemia (ALL) has improved by the use of pediatric-like approaches, erasing the poor prognostic impact of numerous variables. New markers are however still needed to identify poor prognosis patients in prospective trials. The p16INK4A/CDK4-6/pRb pathway and telomerase activity (TA), markers of cell activation and aging, were analyzed in 175 adult ALLs, treated between November 2003 and January 2007 according to the GRAALL/GRAAPH trials, to investigate their prognostic value in this context. Methods The cohort comprised 105 males and 70 females aged between 16 and 59 years (median: 36). Immunophenotype by flow-cytometry allowed for the characterization of 123 B- and 52 T-lineage ALLs, 163 of them being subdivided according to EGIL criteria. Cytogenetic and/or molecular analyses allowed for the detection of BCR-ABL, MLL-AF4 or E2A-PBX1 fusion transcripts, NOTCH1 mutations in T-ALLs, and IKAROS deletions in BCR-ABL-negative B-ALLs. In all cases, cell samples were obtained at diagnosis, before any treatment, from bone marrow aspiration after the patients provided informed consent. Flow cytometric analysis of the DNA content was performed for evaluation of the percentages of cells in S/G2/M phases of the cell cycle. Cell cycle regulatory proteins were examined in 135 samples, by western blot. The TA assay was performed on 156 samples according to a telomeric repeat amplification protocol. Furthermore, in vitro analyses of the p16INK4A/CDK4-6/pRb pathway and TA were carried out in normal peripheral blood lymphocytes before and after stimulation and during lymphocyte long-term culture. Results The p16INK4A/CDK4-6/pRb pathway and TA were analyzed according to the immunological phenotype and molecular characteristics of ALLs. Leukocytosis (p Among sub-groups defined by clinical or biological data, the most significant relationships between the p16INK4A/CDK4-CDK6/pRb pathway or TA and prognosis were observed for BCR-ABL1+ ALLs (31 cases). An above median expression of CDK4 was related to shorter disease-free survival (DFS) (p = 0.031) and overall survival (OS) when taking into account either death due to any cause (p = 0.005), or only disease-related death (p = 0.018). In spite of the small size of this series, this prognostic value remained when the analysis was restricted to patients who did not receive allogeneic stem-cell transplantation (non-Allo SCT) for DFS (p = 0.034), OS (p = 0.019) or disease-related OS (p = 0.05). A shorter DFS was also associated with the CDK4/6 mediated-phosphorylation of pRb (p-pRb/pRb ratio > 0) (p = 0.042), paradoxically, with high expression levels of p16INK4A (p = 0.029), and also with above median TA (p=0.040). Patients with the highest TA (over quartile 75) showed a significantly shorter OS (p = 0.018) especially when considering only disease-related death (p = 0.007). The poor prognostic value of above median TA for OS (p = 0.026) and disease–related OS (p = 0.009) was also confirmed in the non-Allo SCT group. Considering that IKAROS is deleted in most of BCR-ABL1+ ALLs and since survival was similar in BCR-ABL1+ ALLs and BCR-ABL1-/IKAROSdel B-ALLs, these two groups of patients were pooled for analysis (42 cases). A shorter DFS was linked to pRb phosphorylation (p = 0.036), while both a shorter DFS (p = 0.026) and OS (p = 0.055) were noted for patients with high TA. In vitro analyses in normal lymphocytes demonstrated that increased expression of p16INK4A, CDK4, p-pRb and TA is related to cell activation, suggesting that ALL blasts with these criteria could be in an activated stage. Conclusions These data bring new perspectives to the biological characterization of ALLs and associate a poor prognosis in BCR-ABL1+ ALLs with enhanced cell activation. Additional investigations could focus, in a prospective series, on the analysis of the cell activation markers described here and on the development of new therapeutic strategies by proposing the association of lymphocyte activation inhibitors. Disclosures: No relevant conflicts of interest to declare.

Background IA is a major cause of morbidity and mortality for patients with hematological malignancies and has been mainly reported in patients with acute myeloblastic leukemia (AML) and stem cell transplantation. Much less is known about the role of IA during the treatment of lymphoproliferative diseases like ALL. Here, we retrospectively evaluated the characteristics of patients and the incidence of IA occurring during the induction course in adult ALL patients enrolled in the GRAALL-2005 trial. Methods We collected the data of 36 patients with IA during induction chemotherapy. All were included between May 2006 and October 2012 in the multicentric GRAALL-2005 phase III trial. According to ALL characteristics, these patients were treated in 3 different substudies: GRAALL, GRAALL-Rituximab (GRAALL-R) for CD20+ ALL, GRAAPH for Philadelphia (Ph) chromosome-positive ALL. IA was defined retrospectively using the EORTC modified criteria (De Pauw, CID 2008). Results Among the 969 patients enrolled, 36 (3.7%) developed IA during induction therapy. The median age was 30 years for all patients receiving induction and 47 years (range, 18 to 59) for patients with IA. We observed 18 IA (3%) by the GRAALL protocol (593 patients enrolled), 16 (8.3%) by the GRAALL-R protocol (191 patients enrolled), and 2 (0.7%) by the GRAAPH protocol (270 patients enrolled). In the GRAALL-R protocol, IA was diagnosed in 6 patients randomly assigned to receive rituximab and in 10 patients treated in the control group. The median time between first day of induction therapy and IA diagnosis was 20 days (range, -2 to 71). The median time between the first day of neutropenia and IA diagnosis was 18 days (range, 0 to 76). At the time of IA diagnosis, 13 patients were hospitalized in laminar airflow rooms, 10 patients in rooms with overpressure, 1 patient in a room with high-efficiency particulate filter, 5 patients in conventional rooms, 1 patient received home care and the type of hospitalization was unknown for the 6 remaining patients. Four patients with IA (11%) had received antifungal drug for invasive fungal infection (IFI) prophylaxis: one patient received fluconazole, one received caspofungin, and two received liposomal amphotericin B. All patients with IA presented pulmonary symptoms associated with a sinusal or cutaneous localization in 1 and 2 patients respectively. The diagnosis of IA was classified as possible in 7 episodes (19 %), probable in 22 episodes (61%), proven in 4 episodes (11%), and indeterminate in the remaining 3 episodes. Detection of Aspergillus antigen in serum by latex agglutination was positive in 24 cases (67%). Chest CT scans were taken for 33 patients (92%): nodules were found in 19 patients, halo sign in 20 patients, and “air crescent sign” in 1 patient. Bronchoalveolar lavage was performed in 11 patients (31%): culture was positive in 7 of them and Aspergillus antigen was positive in 2 of them. Biopsies where positive in 4 cases: in pulmonary biopsy in 3 patients and in cutaneous biopsy in 1 patient. Of all the Aspergillus isolates identified to the species level, Aspergillus fumigatus was isolated from 4 patients and Aspergillus flavus from 1 patient. Overall and IA-attributable 12-week mortality was 8 (0.8%) and 6 (16.7%) patients respectively. Conclusion IA is less frequently observed during induction therapy in adult ALL as compared to adult AML patients. However, the attributable mortality appears to be high in these patients. They are as at risk patients for IA and should be included in prophylactic and empirical antifungal clinical trials. Disclosures: No relevant conflicts of interest to declare.

Abstract 4791 Acute lymphoblastic leukemia (ALL) multiparameter flow cytometry (MFC) study of bone marrow aspiration after chemotherapy is crucial for determining minimal residual disease (MRD). Hematogones (HGs) have to be distinguishing from leukemic cells in B-cell subtypes and could be quantify during follow-up. To date, the incidence of Hgs in ALL and their prognostic significance have not been investigated. The aim of this multicenter study was to quantify Hgs after chemotherapy in ALL adult patients and to define its prognostic value. We retrospectively analyzed the incidence of HGs in 95 ALL patients, 71 with B-ALL (75%), 24 (25%) with T-ALL in first line treatment. The median age was 37 years [8–71], 20% had t(9;22) cytogenetic abnormality, and 70% had abnormal karyotype. 4/5-color MFC analysis MRD and HGs were performed at different time point (TP) after diagnosis: TP1 (post-induction, day 45 [41–61], n=78), TP2 (post-consolidation, day 111 [94–144] 25, n=42), TP3 (post-intensification/before hematopoietic stem cell transplantation (HSCT), day 179 [125–268], n=58), TP4 (n=11), TP5 (n=17), TP6 (n=9) after a median of 33, 91 and 167 days after HSCT, respectively. A total of 39 patients (41%) relapsed with a median of 26 months [7.7–47.9]. Forty seven patients (50%) received an HSCT in a complete (98%) or partial remission (2%). At TP1, TP2, TP3, TP4, TP5, TP6, the median HGs [range] were as followed: 0.00 [0.00–6.90]%, 0.30 [0.00–11.2]%, 0.98 [0.00–33.00]%, 0.52 [0.00;23.00]%, 5.50 [0.00;25.00]%, 4.60 [0.00;34.00]%, 5.90 [0.32;11.80]%, respectively. Figure 1 showed the percentage of patients with negative MRD (Figure 1A) and detectable HGs (figure 1B) during the follow up of ALL patients. There is a progressive increase of the percentage of patients with detectable HGs during the time of treatment and follow-up. Interestingly, there was no correlation between age and HGs level while in physiological situation the HGs rate decreases with increasing age. There was a negative correlation between positive MRD and detectable HGs at TP1 (p=0.022) but not at TP3 (p=0.88). In univariate analysis positive MRD at P1 and P3, age (/10), the presence of t(9;22) and absence of HGs at TP3 (figure 2) were bad prognostic factors for relapse free-survival (RFS) and overall survival (OS). The presence of HGs at other different time of evaluation was not associated with a significant decrease of relapse or death. However, patients who had a negative MRD at TP1 and detectable HGs in the bone marrow at TP3 exhibited a better RFS and OS (p=0.018 and p=0.065 respectively). Patients who had negative MRD at TP3 and had detectable HGs at TP3 had also a better RFS and OS (p=0.007 and p=0.011, respectively) compared to patients with negative MRD at TP3 and without HGs (figure 3). In patients who had a positive MRD at TP1, detectable HGs at TP3 identified a subgroup of patient with favorable OS compared to patient with positive MRD at TP1 and without detectable HGs (p=0.072). These results should be taken with cautious because of the decreasing number of patients evaluated at different time points. However, HGs analysis could represent a new area of investigation in search of new prognostic factors in the context of adult ALL. Figure 1. Percentage of patients with (A) negative MRD and (B) detectable HGs at different time points after starting of treatment. Figure 1. Percentage of patients with (A) negative MRD and (B) detectable HGs at different time points after starting of treatment. Figure 2. Overall survival according to HGs status at TP3. Figure 2. Overall survival according to HGs status at TP3. Figure 3. Overall survival in patients with negative MRD at TP3 according to HGs status. Figure 3. Overall survival in patients with negative MRD at TP3 according to HGs status. Disclosures: No relevant conflicts of interest to declare.

The Bcr-Abl oncogene is present in 30–40% of adult patients with acute lymphoblastic leukemia (ALL). The Abl kinase inhibitor imatinib-based therapy has become standard for this subset ALL. Acquired resistance to imatinib occurs frequently and is associated with mutations in the tyrosine kinase domain (TKD) approximately in about 80% of patients. In contrast, TKD mutations are uncommon in primary imatinib resistance which appears to be multifactorial, although the underlying mechanisms have been incompletely elucidated. We have established a Ph+ cell line for the analysis of non-mutational resistance mechanisms of imatinib resistance: SupB15RT, a Bcr-Abl expressing lymphoblastic cell line derived from SupB15WT cell line by gradually increasing the exposure to imatinib. SupB15RT shows cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. We have shown that several commonly implicated mechanisms of imatinib resistance do not play a role in conferring the imatinib resistance in SupB15RT cells. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affymetrix- Microarrays, we identified 29 differentially regulated genes. Autotaxin (ATX) is one of the most highly up-regulated genes in imatinib resistant SupB15RT cells, and suggested a contribution to imatinib resistance. ATX is an exo-enzyme (pyrophosphophatase/phosphodiesterase). It plays a role in tumor progression and migration as a tumor cell autocrine motilty factor in various solid tumor cell types. ATX is involved in the synthesis of the signaling molecule, lysophosphatidic acid (LPA) which promotes survival and motility. It was the aim of this study to determine whereas ATX plays a functional role for imatinib resistance in Ph+ ALL. Using RT-PCR we demonstrated that 2 isoforms of ATX are expressed in SupB15RT cells: ATXshort and ATXlong. ATXlong (863 aa) contains highly basic insertion in the catalytic domain (52 residues). We retroviraly transfected BaF3 cells with p185 and/or ATXshort or ATXlong to analyze its influence on growth, adhesion and migration in mouse cell model. In comparison to wild type BaF3 cells the proliferation of BaF3 cells expressing ATXshort is enhanced (1,5-fold), whereas ATXlong expressing BaF3 cells showed no difference in proliferation in comparison to Mock infected cells. The proliferation of p185 expressing BaF3 cells co-expressing ATXshort or ATXlong is not inhibited by the treatment with 1μM imatinib after 3 days in contrast to p185 expressing BaF3 cells. In adhesion experiments, BaF3 cells expressing ATXshort showed a higher attachment independent of p185 expression. We also performed migration experiments using transwell assays. These assays showed more migration with cells co-expressing p185 and ATXlong compared to p185 alone. This is in agreement with our results for SupB15RT vs. SupB15WT with a 3-fold migration increase of SupB15RT. Application of 10% fetal calf serum (FCS) in migration experiments resulted in a 1,5-fold higher migration of the ATXlong expressing BaF3 cells compared to culture without FCS. One explanation for this finding may be that FCS contains lysophosphatidic choline (LPC) which is converted to LPA by ATX. Although expression of both 2 isoforms of ATX is important for the increased proliferation, it seems that the 2 isoforms have different cellular functions in Ph+ lymphoblastic cells. ATXshort seems to enhance adhesion whereas ATXlong plays an important role in motility. Taken together our results indicate a role for ATX in TK- inhibitor resistant SupB15RT cells through LPA signaling via LPA receptors. The ratio between ATXshort and ATXlong probably is important for the intracellular signaling and has to be explored.

The Bcr-Abl oncogene is present in 30-40% of adult patients with acute lymphoblastic leukemia (ALL). Therapy with imatinib has become standard for Ph+ ALL but resistance to the tyrosine kinase inhibitor occurs for the majority of patients. In about 80% of patients with acquired resistance mutations in the tyrosine kinase domain (TKD) have been found. In contrast, primary resistance to imatinib appears to be multifactorial and precise mechanisms have been incompletely elucidated. We have established an imatinib-resistant cell line (SupB15RT) which was derived from the previously well characterized SupB15 cell line (SupB15WT) by gradually increasing the exposure to imatinib. We found that several commonly implicated mechanisms of imatinib resistance, i.e. Bcr-Abl gene amplification, point mutations in the TKD, Bcr-Abl overexpression, up-regulation of multidrug resistance gene proteins or ineffective inhibition of Bcr-Abl tyrosine phosphorylation do not play a role in conferring the imatinib-resistant phenotype in SupB15RT cells. Thus, the SupB15RT cells represent a suitable model for the analysis of resistance mechanisms in Ph+ ALL with primary imatinib resistance. Interestingly, SupB15RT cells show cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. Analysis of signal transduction pathways downstream of Bcr-Abl revealed that imatinib exposure was not associated with down-regulation of pSTAT-5 and pErk in the imatinib-resistant SupB15RT cells, in contrast to SupB15WT. Phosphorylation of Akt was inhibited by 0.5μM imatinib in SupB15WT cells, whereas imatinib in concentrations up to 5μM failed to suppress Akt phosphorylation in SupB15RT cells, indicating constitutive activation of Akt kinase during imatinib treatment. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affimetrix-Microarrays, we identified 29 differentially regulated (at least 3-fold) genes. One of the most highly up-regulated genes in imatinib-resistant SupB15RT cells was Autotaxin (ATX), a nucleotide pyrophosphatase/ phosphodiesterase 2. This exo-enzyme was originally identified as a tumor cell autocrine motility factor, which is involved in tumor progression and migration in various tumor cell types. ATX is a lysophospholipase D which is involved in the synthesis of lysophosphatidic acid (LPA), a signaling molecule that promotes survival, growth, differentiation, and motility. We investigated if LPA imparted imatinib resistance in SupB15WT cells by modulation of growth, survival and migration. When SupB15WT cells were treated with LPA, alone or in combination with imatinib, SupB15WT cell proliferation was increased both in the absence as well as in the presence of imatinib. The dose-dependent increase of proliferation after LPA treatment was 1.9–2.6-fold (1–10μM LPA) in the presence of 1μM imatinib. In addition we performed migration experiments using Transwell assays. We detected a 3-fold increase in migration of SupB15RT vs. SupB15WT cells. We found no influence on apoptosis in imatinib treated SupB15WT cells treated with LPA compared with cells not treated with LPA. Taken together, our results indicate a role of ATX in imatinib-resistant SupB15RT cells, preferentially by stimulating proliferation and migration through LPA signaling via LPA receptors and activation of PI3K and Akt.

Bortezomid (VELCADE®) is a potent proteasome inhibitor used in patients with relapsed and/or refractory multiple myeloma (MM). Numerous undesirable effects are known including thrombocytopenia, neuropathy, various gastrointestinal disorders, fatigue, or rare pulmonary complications. Cutaneous complications have also been described. We reported here a case of Bortezomid-induced Sweet syndrome. A 72-year-old man received Bortezomid for a second relapse of MM previously treated with VAD (vincristine, adriamycin, dexamethasone), double high dose melphalan and autologous transplant, and thalidomide. Bortezomid was given at the dose of 1.3 mg/m2 on days 1, 4, 8, and 11 every 21 days. Dexamethasone was associated with the first cycle at the dose of 20mg/d given on days 1, 2, 4, 5, 8, and 9. On day 11 of the third cycle, a partial response was observed. However, the patient presented a painful skin eruption without any fever. Centimetric erythematous, oedematous papulo-nodular plaques were observed on his back and a periorbital oedema. Spontaneous partial regression was noted on the first day of the following cycle but major analgesic drug were necessary. These cutaneous lesions on the back and the dorsal of fingers recurred on the fourth and fifth cycles with a maximum on day 11. Histopathological examination of a lesion showed oedeme of the dermis and a perivascular polymorphic dermal infiltrate of lymphocytes, neutrophils and eosinophils with some leukocytoclasia, but without parietal necrosis or vasculitis. This findings were consistent with neutrophilic dermatosis. During the sixth Bortezomid cycle, the administration of dexamethasone 20 mg/d on days 1, 2, 4, 5, 8, 9, 11, and 12 prevents the reoccurrence of the skin lesions but the periorbital oedema persists. A variety of cutaneous manifestations has been described in MM. Sweet syndrome has been described in two untreated MM or in some patients receiving granulocyte-colony stimulating factor. Bortezomid has been associated with rash, pruritus, erythema, periorbital oedema, urticaria, alopecia, skin nodules. Few cases of non-necrotising cutaneous vasculitis with perivascular lymphocytic infiltrates have also been reported. Bortezomid-induced Sweet syndrome has been reported in three other patients at the second and third injection of the second cycle for two of them while the eruption occurred after the first injection in the third case. The recurring skin lesions after each bortezomid cycle strongly suggests Bortezomid responsibility’s in the occurrence of Sweet syndrome. Unlike classic hypersensitivity type reactions, this vasculitis rash may not necessary prompt cessation of drug as the administration of dexamethasone prevents its recurrence.

Between March 1986 and May 1998, 28 patients (pts) (60,7% M, 39.3% F) with localized high-grade PBL were enrolled in the 02 (< 60 years, 20 pts) or 03 (≥ 60 years, 8 pts) GOELAMS trial. The aim of the study was to evaluate the OS, DFS and FFR after 3 VCAP courses (eldisine 3 mg/m² d1, doxorubicin 60 mg/m² d1, cyclophosphamide 1500 mg/m² d1, prednisone 80 mg/m² d1–5 every 3 weeks) in younger pts and after 3 VCEP-Bleo courses (eldisine 3 mg/m² d1, cyclophosphamide 750 mg/m² d2, farmorubicin 80 mg/m² d1, prednisone 50 mg/m²/d d1–7, bleomycine 10 mg d1 and 5, every 3 weeks) in older pts. Involved field radiotherapy (40 Gy) was performed for every patient. The median age was 46 years (17–69) and 70 years (65–75) respectively. Four patients between 60 and 69 with a good performance status (PS) were included in the 02 trial. In contrary to previous reports, the main localization were axial skeleton (20, 71%) including vertebrae (13), pelvis (5) and ribs (2) while skull and extremities were involved in both 4 patients (mandible 3, occiput 1, tibia 1, finger 1, humerus 2, one of them with including scapula involvement). Histological subtype included diffuse large cell lymphoma (68%), diffuse mixed cell lymphoma (14%), immunoblastic (10%), anaplastic Ki1+ (3%) and unclassified (3%). Immunophenotype were B (53.6%), T (3.6%), anaplastic (3.6%) or undone (39.3%). Ann Arbor classification included 23 stage I (82%) and 5 stage 2 (18%). Skin and subcutaneous tissues were involved by extension in 2 pts. Spinal compression, epidural involvement and paraplegia were observed in 8 pts. B symptoms were present in 5 pts (including 2 stage I). LDH were elevated in 5/23 pts. The IPI score was 0 for 18%, 1 for 39%, 2 for 14%, 3 for 10% and undetermined for 18%. PS >2 and bulk (≥ 5 cms) were observed in 43% and 39% of pts respectively. Besides age, VCAP and VCEP-Bleo groups were comparable for histological type, localization, Ann Arbor classification, B symptoms and LDH. All but one patient (96%) achieved CR. A 63 years old patient with costal localization, resistant to chemotherapy, died of progression after 15 months. Relapses occurred in 9/27 pts (33%, 3/8 for older pts, 6/20 for younger pts) at a median time of 2.3 years (0.4–6.5) from CR. Four of these relapses occurred before 2 years of CR, 4 between 2.1 and 5 years and 1 occurred 6.5 years after CR. Amongst 8 pts with spinal compression, 1 died in CR, 4 relapsed (1 alive in CR2) and 3 are in continuous CR. The relapse rate of pts without epidural involvement was 25% (5/20). With a median follow up of 8 years (1.2–17), OS, EFS and FFR were 62% (±12), 56% (±10) and 60% (±10), respectively. OS, EFS and FFR were 66%, 54%, 63% and 56%, 62%, 62% in VACP group and VCEP-Bleo group respectively. In univariate analysis, PS >2 significantly decreases OS, EFS and FFR (P = .036, .013 and .064). Epidural extension significantly decreases EFS (P = .009) but not OS. In multivariate analysis, poor PS but not epidural extension significantly decrease EFS and OS (P=.02 and P = .03 respectively for PS). The survival results of PBL are very similar to survival of 325 localized aggressive NHL treated in the 02-GOELAMS trial reported previously (Desablens ASH 2002). The poor prognostic value of PS and epidural extension of PBL should be underlined.

Abstract 1697 Background Abnormal and clonal hematopoiesis resulting in peripheral blood cytopenias, and risk of progression to acute myeloid leukemia (AML) are the main characteristics of myelodysplastic syndromes (MDS) which present a high diversity of somatically mutated genes. Recently, mutations targeting genes whose products participate to the early steps of RNA splicing (SF3B1, SRSF2, ZRSR2, and U2AF35) have been reported. Mutations in ASXL1, DNMT3, EZH2, IDH1/2 and TET2, suggest deregulation of the epigenetic control of transcription. Additional genes known to be mutated in MDS include RUNX1, TEL/ETV6, TP53 and NRAS The alteration of some of these genes may carry prognostic value. Next-generation sequencing analyses of AML and related disorders recently identified mutations in two chromosome X genes, BCOR and BCORL1, that code for related transcriptional co-repressors interacting with histone deacetylases and presenting specific properties. BCOR interacts with BCL6 and constitutional inactivating mutations have been described in the Oculo-Facial-Cardio-Dental syndrome. BCOR is also affected by mutations or translocations in retinoblastoma and sarcoma. BCORL1 has been implicated in chromosomal rearrangements in hepatocellular carcinoma. In this study, we have investigated BCOR and BCORL1 gene by Sanger sequencing in a cohort of 221 MDS samples. Methods The 221 MDS samples were collected at diagnosis in multicenter clinical trials in France between 1999 and 2011. The coding sequences of BCOR (ENST00000378444) and BCORL1 (ENST00000218147) were analyzed by Sanger sequencing. Mutational analyses of ASXL1, CBL, DNMT3A, ETV6, EZH2 IDH1/2, JAK2, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF35, and ZRSR2 were previously reported. Non tumoral material (buccal swab or CD3+ cells) was analyzed for the presence of the identified variations of BCOR or BCORL1 when available (n=9). The prognostic impact of BCOR mutations was evaluated in MDS patients with available follow-up information (n=203). Results BCOR and BCORL1 coding sequences were analyzed in the cohort of 221 MDS patients and we found mutations in 8.6% (n=19) and 2.3% (n=5) respectively. Alterations were distributed all over the coding regions. Strikingly, two patients presented concomitant inactivating mutations of BCOR and BCORL1. Among the 19 BCOR alterations, 9 were missense, 3 nonsense, 5 frameshift and 2 splice site mutations. No significant difference in age, sex, karyotype, blood counts or bone marrow blasts between BCORmut and BCORwt patients was observed. Mutations were found in patients of all IPSS risk-groups and WHO subtypes. Comparison of cytological bone marrow reports revealed a trend for a higher rate of dysgranulopoiesis in BCORmut patients (P=0.06). Because only truncating mutations (splice, frameshift and nonsense mutations) are unambiguously expected to affect BCOR function, statistical analyses were restricted to truncating BCOR mutations (n=10/221, 4.5%). Truncating BCOR mutations were frequent in RUNX1mut patients (19% versus 3% in RUNX1wt patients; P=0.027). They were by trend associated with SRSF2 and DNMT3A mutations (P=0.09) and were exclusive with IDH1/2, JAK2, NRAS, TP53, and ZRSR2 mutations. In univariate analysis, Overall Survival (OS) and AML transformation rate did not differ between patients with either missense or truncating BCOR mutations and BCORwt patients (Figure A&B). However, inferior OS (P=0.034) and higher AML transformation rate by trend (P=0.051) were observed for patients with truncating BCOR mutations (Figure C&D). Multivariate analysis demonstrated that a truncating BCOR mutation was an independent unfavourable prognostic factor for OS (HR 3.3; 95%CI 1.4 – 8.1; P=.008). The low number of BCORL1 mutated patients precludes any statistical analyses but clinical follow-up was available for 4 out of 5 patients. Three patients died 7, 17 and 27 months after MDS diagnosis. The fourth patient received allogeneic bone marrow transplantation after AML transformation and is in complete remission 5 years after transplantation. Conclusion In summary, truncating mutations of BCOR were independently associated with a worse OS in MDS. These data support the idea that BCOR mutations appear as associated with RUNX1 and DNMT3A mutations as reported in AML. Moreover, and despite a relatively low mutation frequency in MDS, BCOR might be considered as a key gene in risk stratification. Disclosures: No relevant conflicts of interest to declare.