ALBERT

Description: Determination of the membrane seal capacity of deformation bands is critical for managing geologic reservoirs in porous sandstones. In this study, we have analyzed a cataclastic shear-band network developed in uncemented porous sandstone in Provence, France. Geometrical analyses of the bands show significant differences between three types of bands (single strand, multistrand, and band cluster), sorted by their number of strands, their amount of shear displacement, and their thicknesses. At the microscopic scale, the image-analysis porosities and the grain-size distributions allow definition of three different types of microstructural deformation: damage zone, protocataclastic, and cataclastic. Whereas damage zone and protocataclastic deformations are observed in each type of band, cataclastic strands are observed in clusters and, sometimes, in multistrands. Cataclastic strands are characterized by a porosity reduction of 10 to 25% and a permeability reduction of three to five orders of magnitude compared to the host rock. Field observations of iron hydroxide precipitations around the bands suggest that cataclastic strands were membrane seals to water flow under vadose condition. This study therefore highlights the importance of the degree of cataclasis in shear bands as membrane seals to subsurface fluid flows in sandstone reservoirs.

Description: Introduction Isolated trisomy 8 is a frequent cytogenetic abnormality in MDS, but hematological characteristics of MDS with isolated trisomy 8 have not been reported in detail. Patients and Methods This was a retrospective analysis of cases of MDS with isolated trisomy 8 diagnosed in 6 French centers of the Groupe Francophone des Myélodysplasies (GFM) between 2003 and 2013. Only patients with isolated trisomy 8 diagnosed as MDS or MPN/MDS (other than CMML) according to WHO were eligible, excluding AML, well characterized MPN (PV, ET) and CMML. Myeloproliferative (MP) features were defined by repeated presence (in the absence of infection) of one of the following: WBC 〉 10G/L, circulating immature granulocytes (myelemia ) 〉 2%, or palpable splenomegaly. Results 103 patients with isolated trisomy 8 were identified, with a median age of 75 years, and M/F 1.7. At diagnosis, median WBC count was 4.1 G/L, with WBC ≥ 10 G/L in 13 patients (12.6 %), myelemia ≥ 2% in 27 patients (26.2 %), palpable splenomegaly in 9 patients (8.7 %). WHO diagnosis included 20 RA, 2 RARS, 22 RCMD, 1 RCMD-RS, 1 RCUD, 21 RAEB-1, 18 RAEB-2, 7 MDS-U, 10 MDS/MPN, 1 hypoplastic MDS. IPSS was intermediate 1 (72.2 %), intermediate 2 (19.6 %), high (8.2 %) ; IPSS-R was low (37.1 %), intermediate (29.9 %), high (22.7 %), very high (10.6 %). MP features were found in 50 patients (48.5 %): 31 at diagnosis, 19 during evolution (in patients without MP features at diagnosis). Bone marrow morphological features could be reviewed in 15 MP cases, showing hypercellular marrow in 60 % cases, granulocytic hyperplasia (E/G

Description: Purpose: Major initial prognostic factors predicting chronic myeloid leukaemia (CML) response to imatinib mesylate (IM) therapy are represented by hema-tological status, Sokal score and duration of disease before IM. Before the era of IM, CML patients harbouring der(9) deletion classically had a worse outcome when compared to non deleted pts, but some preliminary studies suggest that this adverse factor might be reversed by IM. Additionally der(9) deletion seems to be more frequent in Variant Ph chromosome (vPh) patients (up to 30%) whereas its frequency is about 10% in classical Ph pts. We evaluated in this retrospective multicentric study the response to IM in vPh population and the possible impact of der(9) deletion on this response. 92 vPh patients treated by IM were selected from 11 hematology departments within the country. vPh chromosome was assessed by conventional karyotyping analysis and der(9) deletion by FISH. IM resistant patients were screened for ABL kinase site mutation by direct sequencing. Statistical analysis of survival and descriptive parameters were performed according to Kaplan Meyer method, log-rank test, Chi-2 and non parametric Kruskal Wallis tests. Results: among the vPh population 75% pts had a simple translocation and 25% had a more complex one (chromosomes 9, 22 and at least two other break-points). Initially 95% pts were in chronic phase (CP), 2.5% in accelerated phase (AP) and 2.5% in blastic crisis (BC).19 pts (33.3%) were scored in each Sokal score category (low, intermediate and high). At IM initiation: 83% were in CP (〉35% Ph+ ), 8% were in major cytogenetic response (after interferon) and 9% were either in AP or in BC. 74 pts were screened for der(9) deletion, which was present in 16.2% of them. IM response and survival data were available for 53 pts with CP (with 〉 35% Ph+ ) at IM initiation. Median age of this CP population at diagnosis was 50.8 (19.5–79.4), median disease duration before IM initiation : 2 Mo (0–92.4), median follow-up after IM initiation : 24.8 Mo (6.1–69.9). 71.7% achieved RcyM including 67.9% in RcyC. 19 pts presented IM failure or suboptimal response (European LeukemiaNet consensus). 1 pt stopped IM for liver intolerance. We observed 9 (16.9%) primary resistance and 10 (18.9%) loss of response. 12% pts progressed towards AP/BC. 12 resistant pts were screened for ABL kinase site mutations : 3 of them harboured mutations. We found no difference for the overall survival between the populations with (n=9) and without (n=33) der(9q) deletions (median of survival not reached ). Respectively 1 pt (11%) and 3 pts (9%) died in these two cohorts. We found a trend for an increased rate of resistance in the der(9) deletion cohort (55.6% vs 30.3%) but the difference was not significative (p=0.16). We only found significant differences in the repartition of the Sokal score between these two cohorts with more frequent high scores for vPh patients (p=0.03). Resistance in vPh IM treated pts is high (35.8%) but the disease duration before IM was heterogenous. Der(9) deletions slightly increases the rate of IM resistance but we found no difference of survival between the two cohorts.

Description: Abstract 2929 Background: Azacitidine (AZA) has changed the outcome of patients (pts) with myelodysplastic syndromes (MDS) or acute myeloid leukemia with multi-lineage dysplasia (AML-MLD) unfit for intensive chemotherapy. AZA is a hypomethylating agent providing about 50% of responses in MDS and AML with low blast count (Fenaux et al., Lancet Oncol 2009, JCO 2010). To date, no consensus genetic predictor of response has been reported. Methods: In MDS (including RAEB-t) and AML-MLD (〉30% blasts) patients treated by AZA in 5 centers, we performed genomewide single nucleotide polymorphism (SNP) analysis using SNP 6.0 arrays (Affymetrix, High Wycombe, U.K.) on bone marrow (BM) samples. Patients having received ≥ 1 cycle of AZA and who had bone marrow evaluation after ≥ 4 cycles, or who died or progressed before completion of 4 cycles were considered evaluable (the last 2 groups were considered as treatment failures). Responses were scored according to IWG 2006 criteria for MDS and to Cheson et al. (JCO 2003) for AML. DNA were prepared for hybridization according to the manufacturers' recommendations. Affymetrix CEL files for each sample were analyzed using the Genotyping Console software (v3.0.2). Genotyping was performed using Birdseed V2 algorithm. Unpaired Copy Number and LOH analysis was performed with Regional GC correction. Copy number and UPD were also analyzed using the Copy Number Analyzer for GeneChip (CNAG version 3.3.0.1) algorithm (http://www.genome.umin.jp/CNAGtop2.html). Results: The study population included 92 pts: F/M: 41/51; median age 72 (range 35–88). Diagnosis at AZA onset was MDS in 54 (RAEB-1 n=6, RAEB-2 n=37, RAEB-t=11, IPSS int-1 in 7, int-2 in 15, high in 32, undetermined in 2) and AML-MLD in 38 pts. Cytogenetic according to IPSS was favorable in 33, intermediate in 19, unfavorable in 28, unknown in 11. Median number of cycles was 6 (range 1–41). All pts received the approved (75 mg/m2 for 7 days every 4 weeks) or a reduced AZA schedule (75 mg/m2 for 5 days every 4 weeks). Median overall survival (OS) of our cohort was 22 months. DNA samples from 52 patients were available for SNP analysis. There were no significant differences between the SNP and no SNP subgroups in term of median age, sex ratio, disease status at AZA onset, cytogenetic according to IPSS, median number of cycles, responses and OS. We listed aneuploidies (CNA) and uniparental disomies (UPD) detected by SNP analysis in the samples, and focused on 18 chromosomal bands (1p13.2, 2q34, 3p14.2, 3q26, 4q24, 5q33.1, 5q35, 6p21.3, 7q36.1, 9p21.3, 11p13, 11q23.3, 13q12.2, 15q21, 15q26.2, 20q11.21, 21q22.1) containing genes implicated in MDS or AML. Preliminary results show correlations between some CNA/UPD and response to AZA and OS, such as 1/a UPD at 9p21 (6% of patients) associated to a better hematologic improvement (X2, p=0.037), 2/an amplification at 20q11.21 (8% of patients) correlated with a poorer response (X2, p=0.048) and a trend toward poorer OS (Log-Rank, p=0.091), and 3/deletion and UPD at 15q21 (6% of patients) strongly correlated to poorer OS (Log-Rank, p=0.001). Several additional CNA and UPD of potential interest are presently under investigation. Conclusion: SNP analysis using SNP 6.0 is a powerful tool to decipher genome complexity in BM samples of MDS and AML patients treated with AZA. Our data suggest that this approach could allow characterizing profiles of responder versus non responder pts. Our study must be extended to a larger cohort and relevant anomalies must be confirmed by more sensitive techniques such as high-scale sequencing. Disclosures: Cluzeau: Celgene: Consultancy. Raynaud:Celgene: Consultancy.

Description: Abstract 1817 Azacitidine (AZA) is the first line treatment for IPSS (index prognostic scoring system) high-risk myelodysplastic syndrome (HR-MDS). To date, only Khan et al. (Exp Hematol. 2008) and Hollenbach et al. (PLoS One. 2009) have reported apoptosis as a mechanism of AZA effect on MDS cell lines. Nevertheless, approximately 40% of patients treated with AZA are refractory to this molecule. To investigate the possible mechanisms of AZA resistance in MDS cells, we developped AZA-resistant cell clones (AZA-R) from the well-characterized MDS cell line SKM1. The bulk resistant SKM1 cell line (AZA-R) was obtained following long time exposure of cells to iterative and increasing doses of AZA ranging from 0.1 to 8mM. We first showed that AZA triggered loss of cell metabolism in SKM1 parental cells but not in their AZA-resistant counterpart at a maximally effective dose of 1mM. AZA-mediated loss of cell metabolism accounted mainly for induction of apoptosis as judged by both an increase in caspase 9 and 3 activities triggered by this compound and by a significant protection in the presence of the pan-caspase inhibitor Z-VAD-fmk in SKM1 parental cells. Conversely, no or very few activation of caspases and apoptosis were detected in AZA-R cells strongly suggesting that apoptosis is impaired in AZA-R SKM1 cells. Finally, unlike in SKM1 cells, AZA failed to induce mitochondrial membrane permeabilization in AZA-R SKM1 cells. Importantly, basal autophagy was increased in AZA-R versus AZA-S cells as shown by LC3-I cleavage into LC3-II, p62/SQSTM1 protein expression, cathepsin B activation, mTOR and S6 ribosomal protein dephosphorylation and finally electronic microscopy experiments. In addition, Acadesine, an adenosine derivative and AMPK agonist which targets autophagy was capable to circumvent AZA resistance in both AZA-R SKM1 cells and in medullary cells from five MDS patients resistant to AZA after 6 cycles of Azacitidine. In conclusion, targeting autophagy appears as an attractive therapeutical strategy to circumvent AZA resistance in both established MDS cell lines and cells from MDS patients. Therefore, drugs capable of inducing autophagy or autophagic cell death, such as Acadesine (Robert et al., PLoS One. 2009) which is currently in phase II clinical trials for the treatment of Chronic Lymphoblastic Leukemia could be also beneficial for HR-MDS patients resistant to AZA. Disclosures: Cluzeau: Celgene: Consultancy. Raynaud:Celgene: Consultancy.