ALBERT

Description: Double umbilical cord blood (dUCB) allogeneic transplantation following low dose TBI, cyclophosphamide and Fludarabine (TCF regimen)-based reduced-intensity conditioning regimen (RIC) is increasingly used in adults lacking a suitable related or unrelated donor. Currently, there is little data regarding the impact on long-term outcome of CD3+ T cell chimerism (TCC) in this particular setting. Thirty-six adults with various hematological diseases and who receieved dUCB conditioned with TCF were included in this retrospective study. Peripheral blood CD3+ TCC was considered until day +100 post-tranplant in order to determine the impact of full versus mixed chimerism on long-term outcomes. Twenty-nine and 7 patients were documented with full and mixed CD3+ TCC, respectively, within the first 100 days post-transplant. With a median follow-up of 36 months, 3 year-OS, DFS, and relapse incidence were 61%, (95% CI 43-75); 50% (95% CI 32.5-66) and 28% (95% CI 16-44), respectively. In univariate analysis, a full CD3+ TCC was associated with a better 3-year DFS : 59% (95% CI 39-75.5) versus 14% (95% CI 7-46), (HR=0.24 [0.09-0.65], p=0.005) and a lower cumulative incidence of relapse : 24% (95% CI 21.5-57) versus 78% (95% CI 52-99), (HR=0.18 [0.05-0.5], p=0.004). In multivariate analysis, a full CD3+ TCC remained associated with a lower incidence or relapse (HR=0.17, 95% CI 0.028-0.99, p=0.049). CD3+ TCC has no impact on GVHD and NRM in this study. In conclusion, in our study, full CD3+ TCC was independently associated with a lower risk of relapse after dUCB TCF RIC allogeneic transplant in adults, highlighting the need to develop immunotherapy approaches allowing for early conversion to full chimerism after dUCB. Abstract 2479. Table 1 Patients, sustained cord blood and transplantation characteristics. Patients, sustained cord blood and transplantation characteristics Full TCC (n=29) Mixed TCC (n=7) p No.of patients % No.of patients % Patients characteristic Age at transplant, years, median (range) 57 (22-69) 47 (17-64) NS Sex female 14 48 3 43 NS Hematological malignancy : Lymphoid / myeloid 14 / 15 48 / 52 3 / 4 43 / 57 NS Statut at transplant : RC / RP 23 / 6 79 / 21 6 / 1 86 / 14 NS Time to transplant, days, median (range) 395 (137-5645) 216 (92-604) NS Cord blood characteristics Age of cord blood, months, median (range) 31 (9-165) 116 (23-140) NS Matching cordon with patient NS 4/6 10 35 3 43 5/6 19 65 3 43 6/6 0 0 1 14 Number of total nucleated cell 10^8/kg before and after thawing, respectively, median (range) 0,28 (0,16-0,455) ; 0,248 (0,157-0,406) 0,222 (0,135-0,492) ; 0,22 (0,11-0,392) NS Number of CD34+ cell 10^6/kg before and after thawing, respectively, median (range) 0,066 (0,022-0,215) ; 0,043 (0,02-0,2) 0,078 (0,031-0,427) ; 0,041 (0,019-0,259) NS Mismatch between cord blood and patient Sex 14 48 3 43 NS Serology CMV 13 45 3 43 NS ABO 16 55 2 28 NS Rhesus 22 76 6 86 NS Graft Neutrophil count recovery 〉0.5 G/L, days, median (range) 17 (6-32) 11 (7-20) NS Platellet recovery 〉20G/L, days, median (range) 41 (0-164) 31 (0-67) NS Acute GVHD (grade II-IV / grade III-IV) 19 (12 / 6) 65 (41 / 21) 4 (3 / 1) 57 (43 / 14) NS Chronic GVHD (Limited / Extensive) 11 (8 / 3) 38 (28 / 10) 3 (2 / 1) 43 (28 / 14) NS Chimerism Rate, %, median (range) 100 (96-100) 82 (14-94)

Description: Key Points Overall, our results suggest that NOTCH2 and FLT3 aberrant splicing is a common event in AML that correlates with disease status and may correlate with disease outcomes. Selected variants of NOTCH2 and FLT3 transcripts were detected in a significant number of AML patients and could be useful as disease markers.

Description: Landscape analyses of mutational patterns have shown that virtually all myelodysplastic syndromes (MDS) harbor somatic mutations in 〉80% of cases. These molecular alterations provide useful clonality markers with a potential for early diagnosis of MDS when only cytopenia without marked dysplasia is observed. These markers have been proposed as future prognostic tools to guide therapeutic strategies (Bejar et al., 2011; Itzykson et al, 2013; Mufti et al 2013). Mutational analysis is finally a good way to track disease complexity by deciphering oligoclonality in MDS and better understand clonal evolution. Alterations in the TP53 gene are the most common cause of tumor escape from apoptosis. The aim of this study was to identify TP53 mutations in consecutive samples of lower-risk MDS (IPSS ≤1) with del(5q)obtained at follow-up or progression after sequential classical treatments. Next-generation sequencing (NGS) was used to backtrack the mutant clone(s) identified in late samples. The study was performed both by conventional Sanger sequencing and NGS on a GS Junior Instrument (Roche Applied Science, Mannheim, Germany). For each sample, eight exons (4-11) were amplified from 320 ng of DNA with preconfigured primer plates provided within the IRON II study network. PCR reactions were performed using the FastStart High Fidelity PCR System kit (Roche Applied Science). After double purification with Agencourt AMPure XP beads (Beckman Coulter, Miami, FL), exon-specific amplicon pools were generated and quantified using the Quant-iT™ Broad-Range PicoGreen DNA Assay Kit (Invitrogen, Carlsbad, CA). Emulsion PCR was performed with GS Junior emPCR Reagents (Lib-A) (Roche Applied Science) using 5 x 106 beads at a copy per bead ratio of 0.6. Finally, a fraction of 5-7% enriched beads was loaded on GS Junior Titanium sequencing PicoTiterPlate kit (Roche Applied Science). Data were analyzed for sequence alignment and variant detection using the GS Junior Sequencer and GS Amplicon Variant Analyzer softwares, versions 2.7 and 2.9 (Roche Applied Science). The results were further processed using the Sequence Pilot software version 4.0.1 (JSI Medical Systems, Kippenheim, Germany). The sensitivity of variant detection was set to a lower limit of 〉1% for bidirectional reads. This threshold was chosen according to a recent study investigating the assay's lower limit of detection (Grossmann et al., 2013), thus underlining the strength of NGS to identify subclones at a low frequency, not detectable by conventional Sanger analysis. A total of 89 DNA samples were extracted from the cytogenetics pellets of a cohort of 40 MDS with del(5q). TP53 mutation analysis was performed on 40 initial and 49 follow-up or progression samples including serial samples for 23 subjects. The depth of coverage was at least 500X and up to 8,444X per amplicon. Of those samples obtained and analysed at time of last follow-up or progression, 14 (61%) had TP53 mutations, mostly in the DNA-binding domain. Performing backtracking on previously collected serial samples, TP53 mutations were retrieved by NGS in 43% of initial samples (n=6), which is different from what was previously described by Jädersten et al (2011). A complete scenario of clonal evolution was retrieved in 11 cases, evidenced by TP53 mutations and/or cytogenetics. These were always consecutive to treatment with lenalidomide, yet 6 of the 12 cases without clonal evolution were also consecutive lenalidomide. Figure 1 provides the example of a complete follow-up including nine time points. More correlation with treatment will be provided. Although lenalidomide remains the treatment of choice for MDS with del(5q), resistant subclones may survive and culminate even following therapy initiation. This theory was recently suggested by Landau et al. in CLL (2013) and our test results support this. Early detection of emerging subclones could lead to initiation of alternative treatment, and we thus propose that a monitoring of TP53 alleles is performed annually after the onset of therapy for MDS using NGS. Figure 1. Figure 1. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Moreau:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau.

Description: Although most multiple myeloma (MM) cases are characterized by the detection of a monoclonal immunoglobulin in the serum, about 15% of the patients present only immunoglobulin light chains, detected either in the urine or serum or both. These patients are designated as having light-chain (LC) MM. Using fiber-fluorescent in situ hybridization, and in contrast to patients and myeloma cell lines secreting heavy chains (who presented a legitimate functional IgH rearrangement in every case), LC MM never displayed a functional IgH recombination. Interestingly, most LC MM cases presented one IgH allele with a germline configuration (including the DJ region), the second allele being usually involved in an illegitimate recombination. Of note, most of these translocations occurred close to (or at) switch regions, even though in some cases, breakpoints involving nonswitch regions were observed. Thus, this study clearly showed that LC MM is due to the absence of legitimate IgH rearrangement at the DNA level, reflecting possible abnormalities in the IgH gene recombinations during B-cell maturation. Furthermore, it showed that this defect did not prevent the activation of the switch process because most of 14q32 translocations observed in LC MM occurred at switch regions.

Description: Pharmacogenomic profiles of genes involved in bortezomib - dexamethasone response may help to understand resistance and could provide new therapeutic targets as well as contributing to novel prognostic markers in multiple myeloma. We have used gene expression profiling to analyze the complex signaling pathways regulating the response to bortezomib - dexamethasone. Gene expression profiles were established in 9 cell lines, derived from 9 myeloma patients, incubated or not with a combination of bortezomib 10 nM and dexamethasone 1 μM. These concentrations correspond to the ones used for patients in the IFM 2005-01. Cells were collected after 6 hours of treatment. We focused our interest in early response genes, making the hypothesis that the comprehension of early effects would help to better understand the mechanisms of resistance that take place in at least two third of myeloma patients. Supervised analysis with permutations identified significantly up regulated genes involved in stress responses (heat shocks proteins, RTP801/dig2/REDD1/DDIT4), endoplasmic reticulum stress (HERP/HERPUD1, gadd145/CHOP/DDIT3), ubiquitin/proteasome pathway (proteasome 26S subunits PSMB7, PSMC4, PSMD3 and PSMD13), unfolded protein response (such as SQSTM1, ATF4) or redox equilibrium (PLRX, PRDX1). We assumed that these genes might represent a molecular signature of response to bortezomib and provide important insight into the complex mechanisms of action of these drugs. We focused on REDD1 a gene cloned in 2002 that is known to be rapidly induced by a wide variety of stress conditions (arsenic, hypoxia, dexamethasone, thapsigargin, tunimycin and heat shock) and DNA damages (ionizing radiation, ultraviolet radiation, DNA alkylant). We found that both REDD1 gene and protein expression were early and highly induced after bortezomib exposure alone or in combinaison with dexamethasone. This effect was dependent upon cell line: REDD1 was overexpressed within two hours in resistant cell lines in association with a cell size decrease while in sensitive cell lines, neither REDD1 induction nor morphological changes occured. REDD1 induction was associated with the dephosphorylation of S6K1, a key substrat of mTOR, a protein kinase which controls cell growth and cell size in response to various signals. SiRNA studies confirmed that bortezomib lead to a negative regulation of mRTor activity mediated by REDD1: disruption of REDD1 abrogates both S6K1 phosphorylation and early transitory cell size reduction. Our results are in accordance with data obtained in mouse showing an early regulation of mTOR pathway and cellular proliferation induced by REDD1 expression in response to stress. Our study suggests that mTOR regulation could be a resistance mechanism mediated by REDD1 expression. As we found that REDD1 was differentially induced in primary plasma cells from patients, this gene expression could help to predict response to bortezomib. Our objective is now to clarify the pathway that links bortezomib to REDD1 in multiple myeloma and to investigate REDD1 expression in patients enrolled in IFM 2005-01 clinical trial.

Description: Abstract 4981 Background. In the two International Prognostic Scoring Systems of Myelodysplastic Syndrom (MDS), the percentage of BLAST cells in the Bone Marrow is the most important parameter implicated in score: either directly in the IPSS (International Prognostic Scoring System) (1) or indirectly in the WPSS (Who classification-based prognostic scoring system) (2). In these two systems, as in the recommendations of the WHO 2001 (3), the morphological criteria defining blasts cells compared to promyelocyte cells are not specified. In 2005, the IWGM-MDS (International Working Group on Morphology of myelodysplastic syndrome) (4-6) has established morphological criteria defining Blasts cells. PURPOSE OF THE WORK. Our objective in this study is to evaluate the reproducibility, among five observers of our laboratory, of the counting of the marrow blasts of 73 myelodysplasia. Materials and Methods. This study was conducted in several stages. 1st step was to test the implementation of the MDS-Foundation (http://www.mds- foundation.org/virtualmicroscopy) by 5 observers. The 2nd step was to perform correlation test of the selected cells from RAEB (selected and stained in our laboratory conditions), for the 5 observers. Finally Step 3 was to assess the correlation of blast percentage of 73 bone marrow smears from MDS patients, selected due to the presence of an excess of blast cells in the first reading on the bone aspiration. Results. Our results show that the correlation on counting blasts is generally satisfactory (percentage agreement: Test 1 = 86% and test 2 = 94%), while the concordance on the counting blasts of bone marow smears of 73 MDS patients and concordance on the WHO classification seems less satisfactory (agreement 3/5 observers. = 95% but agreed to 4–5/5 observers = 64%). These results can be explained partly by the inter-observer variability and by the variability of some parameters specific to smear marrow (poor quality smears, the staining and/or poverty of smears). Conclusion. The evaluation of the blasts in the MDS must be achieved: (i) at least 500 cells counted (ii) by at least two different observers (iii) by a third observer in discordant case. Despite these recommendations, the assessment of the blasts in myelodysplasia is difficult to achieve in many cases. Disclosures: No relevant conflicts of interest to declare.

Description: TP53 aberrations, including somatic mutations of TP53 gene or 17p deletion leading to the loss of the TP53 locus, are a major predictive factor of resistance to fludarabin based chemotherapy in chronic lymphocytic leukemia (CLL) and remain an adverse prognostic factor in the chemofree era. Therefore, detection of TP53 alteration before each new line of treatment is required for theranostic stratification. In order to better characterize the distribution and combination of the TP53 variants in CLL, we collected the TP53 sequencing data of 343 patients harboring TP53 mutations from centers of the French Innovative Leukemia Organization-CLL (FILO) and established a large data base of 573 TP53 mutations. Mutations were identified through NGS sequencing (covering exon 2 to 11) allowing the detection of low frequency variants down to 1% VAF. Several distinct low VAF mutations were orthogonally confirmed by digital PCR. TP53 variants were analyzed through UMD_TP53 data gathering 90 000 TP53 mutations from all type of cancers. IGHV mutational status and FISH analysis were available for 224 and 176 patients respectively. Using ACMG criteria from the UMD_TP53 database, we confirmed that 523 could be classified as pathogenic, 42 were likely pathogenic and 8 were VUS (Variants of Unknown Significance). As expected, the mutation distribution along the p53 protein exhibited a clustering of variants in the DNA binding domain of the protein. We also confirmed the presence of a specific hotspot at codon 234 (6%) which is noticeable in other CLL cohorts but absent in solid tumors. 431 TP53 variants led to the expression of a mutant protein whereas the remaining 142 led a TP53 null phenotype. For 8 patients without 17p deletion and a mutation VAF larger than 50%, SNP analysis indicate that these tumors had a copy number neutral loss of heterozygosis at 17p with a duplication of the mutant allele leading to homozygous mutations of TP53. When focusing on the allele burden of TP53 mutations, 264/573 (46%) variants had an allele frequency

Description: Abstract 761 Background: AML is characterized by specific chromosomal translocations along with a number of other recurrent mutations. However, it is also becoming clear that AML cells have frequent epigenetic abnormalities that also contribute to transformation. Alternative splicing (AS) is an epigenetic event that is used in normal cells to generate protein diversity from single genes. Using genome-wide screening approaches, we have shown that alternative splicing of numerous genes is aberrant in most cases of AML, and that several common myeloid cell surface proteins may have novel and potentially antigenic epitopes as a result. Methods: Using Affymetrix Human Exon 10ST arrays we performed a genome-wide analysis of AS in bone marrow (BM) aspirates from two cohorts (training cohort including 40 patients and test cohort including 41 patients) of patients with AML (total 81 patients) and CD34+ BM progenitor cells from 8 normal donors (NDs). After principle component analysis, 6 patients were removed from the test group, because they were outliers. In final array analysis for the test group we analysed 35 patients. Aberrant splicing of several selected genes was confirmed through cloning and sequencing analysis followed evaluating expression patterns of cloned novel splice variant transcripts in 193 patients compared to NDs. To evaluate the clinical significance of AS in AML we determined if there were any correlations between novel aberrant splice variant expression and clinical features of patients. Results and conclusions: We found that AS is a common event in AML involving many genes in any given patient. Overall, we identified ∼550 genes that were significantly spliced in patients compared to normal CD34+ cells. Three genes encoding myeloid cell surface proteins, NOTCH2, CD13, and FLT3, were selected for further study. By cloning and sequencing analysis we found that novel splice variants of the selected genes are the results of either exon skipping, usage of cryptic 5' or 3' splice sites on exons and/or partial retention of an intron. We also found that these splice variants encode proteins, as assessed by expressing splice variant transcripts in the HEK293 cell line. To identify the most frequently expressed variants of NOTCH2, FLT3 and CD13 in AML, we performed expression profiling of these transcripts and their variants in an independent validation group of 193 AML patients. These analyses identified NOTCH2-Va (74%), FLT3-Va (50%) and CD13-Va (60%) as the most frequently expressed variants in AML. Expression of these novel splice variants, other than NOTCH2-Vb, was undetectable in BM CD34+ cells, normal monocytes or neutrophils. To examine whether expression of novel variants are associated with disease evolution, we performed longitudinal monitoring of expression levels of NOTCH2-FL, FLT3-FL, and CD13-FL and their splice variants in paired samples of 13 patients taken over the course of the disease. Our study suggests the “splicing profile” normalizes in remission and recurs when patients relapse. Correlation analysis among NOTCH2, CD13 variants and FLT3 splice variants, and clinical features of patients with AML, showed that splice variant NOTCH2-Va expression is significantly negatively associated with overall survival (OS) of patients (P=0.008). Our study also showed that deregulated expression of NOTCH2-Va identifies two subgroups of patients within the intermediate-risk profile patients. We also observed significantly inferior OS of the patients that overexpressed NOTCH2-Va within this group (P=0.007). Thus, NOTCH2-Va expression can be used to stratify patients with cytogenetically determined intermediate-risk profiles. Overall, our study demonstrates that genome-wide study of AS can be used to discover new prognostic markers, and potentially creates new surface antigenic epitopes that could be targeted in AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 652 Long-term survival of patients with acute myeloid leukemia (AML) is poor, and new forms of therapy are needed. Many genetic lesions have been identified and studied, and most patients have chromosome translocations or other mutations that promote self-renewal of leukemic stem cells, block differentiation, enhance growth, and block apoptosis. Only a few of these mutations result in druggable targets (e.g., PML-RARa, Kit, PDGFR, FLT3 for instance). In addition to genetic lesions, epigenetic abnormalities have been shown to be very common in AML, and provide opportunities for novel treatments. Using genome-wide approaches to identify alternative splicing, we have recently shown that AML cells have a high level of aberrantly regulated genome-wide alternative splicing (AS) as a frequent epigenetic event. By comparing samples from 62 AML patients with 10 normal donors (NDs) we identified 428 genes differentially spliced in AML. A list of differentially spliced genes includes 50 oncogenes and 52 tumor suppressor genes, as well as genes encoding proteins involved in cell proliferation and differentiation, and apoptosis. We evaluated splicing event frequency in AML compared to NDs and we observed that on average 527 (range 137–1657) genes were identified as differentially spliced in any given patient, out of 62 analyzed. Also, we found that any given differentially spliced gene, of the 3,108 detected, were spliced on average in 26 (range 1–54) AML patients. Thus, splicing aberrations are highly recurrent in AML patients. To identify the causes of aberrant splicing in AML, we evaluated transcript levels of the 24 major splicing factors (SFs) that are involved in the first and second splicing transesterification reactions. These splicing factors are important proteins involved in spliceosomalassembly. Expression levels of these SFs were evaluated in 20 AML patients exhibiting high levels of AS. Quantitative RT-PCR analysis showed significant (up to 30 fold) upregulation of U2AF2 (P

Description: Key Points Myeloma cells are highly sensitive to PRIMA-1Met, independent of p53. PRIMA-1Met induces myeloma cell death by impairing GSH/ROS balance.