ALBERT

Description: Key Points UDS demonstrated that BCR-ABL KD mutations detectable with conventional methods may just be the tip of the iceberg. The information provided by conventional Sanger sequencing may not always be sufficient to predict responsiveness to a given TKI.

Description: Abstract 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results: BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p 〈 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions: Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Description: Abstract 3136 Deletions of the 9p21 chromosomal region are frequent events for the development of a variety of cancers, including solid tumors and hematological malignancies, such as childhood ALL. This region contains the 40-kb region encoding the p16INK4a/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15INK4b/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. In order to explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate the influence on the prognosis, 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18–76) and the median blast percentage was 90% (range, 18–99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21. PCR amplification and mutation screening of each exon by cloning and subsequent sequencing were also performed. Moreover, gene-expression profiling was performed (Affymetrix Human Exon 1ST Array) to draw a specific signature associated with deletions. At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8–31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm eliminating a large number of genes. In contrast, cases with bi-allelic inactivation were 28% with the majority of deletions (75%) limited to CDKN2A/ARF/CDKN2B genes. FISH analysis was performed using three different BAC clones, but since they overlooked microdeletions we only appreciated a mild fluorescent signal reduction. In order to assess whether 9p21 loss is responsible for progression, the genomic status of this locus was assessed at the time of relapse and an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06) was found. Functionally, deletions led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005), as demonstrated by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. Finally, the mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by point mutations with only the identification of the missense D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5′UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)] and a variation, known as SNP was found in the exon 2 (rs3731249). Finally, 2 SNPs were found in the 3′ UTR region of CDKN2A/ARF (rs11515 and rs3088440). The role of the mutations identified in the UTR is not yet defined, but the comparison with the germline material from saliva samples showed that these mutations were inherited. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort, finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.003) and cumulative incidence of relapse (p= 0.004). In conclusion, the inactivation of the tumor suppressor genes CDKN2A/ARF is a frequent event in Ph+ ALL. The main mechanism of inactivation is represented by genomic deletions, whereas missense mutations are rare. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Baccarani: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Description: Abstract 284 Background and Aims: Selection of drug-resistant mutations in the Bcr-Abl kinase domain (KD) is a critical problem undermining the long-term efficacy of tyrosine kinase inhibitor (TKI)-based therapies in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. Bcr-Abl KD mutation screening is routinely performed by Sanger sequencing (SS). Before the advent of ultra-deep sequencing (UDS) technologies, no method was available that could conjugate the possibility to scan the KD for the so many mutations known to be associated with TKI resistance with a sensitivity higher than that of SS. UDS technologies also allow high throughputness and accurate quantitation of mutated clones and their application in a diagnostic setting is not far to come. We used an UDS strategy for Bcr-Abl KD mutation screening in order to study the dynamics of expansion of mutated clones in Ph+ ALL patients receiving TKI-based therapies and to test the ability of UDS to highlight emerging clones harboring critical mutations. Methods: 72 samples from 25 Ph+ ALL patients who had developed resistance to one or multiple lines of TKI (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) therapy were selected for this retrospective analysis. All the patients had previously been analyzed by Sanger sequencing (SS) and were known to have developed one or more TKI-resistant Bcr-Abl KD mutations on treatment. In order to reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly collected samples was perfomed with UDS on a Roche GS Junior. UDS allowed to achieve a lower detection limit of at least 0.1% (by generating a minimum of 5,000 sequence reads/patient), as compared to 20% of SS. Results: 39 samples were known to harbor one (n=27 samples) or more (n=12 samples) TKI-resistant mutations with 〉20% abundance, as assessed by SS. UDS could successfully detect all the 54 mutations previously identified by SS. In addition, UDS detected one or multiple lower-level (20% abundance. The type of lower-level mutations detected by UDS could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the TKI being administered or to the previous TKI received. Overall, 44 samples turned out to carry multiple (two to five) mutations at any level, distributed in the same and/or in different subpopulations with a complex clonal architecture that UDS allowed to reconstruct. Of note, in 14/25 (56%) patients with molecularly detectable disease but not yet evidence of cytogenetic or hematologic relapse, UDS could identify emerging TKI-resistant mutations 1 to 2 months before they became detectable by SS. These outgrowing mutations were detected at 1–19% abundance in 12 patients and at 0.1–1% abundance in 2 patients. In the remaining 11 patients, dynamics of outgrow of the TKI-resistant mutations (five T315I, two Y253H, two E255K, one E255V and one F317L) was so rapid that not even strict monthly monitoring could allow to pick them up before they became dominant. Conclusions: Now that multiple options are available, Bcr-Abl KD mutation monitoring has become a precious tool for rational decision-making in order to maximize the efficacy of TKI-based regimens as induction or salvage therapy for Ph+ ALL patients. UDS proved as reliable as SS for the detection of mutations with 〉20% abundance and to have comparable costs. As a key advantage, UDS added precious quantitative and qualitative information on the full repertoire of mutated populations, that SS failed to appreciate in more than half of the samples analyzed. TKI-resistant mutations leading to patient relapse were not necessarily preexisting at low levels at diagnosis or at the time of switchover to another TKI, underlining the importance of regular monitoring of patients. Although TKI-resistant populations may arise and take over very rapidly, in approximately half of the patients monthly monitoring with UDS would have allowed to identify them earlier than SS and well in advance of clinical relapse, thus allowing a more timely therapeutic intervention. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Description: Abstract 1496 Introduction: Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 〉 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p 〈 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Description: Introduction . Anti-thymocyte globulin (ATG), has been shown to significantly reduce the incidence and severity of acute and chronic graft versus host disease (GVHD) in pts submitted toallogeneic stem cell transplantation (allo-SCT). Two different ATG formulations, derived from rabbit immunization against T antigens,thymoglobuline-ATG (tATG) andfresenius-ATG (fATG) are usually employed. However, only few retrospective studies are available in order to compare the efficacy and safety of these two drugs. Moreover, the differential impact of the two formulations on immune recovery has been poorly studied. Methods. Clinical and laboratory data of 76 pts receivingfATG (30 mg/kg for 3 days beforeallo-SCT) ortATG (10 mg/Kg for 3 days beforeallo-SCT) as GVHD prophylaxis forallo-SCT from matched unrelated donor (MUD) for hematological malignancies at the Unit of Stem Cells Transplantation ofSpedaliCivili of Brescia between 2009 and 2014, were retrospectively collected. Basic phenotype of circulating lymphocytes was assessed by flow-cytometry at 3-months interval fromallo-SCT. The aims of the study were to compare the two ATG formulations in term of incidence of acute and chronic GVHD, infections, non-relapse mortality, relapse-free survival, overall survival and immune recovery. The study was approved by the local Ethic Committee. Results. Overall, 46 (60%) pts receivedfATG and 30 (40%)tATG as GVHD prophylaxis, in association to cyclosporine andmethotrexate. Baseline characteristics were (median): age, 46 years (range, 17-66); male, 68%; acute leukemia, 53%; complete hematological remission, 51%; HLA full-matched (8/8), 40%; source of stem cells, peripheral blood, 87%;myeloablative conditioning regimen, 49%; median follow-up, 22 months (range, 1-88). No significant differences were observed between the two populations according age, sex, hematological disease, disease status atallo-SCT, HLA match, stem cell source, amount of CD3+ and CD34+ cells infused and conditioning regimen. Overall, 26 (57%)fATGand 19 (61%)tATG-exposed pts developed an acute GVHD (p=0.8); among 18 (24%) pts who developedcGVHD, 2 out of 10 (20%)fATGand 6 out of 8 (75%)tATG-receiving pts evolved towards severecGVHD(p=0.05). In particular, the cumulative incidence of severecGVHD, was 5,2% versus 27,6% at 2 years (p=0.03) infATGandtATGgroup, respectively (Figure 1). Bacterial infections were encountered in 42 (91%) and 25 (81%) (p=0.19), fungal infections in 12 (26%) and 6 (19%) (p=0.58)fATGandtATG-treated pts. CMV reactivation was observed in 28 (61%) and 19 (73%) (p=0.43)fATGandtATG-exposed pts, respectively. However, a trend for a higher incidence of early-CMV reactivation was seen in thetATGgroup, with a cumulative incidence of CMV reactivation at 30 days of 30% versus 52% infATGandtATGcohort, respectively (p=0.09). No differences were observed betweenfATGandtATGpopulations in term of relapse-related mortality (24% versus 40%, p=0.25), non-relapse mortality (28% versus 20%, p=0.48) and overall survival (52% versus 60%, p=0.71). Immunologic reconstitution was evaluated in 48 pts (fATG, 64%). As compared totATG,fATGpts showed a significant lower amount of CD3-/CD16+ NK cells (median, 137/mclvs255/mcl, p=0.05) and a trend for lower CD3+CD4+CD25+CD127- T-regcells (5/mclvs11/mcl, p=0.07) and CD4/CD8 ratio (0.26vs0.57, p=0.08) at 3 months fromallo-SCT. At 6 months CD4/CD8 ratio was significantly lower infATGcompared totATGgroup (0.26vs0.42, p=0.01). At 9 months, lymphoid populations did not differ between the two cohorts. Overall, ATG administration was well-tolerated without grade III-IV adverse events, however an increase in infusion-related events (mainly, fever and chills) was recorded infATGgroup (67%vs32%, p=0.004). Conclusion. This retrospective study, reporting a quite large population ofallo-SCT pts from MUD homogeneously followed in a single Hematology Center, shows thatfATG-treated pts have a lower incidence of severecGVHD compared totATG. On the contrary a slight increase of early CMV reactivation has been observed intATG group. These differences do not result in a higher relapse-related or overall mortality. The distinct immune reconstitution as identified by flow-cytometricanalysis, could justify these observations, that need to be validated by prospective studies. Figure 1 Cumulative incidence of severecGVHD according to type of ATG employed Figure 1. Cumulative incidence of severecGVHD according to type of ATG employed Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4711 Background: Myeloproliferative diseases (MPDs) are a group of hematological malignancies characterized by the abnormal increase of different blood cells in peripheral blood and in other hematological organs. The molecular pathways involved in disease pathogenesis and progression as well as in drug sensitivity and resistance are not fully elucidated yet. Given the physiological similarity to mammals, the zebrafish (ZF) could accelerate the study of the molecular basis of these diseases. In the last decade several human leukaemia associated oncogenes have been transiently over-expressed in ZF embryos in order to perturb the myelo-erythroid compartment, with the aim to create experimental models to discover new molecular pathways that can became potential targets for new chemical compounds. Methodogy: We took advantage of the binary Gal4/UAS system to express oncogenic human HRAS in the ZF hematopoietic compartment. We used the tg(MAZe) driver line, a specific transgenic line that allows random mosaic expression of Gal4-VP16 after heat shock(hs) treatment(1). This driver line was mated with a tg(UAS:eGFP-HRASV12)(2) responder line in order to mimic somatic events leading to human oncogene expression. Results: Surprisingly a single heat shock at 30hpf, (referred here as HRASV12hs) induces human HRASV12 expression in hematopoietic progenitors as judged by the expansion of the hematopoietic tissue. Histological analysis showed an increased number of monocytes/macrophages. Blood smear of HRASV12hs larvae showed the expansion of blasts and myeloid precursors. Quantitative analysis of gene expression highlights a remarkable increase of myelo-erythroid restricted genes associated with a slight increase of staminality markers (pu1, mpx, lmo2, mpl, CD41 and c-myb). Moreover fish raised to adulthood developed hyper-plastic kidney marrow, the site of definitive haematopoiesis, the equivalent of mammalian bone marrow. Conclusions and Future Perspectives: The combination of hyper-proliferation of blood cells associated with the expansion of the hematopoietic compartment that we found in this model reproduces the pathological features of human myeloproliferative disorders. This study shows that it is possible to drive the expression of leukemia associated oncogenes to the hematopoietic compartment in developing ZF thus reproducing some of the features of human blood malignancies in an in vivo model. Our Group is now testing different driver lines in order to induce oncogene expression in earliest hematopoietic progenitors and is focusing on different responder lines expressing human oncogenes involved in hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction The long-term efficacy of allogeneic haematopoietic stem cell transplantation (SCT) relies primarily on the Graft-versus-tumor (GVT) effect, which partially overlaps with Graft versus Host disease (GvHD), the most common cause of morbidity and mortality in SCT. Researches on GVHD-biomarkers are still ongoing and a set of validate markers are still lacking, especially for chronic GVHD. Furthermore, immune parameters that univocally associate with GVHD or GVT have not been identified yet. In this study, lymphocyte subsets together with TCR-repertoire analysis, and index of thymic and bone marrow output were evaluated at different time points, in order to identify possible predictors of GVHD and ineffective GVT. Methods Prospective evaluations of lymphocyte subsets, thymic and bone marrow output were performed in 40 patients before SCT, at 30, 90, 180 days and 1 year after SCT. CD4+/CD8+ naïve, central memory, effector memory, terminally differentiated effector memory (TEMRA) cells, subsets of regulatory T-lymphocytes, immature B cells, naïve, switched and unswitched memory B cells, memory double negative (IgD-CD27-) B cells were analysed by flow cytometry. Analysis of thymic and bone marrow output was performed by detection of T cell receptor excision circles (TRECs) and kappa-deleting recombination circles (KRECs). TRECs and KRECs were simultaneously quantified by a duplex quantitative Real-Time PCR. Heteroduplex assay was used to perform TCR-repertoire analysis. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, to solve the problem of the high number of variables (immunological, patients- and transplant related) in comparison with the relatively limited and heterogeneous pool of patients. Results Twenty patients developed acute GVHD (median time: 28 days, range 19-120). Chronic GVHD was observed in 9 patients (median time: 6 months, range 4-10). In multivariate analysis, acute GVHD correlated positively with pre-transplant percentage of CD4+ central memory cells, and with values of regulatory effector memory T-cells and CD4+TEMRA cell at day +30 (p=0,0006). Pre-transplant percentage of unswitched memory B cells was also associated with acute GVHD, whereas pre-transplant levels of KRECs were inversely correlated (p=0,0005). Chronic GVHD was associated with matched unrelated donor and with (peffector memory), as shown in some experimental models. Increased values of CD8+ effector memory cells could be an early sign of ineffective GVL. Imbalance toward a lymphocyte B-response, and especially toward "senescent" memory (IgD-CD27-) B cells, could promote tolerance to tumor cells. The validation of these clusters of immunological parameters as specific early predictors of GVHD or GVT, even before SCT, could potentially allow the development of pre-emptive and targeted therapies. Disclosures No relevant conflicts of interest to declare.

Description: Background: FLT3 internal tandem duplication (ITD) is frequently detected in AML patients and is an independent predictor of unfavourable outcome, while secondary point mutations in the FLT3 tyrosine kinase domain (KD) are common causes of acquired clinical resistance to FLT3 inhibitors, such as AC220 and Sorafenib. Technologies allowing massively parallel, ultra-deep sequencing (UDS) are currently being evaluated in diagnostic settings since they may conjugate throughputness, sensitivity and accurate quantification of mutated clones. Aim: Since recent whole genome sequencing studies have suggested that FLT3 ITD may evolve from small subclones undetectable at diagnosis by routine PCR, we tested the ability of an UDS strategy for FLT3 mutation screening to highlight small clones harbouring ITD mutations. Furthermore we evaluated if an UDS strategy could highlight in AML patients treated with FLT3 inhibitors emerging clones harbouring critical mutations, anticipating the development of drug-resistance. Methods: 886 AML patients were analyzed in Seràgnoli Institute of Bologna between 2002 and 2013 for a panel of genetic alterations, including FLT3. For our purpose, we retrospectively analyzed five AML (four CN-AML and one t(3;3) AML) who were found negative for FLT3 ITD- at diagnosis by conventional PCR and Sanger Sequencing, but were then found FLT3 ITD+ during follow-up at relapse or disease progression and ten AML (five FLT3+ and five FLT3-) treated with the FLT3 inhibitor AC220. In order to reconstruct the dynamic of mutation emergence, we performed a longitudinal re-analysis of RNA samples with UDS on a Roche GS Junior. UDS achieved a lower detection limit between 0,1% and 1%, depending on the relative number of sequence reads per sample obtained in each run. Results: 886 AML patients were analyzed for a panel of genetic alterations including FLT3 ITD and TKD and 239 of 886 (27%) were FLT3+. In particular 256 were cytogenetically normal (CN) AML and of these 66 (25,8%) were FLT3+ and 192 were FLT3-.UDS strategy revealed that all the CN-AML analyzed already carried at diagnosis a small clone FLT3 ITD+ (allelic ratio 0.2-2%), that increased over time during follow-up, while in the t(3;3) AML the ITD clone emerged only at disease progression. In the three CN patients treated with chemotherapy the ITD+ was a minor clone until complete remission (CR), while after relapse the ITD+ clone expanded; one of these patients carried two rare ITD clones at diagnosis and only one became dominant at relapse, likely through loss of heterozygosity (LOH) of the mutated allele. In one CN patient who was treated only with FLT3 inhibitor AC220 the allelic load of the mutated clone increased over time before treatment and then followed the dynamic of the disease (regression at complete remission). For the five AML FLT3- treated with AC220 we didn’t find any novel FLT3 mutation after treatment, while for the five AML FLT3-ITD+ analyzed, we were able to follow the allelic load of the FLT3-ITD+ clone during treatment and the appearance in two patients of novel TKD mutations after treatment that are able to confer drug resistance (allelic ratio 2-55%). Conclusions: The high sensitivity of UDS technology allows detection of emergence of mutated clones earlier than conventional methods: this is a precious tool to find small clones FLT3 ITD+ that may evolve over time and worsen the prognosis of otherwise good prognosis CN-AML patients and to better calibrate therapy for these patients. Furthermore the prognostic value of determining the presence of FLT3-ITD by UDS is stronger than conventional methods because of the possibility to determine the ratio of mutated versus wild-type allele, the length and the size of insertion within a single analysis. In the setting of FLT3 inhibitors, UDS gives advantage in monitoring MRD by determining exactly the allelic load of mutated FLT3 ITD clones before and after treatment and high sensitivity in highlighting emerging mutated clones during treatment that may confer resistance, giving possibility to switch eventually to other inhibitors before coming out of overt clinical resistance to therapy. For monitoring patients treated with FLT3 inhibitors with UDS we will go on by screening AML treated with Sorafenib, to follow the emergence of any novel critical mutation during treatment. Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Description: Introduction In AML and MDS cases, the genetic lesions inherited or acquired by the hematopoietic stem cells are considered as starting events. Familial AML and MDS, recently recognized in the revised WHO classification (2016) provide a useful model for investigation of predisposing genetic mutations. Genetic analysis of several pure familial leukemia pedigrees led to the discovery of well defined syndromes associated with inherited de novo mutations on germline DNA. Growing clinical awareness as well as a widespread use of NGS have led to an enlarged description of familial MDS/AML cases, and the number of mutations involved, suggesting they are more frequent than those previously recognized. Despite the recent discovery of well-established causative gene mutations (RUNX1, GATA2, ETV6, TERT, TERC, SRP72, ANKRD26, DDX41, CEBPA), many cases remain unexplained (about 80%), suggesting that other inherited mutations could predispose to MDS/AML. It is expected that new sequencing approaches will help to the identification of more cases, more genes as well as novel syndromes. In 2017, we started a multicentric prospective study (Clinical trial.gov NCT03058588) aiming to look for predisposing mutations in patients and relatives affected by Familial AML and MDS syndromes (FAMS) by NGS and to screen for old and new mutations potentially associated with the disease. Methods At present, 12 AML/MDS patients have been enrolled. Leukemic (bone marrow) and germline (buccal swab) DNA were analyzed by NGS gene panel approach based on a 28 genes associated to myeloid leukemogenesis, including the 9 above mentioned genes associated to FAMS. NGS libraries were performed by a Nimblegen (Roche) custom panel based on gene capture strategy and the sequencing was performed by MiSeq (Illumina). Results Ten patients did not reveal any germline mutations and the candidates are undergoing to whole exome sequencing. One presented a germline mutation on RUNX1, and the analysis of the affected relatives is on going. One revealed a new mutation. She was a 70 years old woman affected by RARS and her pedigree was characterized by 9 relatives affected by hematologic and solid neoplasia and trombocytopenia (fig 1). The NGS analysis revealed the mutation c.*514C〉T in 3'UTR of ETV6 with VAF of 50% on tumor DNA. The variant has never been described before, while ETV6 has been already associated with FAMS. Sanger sequencing confirmed the mutation on the germline DNA in heterozygosis. The screening of 2 affected relatives still alive confirmed the presence of the variant in heterozygosis. In silico analysis performed on PolymiRST Database revealed that c.*514C〉T in 3'UTR of ETV6 results in a gain of miRNA binding site: hsa-miR- 4717-3p and hsa-miR- 942-3p. Discussion The variant c.*514C〉T in 3'UTR of ETV6 seems to repress ETV6 due to RNA interference. The new binding miRNAs have been already described as over-expressed in solid and hematologic tumors. Moreover, the down-regulation of ETV6 is associated with alteration of cell growth and hematopoiesis. Due to these evidences, c.*514C〉T in 3'UTR of ETV6 could be considered as a new mutation involved in FAMS predisposition. Disclosures No relevant conflicts of interest to declare.