Abstract: Background:Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy of the elderly with a heterogenous molecular pathophysiology. Whereas mutations in components of the RAS pathways are among the most common somatic mutations in CMML the JAK2 V617F mutation which is a typical finding in polycythemia vera and around 50% of patients with essential thrombocythemia and primary myelofibrosis, respectively, is by far less frequently detected in CMML but can be consistently found in a subgroup of patients in larger series. Due to the fact that JAK2 V617F-positive CMML is a rare disease the clinical, hematological and in vitro growth characteristics of this entity are poorly investigated. In the "Austrian Biodatabase for Chronic Myelomonocytic Leukemia (ABCMML)" we retrospectively and prospectively collect clinical, biologic, and molecular information of patients with CMML from different centers in a real life setting. Aims:Our aim was to characterize the clinical, hematological, molecular and biologic features of CMML patients harboring a JAK2 V617F mutation. Methods:The diagnosis of CMML was established according to diagnostic criteria of the World Health Organization (WHO) classification of 2008 (Vardiman et al, Blood 2009). Clinical and hematological data were obtained from patients records. For molecular characterization we used next-generation sequencing with amplicon-based target enrichment of 39 CMML associated genes. Only mutations with an allele burden of 〉 10% were considered positive in this analysis. Autonomous colony-forming units granulocyte/macrophage (CFU-GM) growth in the absence of exogenous cytokines was assessed using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Results:Up to now targeted NGS data are available in 116 patients and in vitro culture data in 75 patients respectively. We identified 13 CMML patients who had a JAK2 V617F mutation with an allele frequency 〉 10%. Clinical, hematological, and biologic characteristics in these patients were compared with 103 patients who had NGS sequencing and were negative for the JAK2 V617F mutation. As shown in Table 1 JAK2 V617F-positive CMML patients had significantly higher WBC counts, higher hemoglobin values, higher platelet counts and more pronounced splenomegaly as compared to JAK2 V617F-negative patients. On the other hand the percentage on monocytes in peripheral blood and the numbers of CFU-GM growing in vitro without addition of exogenous growth factors were lower in CMML patients with the JAK2 V617F mutation as compared to patients without this mutation. The majority of JAK2 V617F-positive patients had additional mutations that can be also found in JAK2 V617F-negative patients, in particular mutations in genes of epigenetic regulation and RNA-splicing, respectively. As shown in Figure 1 there was a trend towards a better survival of patients with the JAK2 V617F mutation as compared to JAK2 V617F-negative patients (p=0.05). In a JAK2 V617F-positive CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we were able to demonstrate the disappearance of constitutional symptoms and a durable spleen response lasting for over 56 months (Fig. 2). Conclusion:Out data show that CMML patients with the JAK2 V617F mutation have hematological, biologic and clinical characteristics different from JAK2 V617F-negative CMML patients. These findings suggest that JAK2 V617F-positive CMML patients should be regarded as a distinct subgroup which may benefit from specific targeted treatments. Disclosures Geissler: Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celegene: Honoraria, Research Funding.

Abstract: Background: Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy with features of both a myelodysplastic syndrome and a myeloproliferative neoplasm.The pathogenesis of CMML is incompletely understood due to the large heterogeneity of molecular aberrations in genes involved in epigenetic regulation, RNA-splicing and signal transduction including components of RAS and JAK2 signaling. Functional tests may be important to better estimate the contribution of a particular molecular aberration in the pathogenesis of the malignancy. We have originally demonstrated extensive in vitro formation of myeloid colonies (CFU-GM) without addition of exogenous growth factors in a subset of patients with CMML (Geissler et al, Leuk Res 1988). We reported that this spontaneous CFU-GM colony formation in CMML is a GM-CSF dependent in vitro phenomenon (Geissler et al, J Exp Med 1996) and could also show in a small retrospective study that CMML patients with high spontaneous CFU-GM growth ( 〉 100/105 PBMNC) have a worse prognosis compared to patients with low myeloid colony formation (Sagaster et al, Ann Hematol 2004) suggesting a clinical significance of our observation. In juvenile myelomonocytic leukemia, in which molecular aberrations are mainly restricted to the RASopathy genes including NRAS, KRAS, NF1, CBL and PTPN11, spontaneous formation of CFU-GM due to GM-CSF-specific hypersensitivity is a hallmark feature of disease, which has been included in the diagnostic criteria. We therefore speculated that high spontaneous myeloid colony formation in CMML might also reflect hyperactivation of the RAS signaling pathway. Aim: Our aim was to study the correlation between spontaneous myeloid colony formation and the presence of mutations in RASopathy genes in patients with CMML. Moreover the relationship of high autonomous CFU-GM formation with phenotypic features of CMML and its clinical outcome was investigated. Patients and Methods: In this study we included 137 CMML patients of the "Austrian Biodatabase for CMML (ABCMML)" in whom CFU-GM data and/or molecular data were available. CFU-GM growth in the absence of exogenous cytokines was assessed in a central laboratory using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Molecular characterization was also performed in a central laboratory using NGS with amplicon-based target enrichment of 39 CMML associated genes. Assuming that clones that are too small are unlikely to significantly impact hematopoiesis only mutations with an allele burden of ≥20% were considered positive in this analysis. Clinical and hematological data were obtained from patients records. Results:High spontaneous CFU-GM growth (≥100/105 PBMNC) was found in 38/135 (28%) CMML patients, of whom 3 were already transformed into secondary AML at the time of in vitro culture testing. There was a significant correlation between high CFU-GM formation in vitro and the presence of mutations in genes involved in the RAS signaling pathway. The incidence of RAS pathway mutations was 72% in CMML patients with high colony growth and 31% in patients with low spontaneous CFU-GM formation (p 〈 0.0001). As shown in Table 1 high spontaneous myeloid colony formation was associated with increased WBC counts, increased blast cells, increased LDH, more pronounced splenomegaly and inferior survival (Fig. 1). There was no significant difference regarding autonomous CFU-GM growth in CMML patients with molecular aberrations in genes of epigenetic regulation and RNA-splicing, respectively. High spontaneous CFU-GM was never observed in CMML patients in whom the JAK2 V617F mutation was the only molecular aberration in signaling pathways (0/8 patients). Furthermore the in vitro conversion from a growth factor dependent to a growth factor independent phenotype by RAS but not by JAK2 could be demonstrated in BaF3 cells (Fig. 2). Conclusion: Our findings indicate that high spontaneous in vitro myeloid colony formation is associated with the presence of RAS pathway mutations, leukocytosis, splenomegaly and reduced survival. These results suggest that CMML with high spontaneous colony growth is a mainly RAS pathway driven malignancy resulting in myeloproliferation and inferior outcome. This may have clinical implications concerning therapeutic strategies aimed at targeting the hyperactive RAS signaling pathway in these patients. Disclosures Geissler: Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celegene: Honoraria, Research Funding.

Abstract: Background: Aggressive hematological malignancies in relapsed/refractory setting bear a dire prognosis with low cure rates and short survival. Matching these patients to therapies is challenged by complexity due to spatial and temporal tumor evolution and incomplete understanding of genotype to phenotype correlations. Direct functional testing could address these impediments. The EXALT trial is an interventional, one-arm study designed to assess the clinical value of next generation functional drug screening (ngFDS). An interim analysis on 17 patients suggested a clinical benefit (Snijder et al., Lancet Hematol. 2017). Methods: We applied image-based ngFDS to quantify differential ex-vivo sensitivity of primary patient tumor cells to respective non-tumor cells towards 136 small molecule drugs, including EMA approved for any indication or experimental. We screened bone marrow, peripheral blood or lymph node material from 143 patients who suffered from late stage aggressive hematological malignancies (acute leukemias, aggressive B- and T-cell lymphomas) , discussed the results in a multidisciplinary tumor board and recommended treatments to physicians (A). The primary endpoint of this study was the percentage of patients reaching a PFS-ratio (PFS(ngFDS treatment)/PFS(previous treatment)) of ≥1.3 with an H0 hypothesis & lt; 15% patients. The secondary endpoint was overall response rate (ORR) defined as proportion of patients reaching complete remission (CR) or partial remission (PR). Additionally, we performed a post hoc analysis to evaluate the matching of ngFDS to drugs used in actual treatment (matching score of received treatment). Results: 56 (39%) patients were evaluable and treated according to ngFDS based recommendations. With 30 of 56 (54%) ngFDS guided patients experiencing a PFS ratio of ≥1.3, the primary study endpoint was reached. 11 patients (37%) had ongoing response at censoring date (B). The median follow-up was 718 days. The median number of days from sampling to treatment was 21 (range 4-77). The ngFDS treatment regimens consisted of a median of 2 drugs (range: 1-6). ORR was 55% for all evaluable ngFDS treated patients, 60% for the lymphoid subgroup and 41% for the myeloid subgroup. Patients on ngFDS guided treatment with performance status ECOG ≤ 1 had a median PFS of 207 days compared to a median PFS of 29 days for patients with higher ECOG (p & lt; 0.001, C). 24 of 39 (62%) patients with ECOG ≤ 1 had a PFS ratio of ≥1.3 (D). In disease specific subgroup analysis median PFS of T-cell lymphoma patients was 235 days versus 60 days for B-cell lymphoma patients (p = 0.018, E). Age (≤60 vs. & gt;60), sex, lineage (myeloid vs. lymphoid), number of previous treatment lines (≤2 vs. & gt;2), and clinical presentation (leukemia vs. lymphoma) did not have an impact on PFS of ngFDS guided treatment. Post hoc analysis including additional 17 non-ngFDS treated patients demonstrated that only patients receiving treatment with a positive ngFDS matching score demonstrated clinical benefit (HR: 0.53, p=0.005; vs. HR: 1.4, p=0.4). ngFDS matched treatments resulted in higher PFS for patients with tumor samples that had a cancer cell fraction of 10-50% in comparison to patients with samples of lower or higher cancer cell percentage (HR:0.35, p=0.01). Conclusion: ngFDS could be integrated in the routine clinical work flow. ngFDS guided treatments led to high rates of PFS prolongation compared to previous treatments of individual patients. ngFDS guided treatment is feasible and effective in patients with late stage aggressive hematological malignancies. These results prompted a prospective randomized trial comparing treatment guidance based on ngFDS or comprehensive genomic profiling or physician's choice (EXALT-2 trial, NCT04470947). Figure Disclosures Vladimer: Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Jaeger:Karyopharm: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; True North: Honoraria, Research Funding; Miltenyi: Consultancy, Honoraria; CDR Life AG: Consultancy, Research Funding; F. Hoffmann-La Roche: Honoraria, Research Funding; Infinity: Honoraria; Takeda: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria. Krall:Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Valent:Allcyte GmbH: Research Funding; Cellgene: Honoraria, Research Funding; Pfizer: Honoraria. Wolf:Celgene: Honoraria, Research Funding. Zielinski:MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Imugene: Consultancy, Honoraria, Speakers Bureau; Ariad: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Merrimack: Consultancy, Honoraria, Speakers Bureau; Merck KGaA: Consultancy, Honoraria, Speakers Bureau; Fibrogen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Tesaro: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Eli Lilly: Consultancy, Honoraria, Speakers Bureau; Athenex: Consultancy, Honoraria, Speakers Bureau. Superti-Furga:Allcyte GmbH: Current equity holder in private company, Other: Founder. Snijder:Allcyte GmbH: Current equity holder in private company, Other: Founder. Staber:Roche: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria; msd: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Abstract: Central nervous system (CNS) relapse in chronic myeloid leukemia (CML) is rare and if recorded is usually found to occur in patients with lymphoblastic transformation or in those with a generalized myeloid relapse. The BCR/ABL tyrosine kinase (TK) inhibitor imatinib is highly effective in patients with CML, but hardly crosses the blood-brain-barrier. We report on two CML patients who developed a myeloid CNS relapse during treatment with imatinib. One patient was in major cytogenetic response at the time of CNS relapse. In both cases, the myeloid origin of neoplastic cells in the cerebrospinal fluid (CSF) was demonstrable by immunophenotyping, and their leukemic origin by detection of the BCR/ABL oncoprotein. No BCR/ABL kinase domain mutations could be detected. Both patients received intrathecal liposomal cytarabine (DepoCyte®) (50 mg each cycle; 6 cycles). In one patient, additional CNS radiation was performed, whereas in the other patient, consecutive treatment with dasatinib (70 mg per os twice daily) was started. In response to therapy, the clinical symptoms resolved and the leukemic cells in the CSF disappeared in both patients. After four months of observation, both patients are in complete cytogenetic and major molecular response, without evidence for a systemic or a CNS relapse. In conclusion, ‘anatomic’ resistance against imatinib in the CNS can lead to an (isolated) myeloid CNS relapse. Liposomal cytarabine with or without radiation is effective as local therapy in these patients. For treatment of patients with a systemic relapse involving the CNS and for prophylaxis, second-generation BCR/ABL TK inhibitors crossing the blood-brain-barrier such as dasatinib should be considered.

Abstract: Abstract 2676 Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) has improved the treatment results in DLBCL substantially. With more patients being cured from the lymphoma long term toxicity becomes an even more important issue. By replacing doxorubicin with non-pegylated liposomal encapsulated doxorubicin in the R-CHOP regimen (R-COMP) we tried to reduce the cardiotoxicity of R-CHOP in the 1st line treatment of DLBCL. We randomized 88 patients with untreated DLBCL to one of two treatment arms. R-CHOP consisted of rituximab 375 mg/sqm, cyclophosphamide 750 mg/sqm, doxorubicin 50 mg/sqm, vincristine 2 mg, each iv. day 1 and prednisolone daily po for 5 consecutive days. Six cycles of chemotherapy and 8 cycles of rituximab were planned. In the R-COMP arm doxorubicin was replaced with non-pegylated liposomal encapsulated doxorubicin 50 mg/sqm iv day 1. Forty and 39 patients were eligible in the R-COMP and R-CHOP arm, respectively. The two arms were well balanced with respect to age, smoking status, heart function, hypertension, and international prognostic index. The primary endpoint of the study was the left ventricular ejection fraction (LVEF) measured by the Simpson method at randomization, after each cycle and 8 weeks after the end of treatment. Mean and standard error were compared by the two-sample t test. Mean LVEF was significantly lower in the R-CHOP arm (62.29%) than in the R-COMP arm (63.56%) (P=.0333). Out of all LVEF measurements 10 (4.6%) vs. 31 (15.8%) were 〈 55% in the R-COMP arm and R-CHOP arm, respectively (P 〈 .001). The N-terminal proB-type of the natriuretic peptide (NT-proBNP) is a strong marker for heart failure. Levels of NT-proBNP began to rise in the R-CHOP arm after the 5th cycle and were significantly different after the end of treatment (median 73 pg/ml vs. 188.2 pg/ml) in the R-COMP arm and R-CHOP arm, respectively. Three (7.5%) and 12 (33.3%) patients had a NT-proBNP 〉 450 pg/ml in the R-COMP arm and R-CHOP arm, respectively (P=.005). Side effects were lower in the R-COMP arm. We observed 26 and 40 severe adverse events in the R-COMP and R-CHOP arm, respectively (P=.029). Most of those were due to infection. The rate of grade 3 and 4 neutropenias was comparable in both arms giving evidence, that non-pegylated liposomal encapsulated doxorubicin was not under-dosed. Although the study was not powered to show differences in efficacy, we had no signal of lower efficacy. The remission rate was 97.5% (CR+CRu 75.0%) and 82.0% (CR+CRu 69.2%) with R-COMP and R-CHOP, respectively. The 3 cases progressing during treatment were in the R-CHOP arm. Clinical heart failure usually appears after several years of treatment. The difference in LVEF may translate in a lower rate of clinical heart failure with longer follow-up. Disclosures: Fridrik: Cephalon: Research Funding. Off Label Use: Non-pegylated liposomal encapsulated doxorubicin in the treatment of lymphoma. Willenbacher:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sandoz: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AESCA: Honoraria, Research Funding. Jaeger:Cephalon: Membership on an entity's Board of Directors or advisory committees. Greil:Cephalon: Research Funding.

Abstract: Acute lymphoblastic leukemia (ALL) is a life-threatening hematopoietic neoplasm characterized by abnormal growth and accumulation of lymphatic blast cells in various hematopoietic tissues. In a substantial number of patients, the Philadelphia (Ph) chromosome and the related oncoprotein BCR/ABL, are detectable. Despite recent advances in the management and therapy of patients with ALL, including the use of BCR/ABL1 tyrosine kinase inhibitors (TKI), the prognosis is still poor. Therefore, several attempts have been made to improve targeted treatment approaches in ALL. One strategy is to identify markers and targets expressed on leukemic stem cells (LSC) in these patients and to apply targeted drugs in order to eliminate LSC. In patients with Ph+ ALL, the leukemia-initiating cell-population is considered to reside within a CD34+/CD38- fraction of the clone. In the present study, we examined the expression of various stem cell markers and target antigens in CD34+/CD38- stem cells and in more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL (n=12), Ph- ALL (n=13), Ph+ CML (n=20), and in control bone marrow (BM) samples (unexplained cytopenia, n=10). Surface expression of target antigens was analyzed by multicolor flow cytometry, and mRNA expression levels by qPCR. As assessed by flow cytometry, CD34+/CD38- cells were found to co-express CD19, the stem cell-homing receptor CD44, the Campath-1 antigen (CD52), AC133 (CD133), FLT3 (CD135), and CXCR4 (CD184) in all ALL patients examined. In a majority of the ALL patients tested (14/25), LSC also expressed Siglec-3 (CD33). In CML, LSC were found to express a similar profile of antigens, including CD33, CD44, CD52, CD133, CD135, and CXCR4, but these cells did not express CD19. In control BM samples, CD34+/CD38- cells expressed a similar phenotype, but the levels of CD33 and CD52 were lower compared to LSC in ALL and CML. The IL-1RAP was found to be expressed on LSC in patients with Ph+ CML and Ph+ ALL, but not on LSC in Ph- ALL or in normal BM stem cells. By contrast, the SCF receptor KIT (CD117) was found to be expressed on LSC in Ph+ CML but was hardly detectable on LSC in patients with Ph+ ALL or Ph- ALL. The IL-2RA (CD25) and the SDF-1-degrading surface enzyme dipeptidyl-peptidase IV (DPPIV=CD26) were expressed on LSC in patients with CML and in all patients with Ph+ ALL exhibiting BCR/ABL-p210, whereas in Ph+ ALL with BCR/ABL-p190, LSC variably expressed CD25, and did not express CD26. In patients with Ph- ALL and in the normal BM, CD34+/CD38- cells did not express CD25 or CD26. The target receptor CD20 was detectable on ALL LSC in 7/18 patients examined. All target receptors tested were also detectable on more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL and Ph- ALL. In consecutive studies, expression of target antigens was confirmed at the mRNA level by qPCR analyses of highly enriched ALL LSC. Finally, we were able to show that the CD52-targeting drug alemtuzumab induces rapid lysis of CD34+/CD38- ALL LSC in all patients examined (Figure). In summary, our data show that LSC in Ph+ ALL and Ph- ALL express a unique phenotype, including clinically relevant cytokine receptors and cell surface target antigens, including the Campath-1 antigen, CD52. In Ph+ ALL with BCR/ABL-p210, the phenotype of ALL LSC largely resembles the phenotype of LSC in Ph+ CML, confirming the close relationship and similar pathogenesis of these two types of leukemias. Ficoll-isolated MNC of 4 patients with Ph+ ALL were incubated in control medium (Co) or in various concentrations of alemtuzumab (10-300 µg/ml) in RPMI 1640 medium in the presence of 30% human serum at 37°C for 1 hour. After washing, cells were stained with fluorochrome-conjugated mAb against CD34, CD38 and CD45 for 15 minutes. DAPI-staining was used to evaluate the percentage of viable cells. Cells were analysed using a FACSCanto II and FlowJo software. Results show the numbers of viable CD34+/CD38- cells and are expressed as percent of control (Co). Values represent the mean±S.D. of four independent experiments. Asterisk (*): p 〈 0.05 compared to control. Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding.

Abstract: There is growing evidence that the anti-apoptotic PI3-K/Akt pathway is involved in pathogenesis and progression of different types of cancer. We have evidence that PI3-K inhibitors such as LY294002 and wortmannin selectively induce apoptosis in CLL cells (Shehata et al Ab. Blood 2006). Recently, a new orally available PI3-K inhibitor, NVP-BEZ235 has been developed. This competitive ATP binding imidazo-quinoline derivative is already in phase I trials against solid tumors. Here we show, for the first time, the effects of NVP-BEZ235 on the viability of CLL cells in vitro. Primary CLL cells from 37 patients were investigated. Sixteen patients were in Binet stage C, 14 in B and 7 in stage A. Seventeen patients had mutated IgVH genes, 15 had unmutated IgVH and mutation status from 5 patients was not available. Fluorescence in situ hybridization (FISH) analysis showed that 23 patients had del(13q), 9 had del(17p), 8 had del(11q) and 4 patients had trisomy 12. Nineteen patients were untreated and 18 patients were previously treated. To overcome the experimental artifact due to the spontaneous apoptosis of CLL cells in vitro, which may mask the actual effect of the tested drugs, we applied a co-culture model using human bone marrow stromal fibroblasts which supports survival of CLL cells ex vivo. CLL cells were exposed to NVP-BEZ235 at different concentrations (1 nM-10 μM) and incubation times (1, 3, 7 days). Cell viability was assessed by annexin-V/propidium iodide staining, flow cytometry and MTT assays. The results showed that cell viability was significantly higher in co-cultures compared to suspension cultures (the percentage of apoptotic cells after 3 days in co-culture was 5±4 compared to 23±12 in suspension cultures, p & lt; 0,01). NVP-BEZ235 induced apoptosis in the majority of CLL samples under both experimental conditions. However, this effect tends to be more remarkable in co-culture than in suspension: 4-10 fold versus 3-fold increase in apoptosis rate respectively. The pro-apoptotic effect was dose and time dependent and could be observed within 16 hours after incubation at 10 nM. A maximum effect was obtained at a concentration of 5–10 μM. The IC50 values varied between patients and were in a range of 250–750 nM. At these concentrations, NVP-BEZ235 was significantly more effective in induction of apoptosis than LY294002. NVP-BEZ235 inhibited the adhesion of CLL cells to stromal cells suggesting that it may interfere with the survival signal provided by the lymphoid microenvironment in addition to its direct effect on the leukemic cells. FACS analysis demonstrated that NVB-BEZ235 specifically targets the leukemic CD19+ cells while a minimal effect on the viability of T cells and monocytes could be observed. The pro-apoptotic effect of NVP-BEZ235 was independent from the mutational status and cytogenetics. In addition, it induced apoptosis in vitro in CLL cells from patients resistant to fludarabine treatment. In parallel to induction of apoptosis in CLL cells, western blotting demonstrated that NVP-BEZ235 significantly inhibited Akt phosphorylation at Ser-473. This effect was also associated with dephosphorylation (activation) of the tumor suppressor PTEN at Ser-380. DNA microarray analysis using Affymetrix U133A Plus 2.0 GeneChips revealed more than 200 genes which were at least 2 fold up- or down-modulated by NVP-BEZ235 in vitro. These genes include LY9, DUSP10, CCR6, RGS2, IRS2, PI4K2A, ISG20, TFRC, EGR1, HSP90, LCK, TNFRSF17, LYZ, TGFBI and TLR10. In conclusion, the results demonstrate a significant and selective pro-apoptotic effect of NVP-BEZ235 in CLL cells. The data point also to the validity of PI3K-pathway inhibition as a novel therapeutic concept for CLL which should be evaluated in clinical trials.

Abstract: Background. Patients with aggressive hematologic malignancies failing at least two lines of therapy are without further standard treatment options and have a poor prognosis. Identifying effective therapies with genomic-based precision medicine is hampered by intratumor heterogeneity and incomplete understanding of the contribution of various mutations within specific cancer phenotypes. Next-generation functional drug screening (ngFDS) in patient samples promises to overcome these challenges, however, proof of its clinical utility is limited. Methods. We investigated the feasibility and clinical impact of ngFDS measured at single-cell resolution using high-throughput automated microscopy in blood, bone marrow, pleural effusions, or excised lymph node biopsies (Figure 1A and Valdimer G et al. Nat Chem Biol. 2017). First, the accuracy of ngFDS to predict clinical outcome was evaluated in a retrospective cohort of 20 previously untreated patients with acute myeloid leukemia (AML). Then, 48 patients with aggressive hematologic malignancies failing at least two lines of treatment were prospectively evaluated for ngFDS guided therapy, of which 17 could receive ngFDS-guided treatment. Individual ngFDS-guided treatment regimens were selected by a committee (EXALT-board) of hemato-oncologists, pathologist, and pharmacists based on top-candidate treatments identified by ngFDS, considering drug availability as well as safety profiles of single agents and previously reported combinations (Figure 1B). The majority of these patients (12/17) presented with aggressive lymphoma and had seen in median three (2-7) prior treatment lines (Figure 2A). Overall response rate (ORR) and progression-free survival (PFS) of ngFDS-guided treatment were compared with ORR and PFS for the most recent regimen (MRR) on which patients had previously progressed. Results. ngFDS accurately predicted individual clinical response of AML patients to initial therapy. From prospectively analyzed patients receiving ngFDS guided treatment the ORR-ngFDS was 88% (15/17) compared to ORR-MRR of 24% (4/17; P & lt;0.0004). Twelve (70%) of 17 patients had a PFS ratio of ≥1.3 and the mean PFS increased 3.9-fold, from 5.7 weeks to 22.6 weeks (P & lt;0.007) (Figure 2B). Furthermore, analysis of an independent cohort revealed that ngFDS could positively and negatively predict patients' outcome to drug treatment. Conclusions. Automatedmicroscopy-based ngFDS is feasible and accurately predicts clinical response. It can successfully guide personalized treatment of aggressive refractory hematological malignancies. Figure 1 Figure 1. Disclosures Staber: Takeda: Honoraria; Abbie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria; Amgen: Honoraria; Gilad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees. Snijder: Allcyte: Equity Ownership, Other: founder and shareholder of Allcyte GmbH that holds a worldwide exclusive license for and commercializes the Pharmacoscopy high content imaging technology.. Vladimer: Allcyte Gmbh: Equity Ownership. Krall: Allcyte Gmbh: Equity Ownership. Hoermann: Ariad: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Research Funding. Sperr: Teva: Honoraria; Meda: Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Phadia: Research Funding; Novartis: Other: Register. Gisslinger: Janssen Cilag: Honoraria; Takeda: Honoraria; Shire: Honoraria; PharmaEssentia: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan Pharmaceuticals AG: Consultancy, Honoraria. Valent: Incyte: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Deciphera: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Teva: Honoraria; Celgene: Honoraria, Research Funding; Blueprint: Research Funding. Jaeger: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses. Superti-Furga: Allcyte GmbH: Equity Ownership.