ALBERT

Description: Abstract 773 Peripheral T-cell lymphomas (PTCLs) are rare and heterogeneous tumors whose biology is largely unknown. Interestingly, the commonest subtypes (i.e. PTCL not otherwise specified, NOS; angioimmunoblastic T-cell lymphoma, AITL; and anaplastic large cell lymphoma, ALCL) present on one hand few disease-specific molecular features and, on the other hand, several apparently common abnormalities. So far, no data are available regarding miRNA expression in these tumors. In order to identify miRNA deregulated in PTCLs, we performed an extensive miRNA profiling (by studying 379 targets on the TaqMan Array MicroRNA Cards) of 44 PTCLs (including 23 PTCLs/NOS, 12 ALCLs, and 9 AITLs) and 13 sample representative of normal T-cell sub-populations (CD4+ and CD8+, both resting and activated). In addition, for all these cases, gene expression profiles (GEPs) were generated by the Ilumina whole genome DASL-assay. TaqMan Quantitative-PCR (qPCR) was then used for validation. First, we found that PTCLs and normal T-cells could be easily distinguished based on their miRNA profile, by both unsupervised and supervised analysis. Specifically, the latter identified 91 miRNA differentially expressed in PTCLs vs. T-cells with a fold change ≥2 and a pvalue

Description: Abstract 2727 CHOP and CHOP-like chemotherapy remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL) and peripheral T-cell lymphomas (PTCL) given their marked single agent activity in these diseases. The interaction between the HDACIs (depsipeptide (R) and belinostat (B)) and a DNMT inhibitor (decitabine (D)) was investigated in vitro, in vivo and at the molecular level in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (P12, PF-382). For all cytotoxicity assays, a luminescence based cell viability assay was used (CellTiter-Glo™) followed by acquisition on a Biotek Synergy HT. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR

Description: Abstract 4631 Background Peripheral T cell lymphomas (PTCLs) are a heterogeneous group of tumors representing approximately 12% of lymphoid neoplasms, basically subdivided into specified and not specified (NOS) forms. PTCL/NOS, corresponding to about 60%–70% of PTCLs, cannot be further classified on the basis of morphology, phenotype, or conventional molecular studies. Clinically, PTCLs/NOS are highly aggressive lymphomas, with a poor response to therapy, and dismal overall survival (20-30%). Their pathobiology is poorly known, though recent gene expression profiling (GEP) studies have provided some hints for better understanding their pathogenesis. In particular, GEP and immunohistochemical studies on tissue-microarrays (TMAs) demonstrated PDGFRA to be systematically activated in almost all PTCLs/NOS, by nominating it as potential therapeutic target. Aims In this study, we aimed to identify the determinants of PDGFRA activation in PTCL/NOS. Specifically, we studied PDGFRA locus in order to identify possible mutations, translocations, or copy number variations and we explored the possible existence of an autocrine/paracrine loop sustaining an otherwise integer kinase. Methods The PDGFRA locus (4q1.1-4q1.3) was studied by FISH and wide-genome SNPs analysis (Affymetrix 500K Array). Direct sequencing of all PDGFRA exons and introns as well as of the promoter region was also performed in 90 cases. IHC and ELISA were adopted in order to study the expression of PDGF-A, PDGF-B and PDGF-C on tissue sections and in supernatants from PTCL/NOS cell cultures, respectively. Finally, the expression of PDGFRA and its activated (phosphorilated) form, p-PDGFRA, was assessed by IHC on TMAs, and by flow-citometry in PTCL/NOS cultured cells as well as in a FIP1L1-PDGFRApos chronic eosinophilic leukemia cell line (EOL-1) before and after the exposure to an anti-PDGF ligand neutralizing antibody (R&D System), given at various concentrations (20-40-60-80 ug/mL). Vitality assessments, proliferation/cell cycle assay (by In Situ Cell Proliferation kit, FLUOS – Roche) and evaluation of PDGFRA and p-PDGFRA were performed at 24, 48, 96 hours. A human PDGF peptide (R&D Sytems) was added to cultured cells for 6 hours to evaluate whether PDGFRA de-phosphorilation was really due to PDGF ligand remotion. Results First, FISH, SNPs analysis and direct sequencing showed preserved integrity of PDGRA locus. Thus we tested the hypothesis of an autocrine/paracrine stimulation. PDGF-A, PDGF-B and PDGF-C were found to be expressed by neoplastic cells at IHC in 93-95% of cases. In addition, PDGF-AA was found to be secreted by cultured neoplastic cells by ELISA. Notheworthy, PTCL cells secreted much more ligand than any other cell taken as control. We then tested whether PDGFRA phosphorylation was actually due to the presence of a PDGF ligand. Indeed, PTCL cells treated with anti-PDGF ligand neutralizing antibody at various concentrations showed PDGFRA dephosphorilation ranging from 30% up to 90% in a time dependent manner. Notably, the effect was specific as in EOL-1 PDGFRA phosphorylation was not modified at all. In addition, PTCL cells treated with a minimum of 20ug/mL of anti-PDGF ligand neutralizing antibody for 48h showed a 70% blockade of proliferation in comparison to untreated cells (BrdU assay). A further addition of 20 ug/ml of inhibitory antibody at 48 hours, increased the proliferation arrest up to 80% at 96 hours. Finally, the addition of a natural human PDGF peptide to cells previously treated with the anti-PDGF antibody, could restore PDGFRA phosphorylation confirming that PDGFRA de-phosphorilation was due to ligand remotion. Conclusions Taken together, our data demonstrate that PDGFRA activity is sustained by an autocrine loop in PTCL/NOS. In fact, though, in vivo, a possible additive paracrine effects mediated by reactive components cannot be excluded, we provide evidence that the phenomenon is largely due to neoplastic cells. Importantly, as PDGFRA signaling abrogation was associated to proliferation arrest, PDGFRA was confirmed as potential therapeutic target. Acknowledgments: this work was supported by Centro Interdipartimentale per la Ricerca sul Cancro “G. Prodi”, BolognAIL, AIRC, FIRB, RFO, Fondazione Cassa di Risparmio in Bologna, Fondazione della Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006, and Vanini-Cavagnino grant. Disclosures: No relevant conflicts of interest to declare.

Description: Diffuse large B-cell lymphoma (DLBCL) overexpressing MYC and BCL-2 have a poor outcome with standard therapies irrespective of the cell of origin (COO), representing an unmet medical need. Furthermore, precision therapy approaches specifically designed for MYC/BCL-2 positive DLBCL are lacking. Recently we reported that MYC positive DLBCL harbor high levels of inherent DNA damage and display constitutive activation of the DNA damage response (DDR) pathways, these alterations being correlated with poor outcome (Derenzini et al. Oncotarget 2015). In an effort to design specific therapies for MYC/BCL-2 positive DLBCL, we integrated nanostring-based gene expression profiling and functional characterization of replicative and oxidative stress biomarkers in 2 independent DLBCL cohorts treated with chemoimmunotherapy: 69 patients from the DLC04 study (Chiappella et al. Lancet Oncol 2017), and 66 patients treated in real life clinical practice (total 135 patients). We used a digital multiplex gene expression profiling (Nanostring technology) panel containing 28 genes: 20 genes used to assign COO subtype according to LST algorithm (Scott et al. Blood 2014), and 8 additional genes employed to assess the MYC and BCL-2 status and to detect potential therapeutic targets (STAT3, TP53, NFKBIA, PTEN, PIK3CA, CD68). Immunohistochemistry (IHC) profiling for MYC, BCL-2, the phosphorylated form of H2AX at S139 (γH2AX: a biomarker of DNA damage and DDR activation) and for 8-hydroxy-2'-deoxyguanosine (8-OHdG) (an oxidative DNA damage marker) was available in all patients. MYC/BCL-2 status evaluated by nanostring outperformed IHC for prognostic stratification in both cohorts (patients were stratified in high and low MYC or BCL-2 expressors based on the median normalized MYC and BCL-2 mRNA levels in the respective cohorts). The 2 cohorts displayed similar patterns of γH2AX and 8-OHdG expression (cut off value 50% of positive cells). 8-OHdG negative cases showed very low levels of γH2AX whereas high 8-OHDG expressors displayed significantly higher levels of γH2AX phosphorylation. In line with this, γH2AX positivity was significantly associated with increased 8-OHdG levels in both cohorts (Figure 1A). These data suggest that oxidative stress could be a major source of inherent DNA damage contributing to constitutive DDR activation in DLBCL. Dual positivity for γH2AX and 8-OhDG was significantly associated with adverse outcome in both cohorts. However, this difference was even more pronounced when focusing the analysis on MYC/BCL-2 positive DLBCL (evaluated by nanostring): in fact dual positivity for H2AX and 8-OHdG identified a subgroup of MYC/BCL-2 positive cases (MYC/BCL-2ox) characterized by a dismal outcome. Interestingly MYC/BCL-2 positive cases without oxidative DNA damage had a very favorable outcome, with similar overall survival (OS) compared to MYC/BCL-2 negative cases. Similar trends were observed in both cohorts and a cumulative analysis confirmed a significantly worse OS for the MYC/BCL-2ox subgroup, compared to all other subgroups (Figure 1B). We found an increased proportion of low NFKBIA expressors in the MYC/BCL-2ox subgroup (with no differences in the COO subtyping and in the fraction of double hit cases). Since NFKBIA is an endogenous inhibitor of NF-kB signaling, these data could indicate a compensatory NF-kB activation in MYC/BCL-2ox patients, in line with the role of NF-kB signaling in driving antioxidant responses. These findings suggest a model of oncogenic cooperation where constitutive NF-kB activation and BCL-2 overexpression could counteract the deleterious effects of inherent oxidative DNA damage driven by MYC. To test this hypothesis and to determine the therapeutic implications of these findings we run a combinatorial high-throughput drug screening (HTS) investigating combinations based on 2 different inhibitors of the DDR (the CHK1/2 inhibitors AZD7762 and MK8776) in a large panel of DLBCL cell lines of ABC and GCB origin (n=10) (Figure 1C). Both CHK1/2 inhibitors showed synergistic interactions with BCL-2 inhibitors (venetoclax) and with drugs inhibiting NF-KB pathway such as PI3K, HDAC, and BET inhibitors. Taken together these data suggest synthetic lethal interactions between MYC, BCL-2, NF-kB and the DDR in DLBCL, providing the rationale for targeted therapeutic interventions in poor prognosis MYC/BCL-2 positive subgroups. Disclosures Chiappella: Nanostring: Other: lecture fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Roche: Other: lecture fees; Teva: Other: lecture fees; Amgen: Other: lecture fees. Zinzani:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Background. Recently, gene expression profiling (GEP) indicated histone-deacetylases (HDAC) as potential therapeutic targets in peripheral T-cell lymphomas (PTCL) not otherwise specified (NOS), the commonest PTCL type. Consistently, phase II trials demonstrated the efficacy of some HDAC inhibitors (HDACi), including SAHA, which was approved for cutaneous T-cell lymphomas (CTCL) treatment. Aims and methods. We investigated the anti-tumour effects of ITF2357 (Italfarmaco, Italy), a novel hydroxamic acid HDACi, on PTCL primarily-cultured cells and cell lines (HH and FEDP), and in a xenografted mouse-model of CTCL. Cultured cells were incubated with different dosages of ITF2357 and SAHA (ranging from 0.5 to 2.5 mM). Cell viability, assessed by trypan-blue exclusion assay, cell-cycle progression, assessed by bromodeossiuridine assay, and apoptotic rate, determined by flow-cytometry analysis of annexin-V binding populations were determined at 48, 72 and 120 hours. Nude mice, injected with HH cells, received ITF2357 (10–20mg/Kg, per os) for 14 days. Micro-PET scan was adopted for disease measurement and treatment response evaluation. Finally, GEP of cell lines exposed to ITF2357 and SAHA were generated to elucidate their mechanisms of action. Results. Cell viability of HH cells treated with ITF2357 ranged from 50% (0.5 mM, at 48 h), to

Description: Outcome for older patients with acute myeloid leukemia (AML) is extremely poor. Intensive induction chemotherapy is often unsuitable. In this phase II study we tested, for the first time, the efficacy of a novel combination therapy with low-dose lenalidomide plus low-dose cytarabine. Further, based on the hypothesis that genetic features might influence treatment response, we aimed at identifying a possible biomarker by studying the global gene expression profiles (GEP). We designed a prospective phase II study to assess the efficacy of the concomitant administration of low-dose lenalidomide and low-dose cytarabine in patients with acute myeloid leukemia (AML) aged more than 70 years. Forty-five patients (median age 76 years, range: 70-85) ineligible for standard therapy, were consecutively treated with low-dose lenalidomide (10 mg/day orally, days 1-21) plus low-dose cytarabine (10mg/m2 twice daily, subcutaneously, days 1-15) every six weeks, up to 6 cycles. Median white blood cell count at diagnosis was 3.2x109/l (range: 0,4-46,8x109/l), whereas median hemoglobin was 8,9 g/dl and median platelet count was 31x109/l. Twenty-three out of 45 patients had an intermediate karyotype (18/23 normal), 18/45 an unfavorable karyotype and 4/45 were not evaluable. Nineteen patients had a de novo AML, whereas 26 patients had a secondary AML (18 after MDS, 3 after a CMPD, 2 after myelofibrosis, 3 after chemo-radiotherapy for a breast cancer). To identify possible biomarkers associated to sensitivity/resistance, global gene and miRNA expression profiling (Affymetrix Transciptome 2.0) was performed on purified AML cells. Induction-period mortality was 17%, with 8 deaths occurring during cycle 1. Thirty-seven patients completed at least one cycle of therapy and are evaluable for response. Overall CR rate was 43% among evaluable patients. Nine out of 16 responding patients are still in CR after a median follow-up of 12 months (range: 2-39). Statistical analysis showed that responding patients had a longer median overall survival than non-responders (428 vs. 74 days, P = .000). Conversely, by studying the global miRNA and gene expression profile we identified a molecular signature, including 114 genes and 18 miRNA associated with the clinical response (CR vs. no CR). Of note, the involved genes belonged to relevant functional categories such as angiogenesis, cell cycle regulation and immune response. Of note, based on the expression of 5 genes, we developed an algorithm to predict treatment response that was successfully validated by showing an 87% overall accuracy. In conclusion, low-dose lenalidomide plus low-dose cytarabine has high clinical activity, predictable by GEP, in elderly AML patients with poor prognosis. The study was registered at EMA (EUDRA-CT 2008-006790-33). Acknowledgments Celgene is acknowledged for providing Lenalidomide for the patients. The study was supported in part by AIL Pesaro Onlus. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1890 Poster Board I-913 Background. Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterised by a proliferation of predominantly megakaryocytes and granulocytes in bone marrow that in fully developed disease is replaced by fibrous tissue. At molecular level, no specific defect has been identified yet. Cytogenetic abnormalities occur in up to 30% of patients, the commonest including del(13)(q12-22), der(6)t(1;6)(q21-23;p21.3), del (20q), and partial trisomy 1q. In addition, approximately 50% of patients with PMF exhibit a single, recurrent, somatic mutation in the gene encoding the cytoplasmic tyrosine kinase Janus kinase 2 (JAK2). However, such mutation is not specific, also occurring in other MPN. Recently a couple of reports dealt with single-nucleotide polymorphism (SNP) array karyotyping of MPD, including some PMF. Importantly, such studies could identify previously uncovered genetic lesions, highlighting the importance of novel high resolution technologies for the detection of formerly unknown, cryptic aberrations. In this study we performed high resolution karyotyping by SNP oligonucleotide microarray by using the most updated Affymetrix array (Genome-Wide Human SNP Array 6.0) in 20 cases of myelofibrosis (MF) in order to identify novel cryptic genomic aberrations. Methods. DNA (500 ng) was extracted from peripheral blood cells (PBMNC) of 14 primary and 6 secondary MF patients. PBMNC were depleted from lymphocytes by magnetic beads. Briefly, CD3+ cells were labeled with anti-CD3 MoAb directly coupled to magnetic microbeads (Miltenyi Biotech), washed and subsequently purified using Mini-MACS technology. After selection, cell present in the positive (CD3) and negative (PBMNC) fractions were counted and submitted to flow cytometry analysis. DNA was processed and hybridized to the Affymetrix SNP arrays 6.0 as for manufacturer instruction. A whole-genome copy number variation (CNV), genotyping, loss of heterozygosity (LOH) and uniparental disomy (UPD) analyses were performed using the Partek Suite 6.0. Ten lab-specific as well as 90 HapMap samples relative to Caucasian healthy donor were used as control reference. Genomic abnormalities were defined as recurrent when occurring in at least 25% of cases. JAK2 mutational status was assessed as reported, by alle-specific PCR. Clinical information and complete follow up were retrieved for all cases. Direct sequencing, FISH, qPCR and immunohistochemistry (IHC) has been chosen for validation. Results. In all patients we could detect several CNV. The median number of CNV was 60 (range, 34-72), including 46 amplifications (A) and 14 deletions (D). All commonest previously described abnormalities were detected. In addition, several formerly uncovered recurrent lesions were identified, mainly involving 1p, 1q, 2p, 4p, 4q, 5q, 6p, 6q, 7q, 8p, 9q 10q, 11p 11q, 12p, 14q, 15q, 16p, 16q, 17q, 18q, 19q, 20p, 22q. The median size of such CNV was 424,582 Kbp (1,379 Kbp-71,277 Mbp). We then compared JAK2+ vs. JAK2− cases. Of note, we found numerous definite aberrations (A or D) distinguishing the two groups and specifically affecting 16q23.1, 1p36.13, 3q26, 14q13.2, 5q33.2, 6q14.1, 7q33, 8p23.1, and 9p11.2. Grippingly, several genes of potential interest for PMF pathogenesis were identified within the involved loci, including RET, SCAPER, WWOX and SIRPB1. Among others, the product of such genes has been selected for validation by IHC. Similarly, many miRNA were recognized, which may deserve further investigation. Conclusions. By using a newly developed highly sensitive array we identified novel cryptic lesions in patients affected by MF. Future studies on larger series, as well as functional analyses will definitely assess their role in the pathogenesis of the disease. Of note, consistent differences were recorded in JAK2+ vs. JAK2−, supporting the hypothesis of different genetic mechanisms occurring in the two sub-groups. Acknowledgments: this work was supported by AIL Pesaro Onlus, Centro Interdipartimentale per la Ricerca sul Cancro “G. Prodi”, BolognAIL, AIRC, FIRB, RFO, Fondazione Cassa di Risparmio in Bologna, Fondazione della Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006.*GV and MRS equally contributed to this work. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3662 The differential diagnosis among the commonest peripheral T-cell lymphomas (PTCLs) (i.e. PTCL not otherwise specified, NOS; angioimmunoblastic T-cell lymphoma, AITL; and anaplastic large cell lymphoma, ALCL) is difficult, the morphologic and phenotypic features being largely overlapping. Noteworthy, recent international studies indicated significant differences in their clinical behavior as well as concerning the presence of potential therapeutic targets. We performed whole genome gene expression profiling (GEP) of PTCLs aiming to identify molecular signatures able to improve their diagnosis. We studied 95 PTCLs, including 73 PTCLs/NOS, 12 ALCLs (6 ALK+ and 6 ALK-), and 10 AITLs. All tissue samples were formalin-fixed and paraffin embedded (FFPE). GEP was performed by Illumina Whole Genome DASL Assay. First, we documented the efficiency of GEP from FFPE tissues by comparing the mRNA levels and the presence of the corresponding protein, including expressed (i.e. CD3) and not expressed (i.e. BCL10) molecules. Secondly, we tried to discriminate different PTCLs basing on their GEPs. By dividing a training (N=47) and a test set (N=48), we found 2 signatures able to differentiate PTCL/NOS vs. AITL and PTCL/NOS vs. ALCL ALK-. Specifically, in the test set the sensitivity (ST) and specificity (SP) of the assays were 100% – 80% (PTCL/NOS vs. AITL) and 100% – 100% (PTCL/NOS vs. ALK- ALCL) (Table 1). Accordingly, the positive (PPV) and negative (NPV) predicting values for the identification of PTCL/NOS were 0.92 and 1 (vs. AITL) and 1 and 1 (vs. ALK- ALCL) (Table 1).Table 1.Accuracy of GEP based signature in differentiating PTCL subtypesSTSPPPVNPVTraining setPTCL/NOS vs. AITL100%80%0.921PTCL/NOS vs. ALK-ALCL100%100%11Test setPTCL/NOS vs. AITL92.50%100%10.77PTCL/NOS vs. ALK-ALCL92.50%100%10.67Validation setPTCL/NOS vs. AITL85%86%0.920.76PTCL/NOS vs. ALK-ALCL96%73%0.960.73 Interestingly, the identified genes represented relevant functional pathways differentially regulated in the 3 tumour types, including protein kinase cascade, proliferation, and cell cycle. When applied to the test set of cases, the assay correctly classified 37/40 PTCLs/NOS (92.5%), 5/5 AITLs, and 3/3 ALK- ALCLs. Finally, we tested our signatures on 133 independent PTCL cases (including 78 PTCL/NOS, 43 AITL, and 12 ALK- ALCL) for which GEP data were available on the GEO database and were originally obtained from fresh/frozen tissues. Interestingly, we could efficiently recognize PTCL/NOS cases vs. AITLs (ST, 85%; SP 86%; PPV 0.92; NPV 0.76) and vs. ALK- ALCLs (ST 96%; SP 73%; PPV 0.96; NPV 0.73). In conclusion, we successfully generated for the first time GEP from routinary FFPE PTCL samples, identifying molecular signatures potentially useful for the clinical practice and, specifically, for the differential diagnosis of PTCL types. Disclosures: No relevant conflicts of interest to declare.