ALBERT

Description: Background: Breast cancer is the second leading cancer worldwide. In Tanzania, though it ranks as the second leading cancer in women after cervical cancer, hormonal receptor status is not carried out routinely in patients. Adjuvant hormonal therapy is given without prior knowledge of hormonal receptors status and patients can incur unnecessary costs and side effects. This study was performed to investigate the expression of hormonal receptors, epidermal growth factor receptors (HER-2) and proliferation index of the breast cancer by Ki-67 in a few selected patients with breast cancer at referral hospital in North-Western Tanzania. The study classified breast cancer subtypes based on hormonal receptors status and the expression of epidermal growth factor receptors. Results: A total of 52 cases of breast cancer were investigated. Patients' mean age at diagnosis was 49 years. The majority of the tumors was invasive ductal carcinoma 47 (90.4%) and 40 (76.9%) were of histological grade III. Thirty-eight (73.1%) of the patient had lymph node metastasis at the time of diagnosis and 36 (69.2%) were at clinical stage III. Only 3 (5.8%) patients were in clinical stage I. There was a tendency of a low level of expression of the receptors, whereby Estrogen Receptor (ER) positive tumors were 17 (32.7%), progesterone receptor (PR) positive tumors were 22 (42.3%), and HER-2 positive tumors were 12 (23.1%). Triple negative tumors constituted 20 (38.4%) of the patients. Most of the tumors (75%) showed high proliferation by Ki-67. Lymph node metastasis was more common in Triple Negative and HER enriched tumors. Conclusion: This study showed a tendency for a low level of expression of hormonal receptors. There was a significant proportion of Triple Negative breast cancers. Routine testing for hormonal receptors in breast cancer is recommended before the initiation of hormonal therapy.

Description: As apple orchards have transitioned to high-density plantings, proper training systems are required to manage increased leaf area. Leaf area index (LAI) is defined as the ratio between leaf area to ground area (m2/m2) and can infer orchard health, light relationships and productivity. New technologies enable rapid assessments of LAI and light interception (LI) in the orchard. In this study, LAI, LI, and productivity were assessed across two training systems (Spindle and V), two rootstocks (Geneva 41® (G41) and Malling 9—Nic29 (Nic29)) and two pruning techniques (“click” and bending) in 2016 and 2017. The objective of this study was to determine a management strategy for “WA38” to meet optimal levels for LAI (1.2–2.0) and light interception (65–75%). Higher light interception was measured in V compared to Spindle and in G41 compared to Nic29 in both years. Minimal differences in LAI and light interception were detected across pruning techniques. In “WA38” the “click” technique maintained more consistent yields than bending. In both years, the Spindle-Nic29-“click” combination maintained optimal thresholds for LAI (1.93 and 1.48), light interception (66% and 68%) and consistent yields. This sequence helps mitigate “blind wood” and alternate bearing, while optimizing leaf area and light in “WA38”.

Description: Orchard-side optimization of fruit quality is experiencing renewed research focus in the fresh fruit industry as new technologies and quality metrics have emerged to enhance consumer acceptance and satisfaction. Fruit dry matter, one such quality index gaining traction among numerous fresh fruit commodities, was targeted for improvement in d’Anjou pear with the application of seasonal pruning cycles (fall, fall and summer, winter, and winter and summer) across two growing seasons in 2016 and 2017 in a mid-aged, traditionally managed commercial orchard in the Columbia basin, Washington, USA. Dry matter was assessed non-destructively on pears using near-infrared spectroscopy at harvest and fruit categorized in to low (16%) dry matter quality categories, revealing that fall pruning positively impacted average predicted fruit dry matter in comparison to winter pruning (15.1 vs. 14.2% in 2016 and 13.7 vs. 13.1% predicted dry matter in 2017 for winter vs. fall pruning, respectively), as well in the abundance of high dry matter fruits. The addition of summer pruning to either fall or winter pruning increased fruit size by up to 13% of proportion of fruits 80 mm or greater in diameter. Further, a tendency for summer pruning to decrease yield (up to nearly 30 kg/tree lower yields), average fruit dry matter (up to 0.5% lower average predicted dry matter), and abundance of high dry matter fruits (up to 11% fewer high predicted dry matter fruits) was observed. Fruit quality classes assembled on predicted dry matter verified the utility of this emerging parameter as a fruit quality metric for pears as demonstrated by more desirable post-harvest eating characteristics such as higher soluble solids content corresponding to greater at-harvest predicted dry matter categories. Targeted seasonal pruning in association with precise at-harvest dry matter fruit sorting may preserve the profitability of pear cultivation through their impact on fruit quality and associated consumer experiences.

Description: Abstract 1770 Chronic lymphocytic leukemia (CLL) is characterized by the expansion of a monoclonal population of mature CD5+/CD23+ B lymphocytes, with a highly variable clinical course. Gene expression profiling studies identified SLAMF-1 (signaling lymphocytic activation molecule aka CD150) as part of the genetic signature characterizing CLL patients with favorable prognosis. Human SLAMF-1 is the prototype member of a family of receptors that act as co-activators through self-interactions on hematopoietic cell surface. SLAMF-1 activation is essential for full T cell functions, including cytokine secretion and development. The role of SLAMF-1 in antigen presenting cells, including B cells, is less well characterized, though it is known that the molecule initiates a signaling pathway that leads to B cell proliferation or CD95-mediated apoptosis. More recently, SLAMF-1 has been attributed a novel function as a microbial sensor that regulates bacterial phagosome functions by recruiting a supra-molecular complex, part of the ubiquitous cellular autophagic machinery. The analysis on the CD19+ fraction of 292 clinically and molecularly characterized CLL patients revealed highly variable levels of SLAMF-1 expression (1–95%). Statistical analyses of the data indicated that patients characterized by a good prognosis (in terms of disease stage at diagnosis or treatment requirements) express higher levels of SLAMF-1 compared to the other subgroups. Moreover, patients with 〉 6% SLAMF-1+/CD19+ CLL cells had a significantly longer treatment free survival (median 6.4 in SLAMF-1+vs 1.2 years in SLAMF-1− patients, P=.002). Consistently, SLAMF-1 expression inversely correlates with CD38 and CD49d, two molecular markers of unfavorable prognosis, and positively associates with the presence of somatic mutations in the IgHV genes. Functional experiments showed that the engagement of SLAMF-1 by an agonistic mAb started a well-characterized signaling pathway. Co-immunoprecipitation experiments demonstrated a direct interaction between the receptor and the adaptor molecule EAT-2, with its consequent phosphorylation and the following downstream activation of Vav-1, p38 and JNK. Moreover, co-crosslinking of SLAMF-1 and sIgM prolonged the phosphorylation of p38 and JNK and resulted in an increased percentage of CLL cells undergoing apoptosis, as compared to either signals alone or the basal condition. Furthermore, the engagement of SLAMF-1 for a period of 6 hours led to an increased appearance of autophagic vesicles, as confirmed by confocal and transmission electron microscopy. The modulation of apoptosis and autophagy was mediated by the sequential phosphorylation of JNK and Bcl-2: the final result is the activation of Bcl-2 (phospho-Ser70) and the release of Beclin-1, an essential member of the autophagic complex. In conclusion, i) SLAMF-1 is expressed at higher levels by patients with a good prognosis, ii) and performs as an immunoreceptor on CLL cells, starting a signaling pathway that involves the adaptor molecule EAT-2, Vav-1 and MAP kinases, like p38 and JNK. iii) SLAMF-1+ CLL patients respond to receptor engagement modulating an autophagic pathway mediated by the phosphorylation of JNK and Bcl-2. Taken together, these results suggest that SLAMF-1 could represent a novel marker for the subset of CLL patients characterized by an indolent clinical course and highlight a hypothetical link between the activation of the autophagic process and a better clinical outcome in CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points CLL lymphocytes show high intracellular and extracellular NAMPT levels, further increased upon activation. eNAMPT prompts differentiation of CLL monocytes into M2 macrophages that sustain CLL survival and reduce T-cell proliferation.

Description: Abstract 4627 Nicotinamide (Nam), is the main precursor of nicotinamide adenine dinucleotide (NAD+). It regulates intracellular levels of NAD+ and consequently activities of four classes of NAD+-consuming enzymes, including NADases, mono-ADP-ribosyl transferases (ARTs), poly-ADP-ribose polymerases (PARPs) and sirtuins. Pharmacological doses of Nam inhibit the physiological activation and proliferation of mouse B lymphocytes, suggesting that this agent might affect also human B cell homeostasis. We approaches this issue by comparing the effects of Nam on normal vs. leukemic B lymphocytes. Chronic lymphocytic leukemia (CLL) was selected as disease model, for testing in vitro the therapeutic potential of Nam, due its intrinsic resistance to apoptosis, mediated by an imbalance in the mechanisms regulating cell death, mainly regulated through the activities of NAD+-dependent enzymes. This study shows that pharmacological doses of Nam (5-10 mM) significantly inhibit proliferation and induce apoptosis of CLL cells. At earlier time points, Nam markedly reduces phosphorylation of multiple intracellular substrates, including ERK1/2. Normal B lymphocytes, used as control, were significantly less sensitive to the action of Nam. We hypothesized that these effects could be explained at least in part as a consequence of the inhibitory effects of Nam on NAD+-consuming enzymes. Attention was focused on SIRT1, a deacetylase that plays a critical role in cancer and that acts as a longevity factor. The results demonstrate that Nam exposure inhibits the activity, and also the expression of SIRT1. This effect is apparent only in leukemic cells, where SIRT1 protein levels are significantly higher than in normal B lymphocytes, obtained from spleen or tonsils, markedly less sensitive to Nam effects. The functional block of SIRT1 induced by Nam is followed by activation of p53, transcription of miR-34a and translational repression of SIRT1 mRNA (p53/miR-34a/SIRT1 functional loop). The endpoint is the activation of apoptosis. The same loop is the target of conventional DNA-damaging drugs, such as etoposide. Thus, addition of Nam to conventional DNA-damaging chemotherapeutics agents, leads to an inhibition of SIRT1 through two independent and synergic pathways, resulting in additive effects on apoptosis. In conclusion this work suggests that Nam represents a potentially useful non-chemotherapeutic agent, characterized by a known and established safety profile, to be associated to conventional cytotoxic drugs in the treatment of selected forms of CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Human SLAMF1 (signaling-lymphocytic-activation-molecule-family1, CD150) is a self-ligand adhesion/co-stimulatory molecule wich belongs to a family of 9 receptors. SLAMF1 is also a microbial sensor, as it regulates Gram- bacterial phagosome functions through an ubiquitous cellular autophagic machinery and serves as a receptor for Measles virus. In this work, we investigated expression and function of SLAMF1 in chronic lymphocytic leukemia (CLL) cells. Results indicate that expression of SLAMF1 is lost in a subset of patients with chronic lymphocytic leukemia characterized by an aggressive form of the disease, with shorter time to first treatment (median 2.2 years in SLAMF1- vs 7.6 in SLAMF1+ patients, P=.001) and overall survival (77.5% survival rate at 10 years in SLAMF1- vs 94.7% years in SLAMF1+ patients, P=.036). Consistently, SLAMF1low CLL patients are characterized by clinical or molecular markers of a more aggressive disease. Stable silencing of SLAMF1 in the CLL-like Mec-1 cell line (constitutively SLAMF1+) modulated pathways related to cell migration, cytoskeletal organization and intracellular vesicle formation/recirculation. Decreased expression of CXCR3 and an increased expression of CXCR4, CD38 and CD44 were maintained at the molecular level, likely explaining why SLAMF1- cells show enhanced chemotactic responses to CXCL12. This phenotype was confirmed in primary cells, by comparing a cohorts of SLAMF1high to one of SLAMF1low patients. Gene expression profiling also indicates profound modulation of pathways connected with vesicle formation and recirculation. Consistently, cross-linking of SLAMF1 with an agonisic mAb in primary cells and in the Mec-1 cell line enhanced the generation of autophagic vesicles and their fusion with the lysosomes. Ligation of SLAMF1 with this agonistic monoclonal antibody promoted the autophagic flux, by increasing accumulation of reactive oxygen species (ROS) and inducing phosphorylation of p38, JNK1/2 and bcl-2. The direct consequence was the formation of the autophagy macro-complex containing SLAMF1, the scaffold protein beclin1 and the enzyme Vps34. In agreement with the observation that many drugs used in CLL have autophagy-mediated effects, including fludarabine and the BH3 mimetic ABT-737, SLAMF1-silenced Mec-1 cells or SLAMF1low primary CLL cells were resistant to treatment with both agents. These results indicate that SLAMF1 plays as a critical role in CLL homeostasis. Loss of SLAMF1 expression modulates genetic pathways that regulate chemotaxis and autophagy and that potentially affect drug responses, thus providing a likely explanation for the unfavorable clinical outcome experienced by this patient subset. Restoring SLAMF1 expression in CLL cells would therefore be of therapeutic value for patients with aggressive CLL. Disclosures Gaidano: Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding.

Description: Extracellular adenosine generated from ATP/ADP through the concerted action of the ectoenzymes CD39 and CD73 elicits potent cytoprotective and immunosuppressive effects mediated by type-1 purinergic receptors. Chronic lymphocytic leukemia (CLL) cells expressing the ectoenzymes CD39 and CD73 can actively produce adenosine, activating an autocrine adenosinergic axis that supports engraftment of leukemic cells in a growth-favorable environment. These effects are mediated by the A2A adenosine receptor, which inhibits chemotaxis and limits spontaneous and drug-induced apoptosis of CLL cells. Following the reported cross-talk between hypoxia and adenosine, we tested the hypothesis of a functional interplay between the adenosinergic axis and hypoxic signals in the CLL microenvironment. Results indicate that culture of CLL cells under hypoxic conditions, such as those observed in lymph nodes from CLL patients, boosts adenosine production, mainly because of the significant increase in the mRNA and protein levels of CD73, the rate-limiting enzyme in adenosine synthesis. CLL also underwent a robust up-regulation of CD26, which functions as an adenosine-deaminase scaffold protein, in keeping with the hypothesis that extracellular nucleotides enter a scavenging pathway, with conversion to inosine and re-uptake by the leukemic cells. Confirmation was obtained using HPLC assays, which showed increased inosine generation under hypoxia. Consistently, expression of membrane nucleoside transporters was also significantly up-regulated. However, hypoxic CLL cells also expressed high levels of the A2A adenosine receptor, which delivered cytoprotective signals and which supported CLL proliferation in response to TLR signaling. Attention was then focused on the stromal and T cell compartments, which are critical to the formation and maintenance of the leukemic niche. Hypoxia enhanced differentiation of circulating monocytes into nurse-like cells, macrophages of the M2 type playing an essential role in nurturing leukemic cells. The enhancement of NLC differentiation under hypoxic conditions relied, at least in part, on the activation of A2A: its engagement by a pharmacological agonist favored NLC generation, with overexpression of indoleamine 2,3-dioxygenase (IDO) and of the M2 macrophage markers CD163 and CD206. Moreover, activation of A2A induced secretion of immunomodulatory cytokines, such as IL-6, IL-10 and CCL18, while pharmacological blockade of A2A under hypoxia prevented NLC differentiation, expansion, expression of immunosuppressive molecules and secretion of cytokines and chemokines. In the T cell compartment, hypoxic cultures were followed by the sharp up-regulation of A2A, without significantly affecting the enzymes that generate adenosine, which were anyway restricted to the regulatory T cell (Treg) compartment. Co-cultures of T lymphocytes and CLL cells under hypoxia resulted in a dramatic decrease of T cell proliferation, partially rescued by A2A receptor antagonists. Furthermore, hypoxic T cells underwent a metabolic switch, with increased expression of nucleoside transporters and enzymes involved in glucose metabolism, suggesting a Warburg effect. This was accompanied by the differentiation of a population of Tr1 cells, characterized by the expression of LAG3 and CD49b and by the secretion of high levels of IL-10 and VEGF. Expression of the PD-1 immuno-inhibitory receptor was enhanced in hypoxic T cells, suggesting that multiple inhibitory mechanisms are activated. We also observed expansion of classical Tregs, defined on the basis of a CD4+/CD25high/CD127low/foxp3+ phenotype. Blockade of the A2A receptor prevented this phenotype, partially restoring T cell proliferation and immune competence. Together, these findings indicate that the adenosinergic and hypoxic axes synergize in shaping the CLL niche, suggesting that pharmacological inhibition of the adenosinergic signals may counteract some of the effects mediated by an hypoxic environment, contributing to disrupt the leukemic niche and to restore the immune system. Disclosures Gaidano: Celgene: Research Funding; MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards.

Description: BACKGROUND: Tumor cell survival critically depends on heterotypic communications with non-malignant cells in the microenvironment. Most of these signals converge on the activation of the transcription factor NF-κB that regulates complex cellular functions, including apoptosis, cell survival and proliferation. Even if NF-kB is constitutively active in most malignancies, including chronic lymphocytic leukemia (CLL), and plays a major role in tumorigenesis, there are no currently approved drugs to target it. IT901 has been recently reported as a novel NF-kB inhibitor, showing efficacy in a non-tumor context1. AIM OF THE WORK: The aim of this work is to test the efficacy of IT901 in CLL and in its more aggressive transformation, Richter syndrome (RS), which represents an unmet therapeutic need. The molecular mechanisms of action of IT901 in leukemic cells are studied, alongside its effects on cells belonging to CLL microenvironment. RESULTS: IT901 induces apoptosis in primary leukemic cells in a dose- and time-dependent manner, showing significant efficacy after 24h of treatment. The apoptotic response is independent of the prognostic subgroup. Conversely, IT901 has minimal impact upon normal B cells. Treatment of CLL cells with IT901 interferes with NF-kB transcriptional activity, resulting in a diminished binding of both p50 and p65 to DNA. Moreover, biochemical analyses indicate a diminished expression of these subunits in the nucleus, as well as of the whole NF-kB complex in the cytoplasm. At the molecular level, compromised expression of NF-kB triggers activation of the Caspase-3 apoptotic pathway, with increased expression of pro-apoptotic proteins (e.g., Bim), paralleled by a diminished expression of the anti-apoptotic ones (e.g., XIAP). Concomitantly, a prominent increase in mitochondrial ROS is evident, providing a link between IT901 effects and induction of apoptosis. Recent data reported the involvement of NF-kB as a transcriptional controller of metabolic pathways promoting oxidative phosphorylation in cancer cells. In line with NF-kB constitutive activation in CLL, dynamic measurement of the energetic profile, indicates a reliance on oxidative phosphorylation, with limited glycolytic capacity. After IT901 treatment, there is a dramatic drop in mitochondrial respiration, with compromised ATP production and a net increase in proton leak, suggesting that primary CLL cells are trying to compensate impaired respiration by shifting to glycolysis. This metabolic response is mediated at the transcriptional levels, as IT901 induces a down-modulation of the genes involved in mitochondrial respiration (e.g., ATP5A1) and a concomitant up-modulation of the ones involved in glucose uptake and lactate transport (e.g., GLUT1). The CLL microenvironment is critical for disease progression and for providing protection from drug-induced apoptosis. Therefore it is important to consider the effects of novel drugs also on non-neoplastic bystander elements. Nurse-like cells (NLC) are a population of monocyte-derived activated macrophages that nurtures CLL cells via soluble and cell contact dependent mechanisms. These interactions are known to activate NF-kB signaling in both partners. Consistently, IT901 inhibited nuclear localization of the p65 subunit in NLC and shifted their polarization towards an M1-phenotype. These results are confirmed using a xenograft model. The Mec-1 cell line was injected into NSG mice and left to engraft for 2 weeks before beginning treatment. Animals treated with IT901 are characterized by decreased tumor growth and leukemic cells diffusion compared to controls, as shown by a diminished number of leukemic cells in kidneys, liver and spleen. Finally, IT901 shows promising effects in a small cohort of leukemic cells obtained from RS patients, inducing significant apoptosis by interfering with the expression and nuclear localization of NF-kB. CONCLUSIONS: Altogether, these results indicate that IT901 blocks NF-kB transcriptional activity. This effect is followed by rapid and marked decrease in genes supporting oxidative phosphorylation, causing mitochondrial damage, ROS release and induction of intrinsic apoptosis. Moreover, IT901 interrupts the support that CLL obtains from the microenvironment. Thus, targeting NF-kB by means of IT901 may be effective for CLL, and possibly even RS patients. 1. Y. Shono et al., Cancer Res76, 377 (Jan 15, 2016). Disclosures Furman: Genentech: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Honoraria; Gilead Sciences: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau.

Description: Abstract 1778 Chronic lymphocytic leukemia (CLL) is characterized by a progressive accumulation of mature B lymphocytes and it is marked by profound defects in T cell function. The mechanisms responsible for T cell dysfunction remain unclear, even if several observations show that T cells from CLL patients express markers of chronic activation. One of this marker is Programmed death-1 (PD-1), a cell surface molecule that inhibits activation of immune cells and it is involved in tumor escape mechanisms through binding of the specific PD-L1 ligand. The aim of this work is to evaluate the expression and function of the PD-1/PD-L1 axis in the CLL context. Using multiparameter flow cytometry, we showed that CD4+ and CD8+ T lymphocytes from CLL patients (n=117) express significantly higher levels of the PD-1 receptor, as compared to the same cell subpopulations purified from age- and sex-matched normal donors (n=33; 52% vs 34%, p