ALBERT

Description: Here, we demonstrate a novel, direct-acting, and synergistic role for 3 hematopoietic stem cell cytokines: stem cell factor, interleukin-3, and stromal derived factor-1α, in controlling human endothelial cell (EC) tube morphogenesis, sprouting, and pericyte-induced tube maturation under defined serum-free conditions in 3-dimensional matrices. Angiogenic cytokines such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) alone or VEGF/FGF combinations do not support these responses. In contrast, VEGF and FGF prime EC responses to hematopoietic cytokines via up-regulation of c-Kit, IL-3Rα, and C-X-C chemokine receptor type 4 from either human ECs or embryonic quail vessel explants. In support of these findings, EC Runx1 is demonstrated to be critical in coordinating vascular morphogenic responses by controlling hematopoietic cytokine receptor expression. Combined blockade of hematopoietic cytokines or their receptors in vivo leads to blockade of developmental vascularization in quail embryos manifested by vascular hemorrhage and disrupted vascular remodeling events in multiple tissue beds. This work demonstrates a unique role for hematopoietic stem cell cytokines in vascular tube morphogenesis and sprouting and further demonstrates a novel upstream priming role for VEGF and FGF to facilitate the action of promorphogenic hematopoietic cytokines.

Description: Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly.

Description: We show that endothelial cell (EC)–generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, α5β1, α3β1, α6β1, and α1β1, which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.

Description: Abstract 1842 Perifosine (Keryx Biopharmaceuticals) is an oral alkylphospholipid that has been shown to have clinical activity in multiple myeloma and Waldenstrom's macroglobulinemia. Pre-clinical data suggest that its activity is due to inhibition of the Akt signal transduction pathway. The Akt pathway is known to be important for viability in CLL, another B-cell malignancy. Therefore, we investigated the in vitro activity of perifosine in freshly isolated primary CLL cells. CLL patients at the Duke University and Durham VA Medical Centers were enrolled in an IRB-approved research protocol for blood sample collection. CLL cells were negatively selected using the RosetteSep B-cell enrichment cocktail (StemCell Technologies) and a ficoll-Hypaque gradient. This method yields greater than 95% purity of malignant lymphocytes, determined by flow cytometry. Prognostic markers such as IgVH mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were determined. We found the 50% effective dose (ED50) of perifosine for inducing cytotoxicity in CLL cells after a three-day incubation using the MTS colorimetric assay to be 510 nM (n = 29, range 120 – 1540 nM). CLL cells were obtained from patients with generally poor prognostic markers: 52% CD38+, 93% ZAP70+, 78% IgVH unmutated, 42% 17p deletion, 8% 11q deletion, 27% trisomy 12, 12% normal, and 12% 13q deletion as a sole abnormality. There were no statistical differences in ED50 between cells obtained from patients in high or low risk prognostic groups. Perifosine induced apoptosis in a dose- and time-dependent manner, measured by Annexin V and PI staining (n = 4). Based upon these pre-clinical results, we initiated a phase II study of perifosine (50 mg orally, twice daily) in relapsed or refractory CLL/small lymphocytic lymphoma (SLL) (NCT00873457). The primary objective of this study is to assess the response rate at 3 and 6 months of perifosine treatment in patients with relapsed or refractory CLL/SLL. Secondary objectives are to monitor toxicity, evaluate overall survival, progression-free survival and response duration, and perform laboratory correlates. Early interim results of this study are presented. Since trial initiation in September 2009, 13 patients have been enrolled. Nine patients had Rai stage III/IV disease at the time of therapy, and 4 patients were fludarabine-refractory. Patients had extensive prior treatment (median 4, range 1 – 11). Many patients had high-risk prognostic features: 9/11 IgVH unmutated, 10/13 CD38+, and 11/13 ZAP70+. Evaluation of interphase and metaphase cytogenetics demonstrated 4 patients with 17p deletion, two with 11q deletion, 2 with trisomy 12, 4 normal, and 4 with other complex cytogenetic anomalies. Of 12 patients who began therapy, 5 patients withdrew from the study prior to 3 months, 6 patients received at least 3 months of therapy, and 1 patient completed 6 months of therapy. Of the patients who received at least 3 months of therapy, there were 5 patients with stable disease, and one patient with partial response, using iwCLL response criteria. Grade 3/4 toxicities included anemia (n=2), fatigue (n=2), dehydration (n=1), febrile neutropenia (n=1), hyperbilirubinemia (n=1), hyponatremia (n=1), cough (n=1), and dyspnea (n=1). One patient required a dose reduction to 50 mg daily and two patients required dose delays due to toxicities. In conclusion, perifosine has potent in vitro activity against primary CLL cells. Preliminary results of this ongoing phase II study of oral perifosine in relapsed or refractory CLL/SLL demonstrate mostly disease stabilization in a group of very high-risk patients and an acceptable toxicity profile. Completion of this clinical study is necessary to determine if perifosine monotherapy has a potential role in the treatment of CLL/SLL. Disclosures: Sportelli: Keryx Biopharmaceutical: Employment, Equity Ownership. Weinberg:Keryx Biopharmaceuticals: Research Funding.

Description: Introduction The current standard to assess chemotherapy tolerability relies on patient self-reporting. However, as the sole mechanism of managing symptom burden, this may be inconsistent and fraught with bias. Mobile wearable health devices have the ability to monitor and aggregate objective activity and sleep data over long periods of time, but have not been systematically used in the oncology clinic. The aim of the study was to assess whether the use of mobile wearable technology establishes patterns of "sleep" and "wake" states in newly diagnosed Multiple Myeloma (NDMM) patients receiving therapy, and whether these patterns differ over time. Methods Patients presenting to the myeloma clinic at Memorial Sloan Kettering Cancer Center (MSKCC) with a new diagnosis of Multiple Myeloma and smart phone or tablet (iOS or Android) compatible with the Garmin Vivofit device were offered to participate in a mobile wearable bio-monitoring study. All eligible participants were required to receive primary chemotherapy treatment at a MSKCC facility. Treatment was determined by physician. NDMM patients were assigned to one of two cohorts (20 in each; Cohort A - patients

Description: Natural killer (NK) cells secrete lytic granules to directly kill virus-infected or transformed cells and secrete cytokines to communicate with other cells. Three-dimensional super-resolved images of F-actin, lytic granules, and IFN-γ in primary human NK cells stimulated through different activating receptors reveal that both IFN-γ and lytic granules accumulated in domains where the periodicity of the cortical actin mesh at the synapse opened up to be penetrable. Ligation of some activating receptors alone (eg, CD16 or NKG2D) was sufficient to increase the periodicity of the actin mesh, but surprisingly, ligation of others (eg, NKp46 or CD2) was not sufficient to induce cortical actin remodeling unless LFA-1 was coligated. Importantly, influenza virus particles that can be recognized by NK cells similarly did not open the actin mesh but could if LFA-1 was coligated. This leads us to propose that immune cells using germline-encoded receptors to directly recognize foreign proteins can use integrin recognition to differentiate between free pathogens and pathogen-infected cells that will both be present in blood. This distinction would not be required for NK cell receptors, such as NKG2D, which recognize host cell–encoded proteins that can only be found on diseased cells and not pathogens.

Description: Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B cell receptor immunoglobulins (BcR IG). Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR IG stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR IG stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. In order to address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29,856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed 'satellites', were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Description: Tumor burden in adult patients with acute leukemia is assessed using the percentage of blast cells in the bone marrow or blood. It is clear, however, that not all blast cells are leukemic cells, especially during rapid marrow regeneration. Similarly, some leukemia cell lines have been shown to differentiate in vitro, and the same process also occurs in vivo. Therefore, the leukemic burden may be due to more differentiated cells as well as to blast cells. The purpose of this study was to investigate whether the human malignancy-associated nucleolar antigen (HMNA) could be used as a marker for leukemic cells and to examine its potential as a diagnostic tool. The proportion of HMNA-positive cells in the bone marrow of patients with acute leukemia was determined by indirect immunofluorescence with antibodies to HMNA and was compared with the differential counts routinely made in the clinic laboratory. The percentages of HMNA-positive cells among the nucleated cells in the marrow of 72 patients with clinical evidence of leukemia were significantly higher (range 9%-98%, median 83%) than those observed for nonleukemic individuals (range less than 0.05%-2.5%, median 1%) or for fractions of marrow cells from normal volunteers enriched for normal early progenitor cells (less than or equal to 2%). Patients with leukemia in remission had a lower percentage of HMNA- positive cells (range 0%-83%, median 3%). The percentage of HMNA- positive cells increased as patients approached relapse. Although the percentage of HMNA-positive cells was related to the percentage of blast cells in the bone marrow of the patients with leukemia, some partially differentiated cells were also HMNA-positive in some specimens, and some blastic cells were HMNA-negative in other specimens. These studies indicate the potential usefulness of HMNA as a marker for leukemic cells.

Description: Background: Induction chemotherapy for acute myeloid leukemia (AML) is more intensive than many other cancer treatments, and may be associated with a different symptom burden. Little is known about the most prevalent symptoms during AML induction, nor how they change over time, and with remission status. Similarly, little is known about the trajectory of quality of life (QoL) and distress scores in this population. We aimed to learn more about the natural history of these issues via a prospective, longitudinal, observational patient-reported outcomes study. Methods: We enrolled 43 inpatients with AML at initiation of induction chemotherapy, and assessed their symptoms, quality of life (QoL), and distress weekly during their month-long hospitalization for induction, and monthly thereafter, using 3 validated instruments: Patient Care Monitor v2.0 (PCM); Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu); and NCCN distress thermometer (DT). We used descriptive statistics and ANOVA to analyze results. Results: Mean age of study participants was 59.4 (SD 13.4); 21 (49%) were female. Patients were mostly high-risk for recurrence, with 25 (58%) being ≥60 years old, 19 (44%) having high-risk cytogenetics, and 10 (23%) having relapsed disease. Among relapsed patients, the mean number of prior treatments was 2.7 (SD 1.3). At the time of this analysis, 5 patients (18%) had gone on to receive a stem cell transplant. As expected, symptoms were most prominent during the second and third weeks of treatment. However, across all 4 weeks of induction patients consistently reported 5 symptoms at a moderate or severe level (scores of 4 to 6, or 7 to 10 out of 10, respectively), including: poor appetite (35%), dry mouth (37%), difficulty sleeping (38%), dysgeusia (44%), and fatigue (56%). Other prominent moderate-to-severe symptoms included diarrhea (35%), daytime sleepiness (30%), and nausea (27.5%), despite standard supportive care. Mean QoL by FACT-Leu worsened substantially from week 1 (121.8, SD 27.6) to week 2 (108.2, SD 26.3), and then slowly recovered thereafter, improving to better than baseline by month 3 and continuing to improve throughout 1-year of follow-up (p

Description: Sera from two patients with granulocytopenia associated with collagen vascular disease caused the destruction of normal human granulocytes by autologous lymphocytes in vitro. Granulocyte cytotoxicity was measured by the release of 51Cr during incubation with test sera and lymphocytes in microtiter plates. Between 8% and 46% granulocytoxicity was produced in granulocytes from 8 normal donors by the sera from these two patients. Less than 6% granulocytotoxicity was seen with the sera from 14 normal subjects and 29 patient controls. Treatment of lymphocyte preparations with carbonyl iron and magnetic separation to remove phagocytic cells or treatment with complement-coated red cells followed by repeated gradient centrifugation to remove complement receptor- bearing lymphocytes did not reduce the granulocytotoxicity. There was a dose-response relationship between the concentration of positive sera and granulocytotoxicity. When these sera were fractionated by Sephadex G-200 gel filtration and by ion-exchange chromatography with DEAE- cellulose, the active component appeared in the IgG-containing fractions. Thus, IgG antibody-dependent, lymphocyte-mediated granulocyte cytotoxicity represents a means of detecting human granulocyte antibodies and is a possible mechanism of autoimmune neutropenia in these two patients.