Abstract 2595
Large Granular Lymphocyte (LGL) leukemia is a chronic lymphoproliferative syndrome that can be broadly classified into two groups depending on whether the expanded cells are T-cells or ...NK-cells. The clinical characteristics of the disease include lymphocytosis, neutropenia, anemia, that can be associated with rheumatoid arthritis and pulmonary arterial hypertension (PAH). Hematologic improvement with immunosuppressive agents such as cyclosporine and low-dose methotrexate has lead to the widely accepted theory that cytopenias are mediated by autoimmune destruction of the hematopoietic stem cell (HSC) compartment or lysis of mature myeloid cells in circulation. We found, however, that autologous HSCs and mature granulocyte populations fail to be recognized or lysed ex vivo by T-LGL leukemia cells suggesting that an alternate mechanism may be involved. In contrast to research done on the T-LGL cells themselves, the role of the bone marrow microenvironment and HSC compartment in T-LGL leukemia patients is completely unexplored. Therefore, bone marrow core biopsies, aspirates, and peripheral blood smears were obtained from 22 patients with LGL leukemia and 14 patients with non-hematological malignancies to serve as controls. Morphology and extracellular matrix composition were examined by H&E and reticulin stains, respectively. Utilizing the European consensus guidelines, grading scale (0 – 3), we determined that bone marrow reticulin fibrosis is present in patients with T-LGL leukemia (Figure 1). The mean fibrosis grade for the LGL group was 2.32 (median of 2.5), whereas, the mean fibrosis grade for the control group was 1.46 (median of 1.50), p-value of 0.01. Our analysis revealed that reticulin fibrosis in LGL leukemia bone marrow was particularly associated with the interstitial stroma and lymphoid aggregates. The degree of fibrosis in T-LGL bone marrow showed no relation to absolute neutrophil counts. However, an in depth analysis of neutrophil morphology revealed several dysplastic features within the neutrophil compartment in T-LGL patients. These features include decreased segmentation, increased numbers of pseudo-Pelger-Huet forms, and an increase percentage of immature neutrophils. Of these, the proportion of immature neutrophils positively correlated with fibrosis grade in T-LGL patients (Spearman r=0.7302; p=0.0002), indicating a possible link between reticulin fibrosis and the quality of hematopoiesis. To explore the pathogenesis of medullary fibrosis, mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of 6 LGL leukemia patients and 5 healthy controls and then expanded ex vivo under non-differentiating conditions. During expansion, healthy MSCs produce cytokines and growth factors necessary for self renewal and for the support of hematopoiesis. However, MSCs from T-LGL patients displayed severely reduced self-renewal potential, reaching a mean of 7 population doublings compared to a mean of 23 for normal MSCs, and were unable to support the proliferation of healthy HSCs in a co-culture proliferation assay. Microarray analysis (H6 V133 plus 2.0) was performed on the MSCs from both control and T-LGL patients with analysis focused on genes regulating basement membrane composition. For normal MSCs, significant reductions in the expression of numerous collagen genes occured as the cells underwent expansion in self-renewal conditions. However, MSCs from T-LGL patients failed to downregulate these genes despite months of culture. The most prominent collagen genes following this pattern were types I (α1, α2), III (reticulin), IV (α1, α2), and V (α1, α2). A combination of qRT-PCR and immunflourescent staining (Figure 2) were utilized to confirm these gene expression changes. Collectively, these results implicate aberrant MSC self-renewal capacity and skewed basement membrane protein expression in the pathogenesis of T-LGL leukemia and suggest that these abnormalities may represent novel targets for future drug discovery.
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No relevant conflicts of interest to declare.
Abstract 4001
Low serum albumin level is known to be an adverse prognostic factor in patients with malignancies such as multiple myeloma. We previously reported that severe hypoalbuminemia (<3.0 ...g/dl) at day +90 post allogeneic hematopoietic stem cell transplant (AHCT) was an independent predictor of non-relapse and overall mortality in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) (Kharfan-Dabaja et al Biol Blood Marrow Transplant. 2010 Jul). In this study we examined prognostic value of serum albumin level in patients with MDS.
Data were analyzed from the Moffitt Cancer Center (MCC) MDS database with chart review verification. The primary objective was to examine the role of serum albumin at time of presentation to MCC as a prognostic marker for overall survival (OS). Patients were divided into 3 groups of serum albumin levels (≤ 3.5, 3.6–4.0 and > 4.0 g/dl). The Kaplan–Meier method was used to estimate median OS. The log rank test was used to compare Kaplan–Meier survival estimates between two groups. Cox proportional hazards regression was used for multivariable analysis.
Between January 2001 and December 2009, 844 patients were captured by the MCC MDS database. The median age was 69 years. MDS subtypes were coded as refractory anemia (RA) (n=98;12%), refractory anemia with ring sideroblasts (RARS) (n=76;9%), del(5q) (n=20;2.4%), refractory cytopenia with multi-lineage dysplasia (RCMD) (n=96;11%), refractory anemia with excess blasts (RAEB) (n=255;30%), therapy related MDS (n=22;2.6%), and MDS-NOS (n=275; 33%). The distribution of IPSS risk groups was: 18.7% Low risk, 42.9% Intermediate-1 (Int-1), 19.9% Int-2, 5.3% High risk, and 13.2% unknown. Baseline characteristics for the three patient groups defined by serum albumin level are summarized in (Table-1). There was no difference in red blood cell transfusion dependency (RBC-TD) rate between the 3 groups (p=0.21). The median OS for all patients was 36 months (95% confidence interval (CI) 31.5–40.5 mo). Age, IPSS risk group, RBC-TD, Serum ferritin were statistically significant prognostic factors in univariable analysis.
The median OS was 19 mo (95%CI= 14.9–23.1 mo), 35 mo (95%CI= 28.7–41.3 mo), and 53 mo (95%CI= 44.7–61.3 mo) for patients with serum albumin levels ≤ 3.5 g/dl, 3.6–4.0 g/dl, > 4.0 g/dl, respectively. (Figure-1) (p= <0.005). After adjustment for age, RBC-TD, OS was statistically significantly inferior among MDS patients with lower serum albumin (Hazard Ratio (HR) = 0.79.; 95%CI= 0.69–0.90; p= 0.001), and higher-risk IPSS group (HR=1.67; 95%CI=1.48-1.87; p= <0.005).
The overall rate of AML transformation was 29.2%. Rate of AML transformation was higher in patients with lower serum albumin, 38% in patients with serum albumin ≤ 3.5 g/dl, 30% for patients 3.6–4.0 g/dl, and 23% in patients with serum albumin > 4.0 g/dl (p-value 0.005).
Among patients in the Low/Int-1 IPSS risk group, the median OS was 28 mo (95%CI=15.7-40.3 mo), 48 mo (95%CI=38.8-58.0 mo), and 60 mo (95%CI=47.6-72.4 mo) for patients with serum albumin levels ≤ 3.5 g/dl, 3.6–4.0 g/dl and > 4.0 g/dl, respectively (p=0.003). Among patients in the Int-2/High IPSS risk group, the median OS was 16 mo (95%CI 13.3–15.7 mo), 22 mo (95%CI 18.0–26.0 mo), and 21 mo (95%CI 8.8–33.2 mo) respectively for patients with serum albumin levels ≤ 3.5 g/dl, 3.6–4.0 g/dl and > 4.0 g/dl, respectively p=0.03).
In this retrospective analysis of a large single institution MDS database, serum albumin is found to be an independent prognostic factor for OS and AML transformation in MDS patients. The prognostic power of low serum albumin was greatest among patients with Low/Int-1 IPSS risk group, but remained an independent variable across all risk groups. Serum albumin may also be a surrogate marker of general health, co- morbidities, and performance status.
Table-1Baseline CharacteristicsSerum albumin ≤ 3.5 g/dlSerum albumin 3.6–4.0 g/dlSerum albumin > 4.0 g/dlAGE (mean)686865IPSS risk Group16 (11%)74 (23.5%)67 (25.6%)Low73 (50.3%)152 (48.3%)132 (49.4%)Int-139 (26.9%)72 (22.9%)57 (21.3%)Int-216 (11.7%)17 (5.4%)11 (4.1%)HighSerum Ferritin (mean)21741067805RBC Transfusion Dependency82 (49.7%)166 (47.6%)377 (47%)
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No relevant conflicts of interest to declare.
Abstract 2925
Antineoplastic vaccine strategies aimed at augmenting immuno-surveillance have been limited by the lack of known tumor antigens and the inability to sustain an immune response. A ...“bystander” vaccine approach has been tested with a K562-GMCSF transfected cell line in combination with autologous cells in acute myeloid leukemia (AML) and in combination with imatinib in chronic myeloid leukemia (CML). Lenalidomide, a thalidomide analogue approved for the treatment of deletion 5q MDS, has stimulatory effects on CD8+ T cells and augments vaccine response in a colon cancer and melanoma model. We present results of a phase I, dose escalation study using a K562-GMCSF-CD40 Ligand transfected, “bystander” vaccine in combination with lenalidomide in International Prognostic Scoring System (IPSS) intermediate and high risk MDS patients who failed hypomethylating agents.
The study was a standard “3+3”, dose escalation phase I design. Eligibility was restricted to patients with a diagnosis of MDS subtypes RAEB-1, and RAEB-2 by World Health Organization classification (WHO), or RAEB-t by FAB criteria. An ANC greater than 500/μl and platelet count greater than 20,000/μl were required. Prior therapy with lenalidomide, secondary MDS, and a diagnosis of proliferative CMML were excluded. GM-CSF and CD40L constructs were transfected into the K562 cell line. Co-transfected clones were selected and subcutaneously injected in the axillary nodal basin of subjects on days 8 and 22 of a 28-day cycle for a total of four cycles. Lenalidomide was administered at a daily dose of 10mg on days 1 through 21 every 28 days until disease progression or limiting toxicity. Toxicities were monitored weekly and BM biopsy repeated after 2 & 4 cycles and at discontinuation of vaccine therapy. The dose escalation of vaccine was 10 × 106 cells in cohort 1, 30 × 106 in cohort 2, 60 × 106 in cohort 3 and 120 cells × 106 in cohort 4. MTD was defined as the highest dose level at which <1 out of 6 subjects experience a DLT. Responses were measured using the international working group criteria (IWG) 2006 for MDS.
Between March 2009 and August 2010, eleven patients were enrolled after signing informed consent. The median age was 74 years, with a male predominance (n=9). According to WHO classification, 4 patients had RAEB-1, 6 had RAEB-2, and 1 had AML (RAEB-t by FAB). The IPSS score at study entry was int-1 (4), int-2 (3), and high risk (4). After four cycles of therapy, no treatment related grade 3 or 4 toxicities were seen in cohorts 1–3 (10-60 × 106 cells). Two participants in cohort 2 (30 × 106) developed grade 3 myelosuppression deemed disease related by the investigator. One participant in cohort 2 died while on this study secondary to a non-treatment related pneumonia. Dose limiting toxicity (DLT) was reached in cohort 4 (120 × 106). One patient developed grade 3 dyspnea and fatigue after one injection of vaccine. This cohort will be expanded to a total of 6 patients. Grade 1 toxicities in all cohorts included rash, myelosuppression, nausea, vomiting, and fatigue. The IWG response rates were complete response (CR) in 2/11 (18%), marrow CR (1/11) (9%), and partial response (PR) in 1/11 (9%). The overall response rate was 4/11 (36%). In all patients who achieved a CR the median duration of response was 367 days (136-490).
Although the MTD has not been reached, this cell-based immunotherapy was well tolerated. Response rates and the duration of these responses in patients who failed hypomethylating agents is encouraging and warrants further investigation. Accrual to the study is ongoing and updated data will be presented.
Lancet:Eisai: Consultancy; Celgene: Honoraria. List:Celgene: Research Funding.
Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin ...receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.
“Single”
T = 1 isometric particles of
Maize streak virus (MSV) have been isolated from infected maize leaves. Biochemical and genetic characterizations show that these particles contain subgenomic ...(sg) MSV DNA encapsidated by the MSV coat protein. The largest sg DNA is 1.56 kb, slightly larger than half genome size, although sg DNAs as small as 0.2 kb were also cloned. The sg DNAs are not infectious, and they do not appear to play a role in the pathogenicity of MSV. This is the first report of sg DNAs for MSV and, to our knowledge, the first time that encapsidated sg DNAs have been characterized at the sequence level for any geminivirus. These data will assist in our investigations into the role of genomic DNA in the formation of the unique geminate capsid architecture of the
Geminiviridae.
Adeno-associated virus (AAV) serotypes 1 to 5 are currently under development as clinical gene delivery vectors for the treatment of human diseases. However, the ubiquitous nature of their cell ...surface receptors, heparin sulfate (AAV2 and 3) and sialic acids (AAV4 and 5), can preclude specific tissue targeting in vivo. Structural studies of AAV4 were initiated to characterize its capsid surface for re-targeting manipulations. Crystals obtained diffracted synchrotron radiation to 3.2 Å resolution. The unit cell is body-centered orthorhombic, I222, with a = 339.6, b = 319.2 and c = 285.0 Å. The virus particle orientation and position have been determined.
Adeno‐associated viruses (AAVs) are actively being developed for clinical gene‐therapy applications and the efficiencies of the vectors could be significantly improved by a detailed understanding of ...their viral capsid structures and the structural determinants of their tissue‐transduction interactions. AAV8 is ∼80% identical to the more widely studied AAV2, but its liver‐transduction efficiency is significantly greater than that of AAV2 and other serotypes. The production, purification, crystallization and preliminary X‐ray crystallographic analysis of AAV8 viral capsids are reported. The crystals diffract X‐rays to 3.0 Å resolution using synchrotron radiation and belong to the hexagonal space group P6322, with unit‐cell parameters a = 257.5, c = 443.5 Å. The unit cell contains two viral particles, with ten capsid viral protein monomers per crystallographic asymmetric unit.
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