ALBERT

Description: No validated biomarkers exist for acute graft-versus-host disease (GVHD). We screened plasma with antibody microarrays for 120 proteins in a discovery set of 42 patients who underwent transplantation that revealed 8 potential biomarkers for diagnostic of GVHD. We then measured by enzyme-linked immunosorbent assay (ELISA) the levels of these biomarkers in samples from 424 patients who underwent transplantation randomly divided into training (n = 282) and validation (n = 142) sets. Logistic regression analysis of these 8 proteins determined a composite biomarker panel of 4 proteins (interleukin-2-receptor-alpha, tumor-necrosis-factor-receptor-1, interleukin-8, and hepatocyte growth factor) that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups in the training set was 0.91 (95% confidence interval, 0.87–0.94) and 0.86 (95% confidence interval, 0.79–0.92) in the validation set. In patients with GVHD, Cox regression analysis revealed that the biomarker panel predicted survival independently of GVHD severity. A panel of 4 biomarkers can confirm the diagnosis of GVHD in patients at onset of clinical symptoms of GVHD and provide prognostic information independent of GVHD severity.

Description: Abstract 2230 Poster Board II-207 Background: Reduced intensity conditioning (RIC) for allogeneic hematopoietic stem cell (HSC) transplantation is a well established therapy for patients with advanced hematological malignancies who are not suitable candidates for fully myeloablative conditioning. The use of alemtuzumab as part of RIC has been associated with decrease in incidence and severity of graft versus host disease (GVHD), particularly in unrelated donor transplants. Methods: This is a single center, prospective study. Patients with relapsed or refractory hematological malignancies who were not candidates for fully myeloablative conditioning regimens received an non-alemtuzumab (nA) containing RIC regimen or an alemtuzumab-containing regimen (A) based on the use of a matched sibling or an allele-matched unrelated donor, respectively. Patients in the nA group were conditioned with either Fludarabine 30mg/m2/day for 5 days and total body irradiation 200cG or Fludarabine 25mg/m2/day for 5 days and Melphalan 70mg/m2/day for 2 days. Patients in the A group received alemtuzumab 20 mg/m2/day for 2 days, Fludarabine 30mg/m2/day for 5 days and Melphalan 70mg/m2/day for 2 days. All patients received peripheral blood HSC grafts. Prophylaxis against graft versus host disease with cyclosporine and mycophenolate mofetil as well as infectious disease prophylaxis and supportive care guidelines were standard across both groups. Results: From January 2003 to March 2009, 56 patients were enrolled in the study and 50 patients who actually received a HSCT are the subject of this analysis. Twenty nine patients were in group nA and 21 in group A. Median age of the patients in the entire cohort was 55 years (range 14-65). Eighteen patients had AML, 12 NHL, 8 MDS, 6 CML, 3 CLL and 3 MM. Twenty-three patients were considered to be at high-risk for post transplant relapse (45% of group nA vs. 47% of group A, P= 0.84). All patients in group nA had hematological engraftment while 2 patients in A (9.5%) failed to engraft. Despite the fact that all patients in group A received a transplant from an unrelated donor, the incidence of severe (grades C or D) acute GVHD was significantly higher in group nA (20.7% vs. 0%, P=0.03). Day 100 mortality was similar between the 2 groups (20.7% for nA vs 33.1% for A, P=0.31). Cumulative incidence of progression did not differ between the two groups (P=0.7) while the cumulative incidence of treatment related mortality was higher in group A (P=0.02, Figure 1). Both median overall survival (OS) (44.1 vs. 5.3 months; P=0.01, figure 2) and progression-free survival (PFS) (8.7 vs 5.3 months, P= 0.02) were superior in group nA. Cox regression analysis including conditioning regimen, age and risk of post-transplant relapse showed that conditioning with alemtuzumab was the only variable significantly associated with inferior OS (RR 2.8, P=0.009) and PFS (RR 2.42, P= 0.01). Group A had a higher incidence of late (after Day 100) infectious complications resulting in death (43% vs.10%, p=0.01). Conclusion: Even though alemtuzumab-based RIC in unrelated HSCT has a protective effect against acute GVHD and risk of D+100 TRM comparable with non-alemtuzumab containing RIC regimens in related donor transplant, its beneficial effect is overcome by excessive late infectious complications. Disclosures: Fouts: Genzime: Consultancy.

Description: Abstract 1856 Poster Board I-882 Background: Lenalidomide is an analog of thalidomide that has shown significant clinical activity in patients with relapsed or refractory multiple myeloma (MM), both as a single agent and in combination with dexamethasone. Bendamustine is a bifunctional alkylating agent that is approved for the treatment of chronic lymphocytic leukemia and indolent non-Hodgkin's lymphoma that has progressed during or relapsed within 6 months following a rituximab-containing regimen. Bendamustine combined with lenalidomide may be an effective treatment option for MM patients, particularly those with preexisting or bortezomib-induced neuropathy. Our primary objective was to determine the maximum tolerated dose (MTD) and safety profile of bendamustine and lenalidomide when administered with dexamethasone for patients with relapsed or refractory MM. Methods: Patients aged ≥18 years with confirmed, measurable stage 2 or 3 MM that was refractory to or progressed after 1 or more prior therapies, including lenalidomide, received bendamustine by intravenous infusion on days 1 and 2, oral lenalidomide on days 1–21, and oral dexamethasone on days 1, 8, 15, and 22 of each 28-day cycle. Treatment was continued until a plateau of best response, as determined by the IBMTR/ABMTR, was reached. Study drug doses were escalated through 4 levels (Table), with 3–6 patients enrolled at each level depending on the rate of dose-limiting toxicity (DLT). After determining the MTD, up to an additional 12 patients will be enrolled in an MTD expansion arm to better evaluate toxicity and clinical activity. Secondary endpoints included preliminary efficacy, as evidenced by objective response, time to disease progression, and overall survival. Results: To date, 11 patients have been enrolled, with a median age of 63 years (range, 38–75 years). The MTD of bendamustine and lenalidomide has not been identified at this point; currently, patients are enrolling on dose level 3 with 100 mg/m2 bendamustine and 10 mg lenalidomide. Thus far, DLT included 1 grade 4 neutropenia at dose level 2. Nine of 11 patients are currently eligible for response assessment. A partial response was observed in 67% of patients, including 1 very good partial response and 5 partial responses (PR). Two patients experienced stable disease and 1 exhibited progressive disease. Grade 3/4 adverse events included grade 3 neutropenia, thrombocytopenia, anemia, hyperglycemia, and prolonged QTC, and 1 grade 4 neutropenia. Conclusions: Bendamustine, lenalidomide, and dexamethasone form a well-tolerated and highly active regimen even in heavily pretreated MM patients, with a PR rate of 67%. Additional updates on response and MTD will be available at the time of presentation. Disclosures: Lentzsch: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cephalon: Consultancy, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Bendamustine is not FDA approved for the treatment of multiple myeloma in the USA. Burt:Millennium: Honoraria; Celgene: Honoraria. Mapara:Resolvyx: Consultancy, Research Funding; Genzyme: Membership on an entity's Board of Directors or advisory committees; Gentium: Equity Ownership; Celgene: Spouse is consultant , has received research funding, and participates on advisory board; Cephalon: Spouse has received funding for clinical trial and participates on advisory board. Redner:Biogen: Equity Ownership; Wyeth: Equity Ownership; Glaxo-Smith-Kline: Equity Ownership; Pfizer: Equity Ownership; Genzyme: Membership on an entity's Board of Directors or advisory committees. Roodman:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; Acceleron: Consultancy. Zonder:Amgen: Consultancy; Pfizer: Consultancy; Cephalon: Consultancy; Millennium: Consultancy, Speaking (CME only); no promotional talks.

Description: High levels of granulocyte/macrophage–colony-stimulating factor (GM-CSF) autoantibodies are thought to cause pulmonary alveolar proteinosis (PAP), a rare syndrome characterized by myeloid dysfunction resulting in pulmonary surfactant accumulation and respiratory failure. Paradoxically, GM-CSF autoantibodies have been reported to occur rarely in healthy people and routinely in pharmaceutical intravenous immunoglobulin (IVIG) purified from serum pooled from healthy subjects. These findings suggest that either GM-CSF autoantibodies are normally present in healthy people at low levels that are difficult to detect or that serum pooled for IVIG purification may include asymptomatic persons with high levels of GM-CSF autoantibodies. Using several experimental approaches, GM-CSF autoantibodies were detected in all healthy subjects evaluated (n = 72) at low levels sufficient to rheostatically regulate multiple myeloid functions. Serum GM-CSF was more abundant than previously reported, but more than 99% was bound and neutralized by GM-CSF autoantibody. The critical threshold of GM-CSF autoantibodies associated with the development of PAP was determined. Results demonstrate that free serum GM-CSF is tightly maintained at low levels, identify a novel potential mechanism of innate immune regulation, help define the therapeutic window for potential clinical use of GM-CSF autoantibodies to treat inflammatory and autoimmune diseases, and have implications for the pathogenesis of PAP.

Description: Abstract 2717 Poster Board II-693 Interactions between chemokines and their receptors play an important role in cancer biology and have been shown to influence several processes such as tumor growth and the development of metastatic lesions. Stromal cell derived factor-1 (SDF-1) is a chemokine that binds to the CXCR4 chemokine receptor and stimulates B cell growth. CXCR4 is frequently over-expressed on tumor cells and the SDF-1/CXCR4 axis has been reported to play a role in promoting survival, angiogenesis and metastasis. The role of the stroma, in particular SDF-1/CXCR4 interactions, in providing a “survival niche” for tumor cells is well described for both hematological and solid tumors. We hypothesized that disruption of the protective microenvironment of tumor cells through antagonism of the SDF-1/CXCR4 axis may enhance the sensitivity of the tumor cells to treatment with anti-cancer agents such as monoclonal antibodies. Mozobil (AMD3100) or its structurally related analog, AMD3465, were used in combination with either Campath (anti-CD52) or Rituxan (anti-CD20) in lymphoma xenograft models to determine whether enhanced tumor protection could be observed. Flow cytometry analysis of lymphoma cell lines confirmed the cell surface expression of high levels of CXCR4 as well as the CD20 and CD52 target antigens. Lymphoma cells were seeded by intravenous injection and tumor-bearing mice were treated with multiple injections of the CXCR4 antagonists AMD3465 or AMD3100 alone or in combination with monoclonal antibodies. Treatment with AMD3465 as a single agent was sufficient to increase survival of the animals compared to untreated controls. In addition, the combination of AMD3465 and Campath showed a synergistic effect with a prolongation of survival greater than that obtained with either agent alone (p

Description: Abstract 43 We have previously shown that TNF Receptor 1 (TNFR1) levels early after myeloablative hematopoietic cell transplant (HCT), especially following unrelated donor (URD) HCT, predict later graft-versus-host disease (GVHD), non-relapse mortality (NRM), and overall survival (OS). Given this data we hypothesized that augmenting standard GVHD prophylaxis with the TNF-inhibitor etanercept would protect against GVHD and NRM while improving survival. We therefore conducted an IRB-approved Phase II trial at two centers to determine whether prophylactic TNF blockade with etanercept decreases the rates of acute GVHD and day 100 NRM seen after high-risk allogeneic matched (n=68, 71%) or single antigen mismatched URD (n=25, 26%) or single antigen mismatched related donor (RD) (n=3, 3%) HCT. Follow-up data are available for 96/100 patients (pts) enrolled between 2005 and 2009. HLA matching used high-resolution DNA-based techniques for HLA-A, B, C, and DR. Patients received tacrolimus, methotrexate (5 mg/m2 on days 1, 3, 6, 11) and etanercept (0.4 mg/kg, max 25 mg) twice weekly from the start of conditioning to day 56. All pts received myeloablative regimens; either TBI/cyclophosphamide (TBI, n=29), fludarabine and 12.8 mg/kg IV busulfan (FluBu4, n=31) or other myeloablative chemotherapy regimens, either BCNU/etoposide/cyclophosphamide or busulfan/cyclophosphamide (BCNU/Busulfan, n=36). The median age of the pts was 45y (range 2-61y). To determine whether the administration of etanercept effectively reduced post-transplant TNFR1 levels we compared day 7 post-transplant TNFR1 levels in the 96 study pts to 132 control pts who received standard GVHD prophylaxis of tacrolimus/methotrexate and were matched for age, conditioning regimen, degree of HLA-match, and disease status. The median day 7 TNFR1 levels in study pts was 2518 pg/ml, significantly lower than the median day 7 level of 3529 pg/ml in control pts (p

Description: Abstract 439 Introduction: Follicular lymphoma (FL) is the most common indolent lymphoid malignancy in North America with approximately 20,000 new cases of this incurable cancer diagnosed each year. In approximately 85% of patients, FL is associated with the reciprocal translocation t(14;18)(q32;q21), which results in a fusion between IGH and BCL2 genes and consequent over-expression of the anti-apoptotic protein BCL2. This translocation likely represents an initiating event for FL, requiring additional mutational events for the onset of clinical disease. To investigate the relationship between genome rearrangements and FL we identified rearrangement locations in the genome followed by detailed, fine-structure analysis of the rearrangements to ascertain their effects on genes and other features of biological interest. Patients and Methods: We used a whole-genome bacterial artificial chromosome (BAC) fingerprint-based approach, termed Fingerprint Profiling (FPP, Krzywinski, M. et al. 2007), to detect genome rearrangements relative to the reference human genome in neoplastic B cells purified from 24 FL patient biopsies. Analysis of 2,640,707 BAC fingerprints revealed 721 candidate genomic rearrangements. To validate these observations and provide base-pair resolution of the rearrangement breakpoints we performed paired-end massively parallel sequencing, on the Illumina Genome Analyzer II platform, of the breakpoint-containing regions captured in the BAC clones. Sequence reads were assembled into contigs using our in-house de novo assembly algorithm ABySS (Assembly By Short Sequences, Simpson, J. et al. 2009) then aligned to the reference human genome. Following manual annotation of the breakpoint junctions PCR primers were designed to assay patient tumour and matched constitutional DNA and thus determine whether the observed genome rearrangements were somatic (acquired) or germline in origin. Results: 727 BACs with apparent large-scale genome rearrangements, representing 354 distinct genome rearrangements across 20 patients, were sequenced in 95 pools, generating 72 Gbp of sequence. The 354 distinct events include 163 deletions, 71 inversions, 27 insertions, 83 translocations and 10 duplications, ranging in size from 3 kb to 67 Mb. PCR assays for 194 of the distinct events have been performed thus far identifying 80 distinct somatic and 114 germline-derived structural variations at base-pair resolution. Of the somatic events 5 are present in two or more of the 20 patients analyzed including a 720 kb inversion of 3q27.3 that results in expression of a BCL6-ST6GAL1 fusion transcript. Identification at base-pair resolution of breakpoint sequences enabled a detailed study of breakpoint and fusion mechanisms. We classified breakpoint junctions into 4 groups; those with microhomology (48%), those with sequence additions (28%), those with blunt fusions (20%) and those with flanking low copy repeats (4%). We were particularly interested in establishing the origin of the observed nucleotide sequence additions in 97 breakpoint junctions. The sequence additions ranged in size from a single nucleotide to 454 bp. In one case we have unambiguously mapped a 53 bp sequence, lying within one of the 3q27.3 inversion breakpoints, to chromosome 5q12.3. This finding is consistent with the recently proposed fork stalling and template switching (FoSTeS) DNA replication-based mechanism and thus represents a novel mechanism in FL lymphomagenesis. Conclusions: We have successfully employed high-throughput clone fingerprinting and sequencing to identify numerous novel somatic and germline genome rearrangements from FL primary tumour samples. Furthermore, base-pair resolution of rearrangement breakpoints provides mechanistic insights. With the complete inventory of somatic and germline events in hand we will be able to propose recurrent structurally altered genes in FL patients for validation in independent datasets and improve our understanding of FL biology. Pathway analyses to identify emerging themes from somatic mutations are also being performed. The PCR assays we have developed will also be of utility in identifying germline predisposition alleles in larger FL patient cohorts. Disclosures: No relevant conflicts of interest to declare.

Description: We conducted a double-blind, randomized multicenter trial to determine whether the addition of mycophenolate mofetil (MMF) improves the efficacy of initial systemic treatment of chronic graft-versus-host disease (GVHD). The primary endpoint was resolution of chronic GVHD and withdrawal of all systemic treatment within 2 years, without secondary treatment. Enrollment of 230 patients was planned, providing 90% power to observe a 20% difference in success rates between the 2 arms. The study was closed after 4 years because the interim estimated cumulative incidence of success for the primary endpoint was 23% among 74 patients in the MMF arm and 18% among 77 patients in the control arm, indicating a low probability of positive results for the primary endpoint after completing the study as originally planned. Analysis of secondary endpoints showed no evidence of benefit from adding MMF to the systemic regimen first used for treatment of chronic GVHD. The estimated hazard ratio of death was 1.99 (95% confidence interval, 0.9-4.3) among patients in the MMF arm compared with the control arm. MMF should not be added to the initial systemic treatment regimen for chronic GVHD. This trial was registered at www.clinicaltrials.gov as #NCT00089141 on August 4, 2004.

Description: The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications.

Description: Emerging, re-emerging and endemic plant pathogens continue to challege our ability to safeguard plant health worldwide. Further, globalization, climate change, increased human mobility, and pathogen and vector evolution have combined to increase the spread of invasive plant pathogens. Early and accurate diagnoses and pathogen surveillance on local, regional, and global scales are necessary to predict outbreaks and allow time for development and application of mitigation strategies. Plant disease diagnostic networks have developed worldwide to address the problems of efficient and effective disease diagnosis and pathogen detection, engendering cooperation of institutions and experts within countries and across national borders. Networking maximizes impact in the face of shrinking government investments in agriculture and diminishing human resource capacity in diagnostics and applied pathology. New technologies promise to improve the speed and accuracy of disease diagnostics and pathogen detection. Widespread adoption of standard operating procedures and diagnostic laboratory accreditation serve to build trust and confidence among institutions. Case studies of national, regional, and international diagnostic networks are presented.