Search

Purpose: Donor lymphocyte infusion (DLI) reliably induces durable remission in 75% to 80% of patients with relapsed chronic myelogenous leukemia (CML) following allogeneic bone marrow transplantation. We previously reported the identification of a high titer–specific immunoglobulin G response against two novel leukemia-associated antigens, CML28 and CML66, which correlated with immune-induced remission. The present studies characterize expression of CML28 and CML66 in primary hematopoietic tissues. Experimental Design: Specific monoclonal antibodies to CML28 and CML66 were developed and used to detect antigen expression in leukemia cell lines and primary leukemia tissue on Western blot and immunohistochemistry. Expression patterns were confirmed by antigen-specific real-time PCR. Results: Both CML28 and CML66 were highly expressed in leukemic blasts from patients with acute myelogenous leukemia and CML blast crisis but barely detectable in normal bone marrow, normal peripheral blood, or leukemic cells from patients with stable-phase CML. In contrast, purified CD34+ progenitors from normal individuals and patients with stable-phase CML expressed high levels of CML28 and CML66 transcript and protein. Immunohistochemical staining for CML66 confirmed rare staining of myeloid precursors in normal marrow and diffuse staining of myeloblastic cells in acute myelogenous leukemia and blast crisis CML marrows. Conclusions: The expression patterns of CML28 and CML66 are strikingly similar and suggest that antigen expression may play a role in shaping the post-DLI antibody repertoire. The CD34+ restricted pattern of expression of CML28 and CML66 is particularly relevant in light of the notion that DLI likely exerts its curative effect by targeting antigens present in self-renewing malignant progenitor populations in CML.

Primary mediastinal large B-cell lymphoma (MLBCL) shares important clinical and molecular features with classic Hodgkin lymphoma, including nuclear localization of the nuclear factor kappaB (NFkappaB) subunit c-REL (reticuloendotheliosis viral oncogene homolog) in a pilot series. Herein, we analyzed c-REL subcellular localization in additional primary MLBCLs and characterized NFkappaB activity and function in a MLBCL cell line. The new primary MLBCLs had prominent c-REL nuclear staining, and the MLBCL cell line exhibited high levels of NFkappaB binding activity. MLBCL cells expressing a superrepressor form of inhibitor of kappa B alpha signaling (IkappaB alpha) had a markedly higher rate of apoptosis, implicating constitutive NFkappaB activity in MLBCL cell survival. The transcriptional profiles of newly diagnosed primary MLBCLs and diffuse large B-cell lymphomas (DLBCLs) were then used to characterize the NFkappaB target gene signatures of MLBCL and specific DLBCL subtypes. MLBCLs expressed increased levels of NFkappaB targets that promote cell survival and favor antiapoptotic tumor necrosis factor alpha (TNFalpha) signaling. In contrast, activated B cell (ABC)-like DLBCLs had a more restricted, potentially developmentally regulated, NFkappaB target gene signature. Of interest, the newly characterized host response DLBCL subtype had a robust NFkappaB target gene signature that partially overlapped that of primary MLBCL. In this large series of primary MLBCLs and DLBCLs, NFkappaB activation was not associated with amplification of the cREL locus, suggesting alternative pathogenetic mechanisms.

We have created a stable transgenic rag2-EGFP-mMyc zebrafish line that develops GFP-labeled T cell acute lymphoblastic leukemia (T-ALL), allowing visualization of the onset and spread of this disease. Here, we show that leukemias from this transgenic line are highly penetrant and render animals moribund by 80.7 ± 17.6 days of life (±1 SD, range = 50-158 days). These T cell leukemias are clonally aneuploid, can be transplanted into irradiated recipient fish, and express the zebrafish orthologues of the human T-ALL oncogenes tal1 / scl and lmo2 , thus providing an animal model for the most prevalent molecular subgroup of human T-ALL. Because T-ALL develops very rapidly in rag2-EGFP-mMyc transgenic fish (in which “mMyc” represents mouse c-Myc), this line can only be maintained by in vitro fertilization. Thus, we have created a conditional transgene in which the EGFP-mMyc oncogene is preceded by a loxed dsRED2 gene and have generated stable rag2-loxP-dsRED2-loxP-EGFP-mMyc transgenic zebrafish lines, which have red fluorescent thymocytes and do not develop leukemia. Transgenic progeny from one of these lines can be induced to develop T-ALL by injecting Cre RNA into one-cell-stage embryos, demonstrating the utility of the Cre/lox system in the zebrafish and providing an essential step in preparing this model for chemical and genetic screens designed to identify modifiers of Myc-induced T-ALL.

The zebrafish is an attractive vertebrate model for genetic studies of development, apoptosis, and cancer. Here we describe a transgenic zebrafish line in which T- and B-lymphoid cells express a fusion transgene that encodes the zebrafish bcl-2 protein fused to the enhanced green fluorescence protein (EGFP). Targeting EGFP-bcl-2 to the developing thymocytes of transgenic fish resulted in a 2.5-fold increase in thymocyte numbers and a 1.8-fold increase in GFP-labeled B cells in the kidney marrow. Fluorescent microscopic analysis of living rag2-EGFP-bcl-2 transgenic fish showed that their thymocytes were resistant to irradiation- and dexamethasone-induced apoptosis, when compared with control rag2-GFP transgenic zebrafish. To test the ability of bcl-2 to block irradiation-induced apoptosis in malignant cells, we compared the responsiveness of Myc-induced leukemias with and without EGFP-bcl-2 expression in living transgenic zebrafish. T-cell leukemias induced by the rag2-EGFP-Myc transgene were ablated by irradiation, whereas leukemias in double transgenic fish expressing both Myc and EGFP-bcl-2 were resistant to irradiation-induced apoptotic cell death. The forward genetic capacity of the zebrafish model system and the ability to monitor GFP-positive thymocytes in vivo make this an ideal transgenic line for modifier screens designed to identify genetic mutations or small molecules that modify bcl-2-mediated antiapoptotic pathways. (Blood. 2005;105:3278-3285)

Human stem cell leukemia-lymphoma syndrome usually presents itself as a myeloproliferative disorder (MPD) that evolves to acute myeloid leukemia and/or lymphoma. The syndrome associated with t(8;13)(p11;q12) results in expression of the ZNF198-fibroblast growth factor receptor (FGFR) 1 fusion tyrosine kinase. Current empirically derived cytotoxic chemotherapy is inadequate for treatment of this disease. We hypothesized that small-molecule inhibitors of the ZNF198-FGFR1 fusion would have therapeutic efficacy. We characterized the transforming activity of ZNF198-FGFR1 in hematopoietic cells in vitro and in vivo . Expression of ZNF198-FGFR1 in primary murine hematopoietic cells caused a myeloproliferative syndrome in mice that recapitulated the human MPD phenotype. Transformation in these assays, and activation of the downstream effector molecules PLC-γ, STAT5, and phosphatidylinositol 3-kinase/AKT, required the proline-rich domains, but not the ZNF domains, of ZNF198. A small-molecule tyrosine kinase inhibitor, PKC412 ( N -benzoyl-staurosporine) effectively inhibited ZNF198-FGFR1 tyrosine kinase activity and activation of downstream effector pathways, and inhibited proliferation of ZNF198-FGFR1 transformed Ba/F3 cells. Furthermore, treatment with PKC412 resulted in statistically significant prolongation of survival in the murine model of ZNF198-FGFR1-induced MPD. Based in part on these data, PKC412 was administered to a patient with t(8;13)(p11;q12) and was efficacious in treatment of progressive myeloproliferative disorder with organomegaly. Therefore, PKC412 may be a useful therapy for treatment of human stem cell leukemia-lymphoma syndrome.

Chromosomal translocations that fuse the mixed lineage leukemia (MLL) gene with multiple partners typify acute leukemias of infancy as well as therapy-related leukemias. We utilized a conditional knockin strategy to bypass the embryonic lethality caused by MLL-CBP expression and to assess the immediate effects of induced MLL-CBP expression on hematopoiesis. Within days of activating MLL-CBP, the fusion protein selectively expanded granulocyte/macrophage progenitors (GMP) and enhanced their self-renewal/proliferation. MLL-CBP altered the gene expression program of GMP, upregulating a subset of genes including Hox a9. Inhibition of Hox a9 expression by RNA interference demonstrated that MLL-CBP required Hox a9 for its enhanced cell expansion. Following exposure to sublethal gamma-irradiation or N-ethyl-N-nitrosourea (ENU), MLL-CBP mice developed myelomonocytic hyperplasia and progressed to fatal myeloproliferative disorders. These represented the spectrum of therapy-induced acute myelomonocytic leukemia/chronic myelomonocytic leukemia/myelodysplastic/myeloproliferative disorder similar to that seen in humans possessing the t(11;16). This model of MLL-CBP therapy-related myeloproliferative disease demonstrates the selectivity of this MLL fusion for GMP cells and its ability to initiate leukemogenesis in conjunction with cooperating mutations.

To develop murine models of leukemogenesis, a series of transgenic mice expressing BCR-ABL in different hematopoietic cell subsets was generated. Here we describe targeted expression of P210 BCR-ABL in stem and progenitor cells of murine bone marrow using the tet-off system. The transactivator protein tTA was placed under the control of the murine stem cell leukemia (SCL) gene 3′ enhancer. Induction of BCR-ABL resulted in neutrophilia and leukocytosis, and the mice became moribund within 29 to 122 days. Autopsy of sick mice demonstrated splenomegaly, myeloid bone marrow hyperplasia, and extramedullary myeloid cell infiltration of multiple organs. BCR-ABL mRNA and protein were detectable in the affected organs. Fluorescence-activated cell sorter (FACS) analysis demonstrated a significant increase in mature and immature myeloid cells in bone marrow and spleen, together with increased bilineal B220+/Mac-1+ cells in the bone marrow. tTA mRNA was expressed in FACS-sorted hematopoietic stem cells expanded 26-fold after BCR-ABL induction. Thirty-one percent of the animals demonstrated a biphasic phenotype, consisting of neutrophilia and subsequent B-cell lymphoblastic disease, reminiscent of blast crisis. In summary, this mouse model recapitulates many characteristics of human chronic myeloid leukemia (CML) and may help elucidate basic leukemogenic mechanisms in CML stem cells during disease initiation and progression. (Blood. 2005;105:324-334)

Prostate tumor cells preferentially adhere to bone marrow endothelial cells (BMECs) compared with endothelial linings from other tissue microvessels, implicating the importance of BMEC adhesion in the predilection of prostate tumor metastasis to bone. E (endothelial)-selectin, which functions as an initiator of leukocyte adhesion to target tissue endothelium, is constitutively expressed on BMECs, suggesting that prostate tumor cells could use this adhesive mechanism to initiate their migration into bone. In this report, we demonstrate for the first time that human bone-metastatic prostate tumor cells roll on human BMECs under physiological flow conditions. We show that these dynamic adhesive interactions are dependent on the expression of BMEC E-selectin and sialylated glycoconjugates on bone-metastatic prostate tumor cells. We also establish the importance of both glycoprotein(s) and glycosphingolipid structures displaying sialyl Lewis X epitopes as potential E-selectin ligands on bone-metastatic prostate tumor cells. Coexpression of sialylated glycoproteins and glycolipids on bone-metastatic prostate tumor cells triggers robust E-selectin binding activity, which is identical to that observed on human hematopoietic progenitor cells. By Western blot analysis, we identify candidate E-selectin glycoprotein ligand(s); distinct sialyl Lewis X (or HECA-452 antigen)-bearing membrane proteins were resolved at Mr 130,000 and Mr 220,000 as well as others ranging from Mr 100,000 to Mr 220,000. Immunohistochemical analysis of HECA-452 antigen expression on normal prostate tissue and on low- and high-grade prostate adenocarcinoma shows that HECA-452 antigen expression is directly associated with prostate tumor progression and may indicate acquisition of E-selectin ligand expression. These findings provide novel insight into potential adhesive mechanisms promoting hematogenous dissemination of prostate tumor cells into bone.

HTm4 is a member of a newly defined family of human and murine proteins, the MS4 (membrane-spanning four) protein group, which has a distinctive four-transmembrane structure. MS4 protein functions include roles as cell surface signaling receptors and intracellular adapter proteins. We have previously demonstrated that HTm4 regulates the function of the KAP phosphatase, a key regulator of cell cycle progression. In humans, the expression of HTm4 is largely restricted to cells of the hematopoietic lineage, possibly reflecting a causal role for this molecule in differentiation/proliferation of hematopoietic lineage cells. In this study, we show that, like the human homologue, murine HTm4 is also predominantly a hematopoietic protein with distinctive expression patterns in developing murine embryos and in adult animals. In addition, we observed that murine HTm4 is highly expressed in the developing and adult murine nervous system, suggesting a previously unrecognized role in central and peripheral nervous system development.

During normal development Notch receptor signaling is important in regulating numerous cell fate decisions. Mutations that truncate the extracellular domain of Notch receptors can cause aberrant signaling and promote unregulated cell growth. We have examined two types of truncated Notch oncoproteins that arise from proviral insertion into the Notch4 gene (Notch4/int-3) or a chromosomal translocation involving the Notch1 gene (TAN-1). Both Notch4/int-3 and TAN-1 oncoproteins lack most or all of their ectodomain. Normal Notch signaling requires gamma-secretase/presenilin-mediated proteolytic processing, but whether Notch oncoproteins are also dependent on gamma-secretase/presenilin activity is not known. We demonstrate that Notch4/int-3-induced activation of the downstream transcription factor, CSL, is abrogated in cells deficient in presenilins or treated with a pharmacological inhibitor of gamma-secretase/presenilins. Furthermore, we find that both Notch4/int-3 and TAN-1 accumulate at the cell surface, where presenilin-dependent cleavage occurs, when gamma-secretase/presenilin activity is inhibited. gamma-Secretase/presenilin inhibition effectively blocks cellular responses to Notch4/int-3, but not TAN-1, apparently because some TAN-1 polypeptides lack transmembrane domains and do not require gamma-secretase/presenilin activity for nuclear access. These studies highlight potential uses and limitations of gamma-secretase/presenilin inhibitors in targeted therapy of Notch-related neoplasms.

Transgenic zebrafish that express GFP under control of the T cell-specific tyrosine kinase ( lck ) promoter were used to analyze critical aspects of the immune system, including patterns of T cell development and T cell homing after transplant. GFP-labeled T cells could be ablated in larvae by either irradiation or dexamethasone added to the water, illustrating that T cells have evolutionarily conserved responses to chemical and radiation ablation. In transplant experiments, thymocytes from lck-GFP fish repopulated the thymus of irradiated wild-type fish only transiently, suggesting that the thymus contains only short-term thymic repopulating cells. By contrast, whole kidney marrow permanently reconstituted the T lymphoid compartment of irradiated wild-type fish, suggesting that long-term thymic repopulating cells reside in the kidney.

Noonan syndrome is a common human autosomal dominant birth defect, characterized by short stature, facial abnormalities, heart defects and possibly increased risk of leukemia. Mutations of Ptpn11 (also known as Shp2), which encodes the protein-tyrosine phosphatase Shp2, occur in approximately 50% of individuals with Noonan syndrome, but their molecular, cellular and developmental effects, and the relationship between Noonan syndrome and leukemia, are unclear. We generated mice expressing the Noonan syndrome-associated mutant D61G. When homozygous, the D61G mutant is embryonic lethal, whereas heterozygotes have decreased viability. Surviving Ptpn11(D61G/+) embryos ( approximately 50%) have short stature, craniofacial abnormalities similar to those in Noonan syndrome, and myeloproliferative disease. Severely affected Ptpn11(D61G/+) embryos ( approximately 50%) have multiple cardiac defects similar to those in mice lacking the Ras-GAP protein neurofibromin. Their endocardial cushions have increased Erk activation, but Erk hyperactivation is cell and pathway specific. Our results clarify the relationship between Noonan syndrome and leukemia and show that a single Ptpn11 gain-of-function mutation evokes all major features of Noonan syndrome by acting on multiple developmental lineages in a gene dosage-dependent and pathway-selective manner.

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with epithelial cell and B-cell malignancies. EBV infection of B lymphocytes is essential for acute and persistent EBV infection in humans; however, the role of epithelial cell infection in the normal EBV life cycle remains controversial. The rhesus lymphocryptovirus (LCV) is an EBV-related herpesvirus that naturally infects rhesus macaques and can be used experimentally to model persistent B-cell infection and B-cell lymphomagenesis. We now show that the rhesus LCV can infect epithelial cells in immunosuppressed rhesus macaques and can induce epithelial cell lesions resembling oral hairy leukoplakia in AIDS patients. Electron microscopy, immunohistochemistry, and DNA-RNA in situ hybridization were used to identify the presence of a lytic rhesus LCV infection in these proliferative, hyperkeratotic, or parakeratotic epithelial cell lesions. These studies demonstrate that the rhesus LCV has tropism for epithelial cells, in addition to B cells, and is a relevant animal model system for studying the role of epithelial cell infection in EBV pathogenesis.

Transcription factors are believed to have a dominant role in acute myeloid leukemia (AML). This idea is supported by analysis of gene-knockout mice, which uncovered crucial roles of several transcription factors in normal hematopoiesis, and of individuals with leukemia, in whom transcription factors are frequently downregulated or mutated. However, analysis of knockout animals has not shown a direct link between abrogated transcription factors and the pathogenesis of AML. Sfpi1, encoding the lineage-specific transcription factor PU.1, is indispensable for normal myeloid and lymphoid development. We found that mice carrying hypomorphic Sfpi1 alleles that reduce PU.1 expression to 20% of normal levels, unlike mice carrying homo- or heterozygous deletions of Sfpi1, developed AML. Unlike complete or 50% loss, 80% loss of PU.1 induced a precancerous state characterized by accumulation of an abnormal precursor pool retaining responsiveness to G-CSF with disruption of M- and GM-CSF pathways. Malignant transformation was associated with a high frequency of clonal chromosomal changes. Retroviral restoration of PU.1 expression rescued myeloid differentiation of mutant progenitors and AML blasts. These results suggest that tightly graded reduction, rather than complete loss, of a lineage-indispensable transcription factor can induce AML.

Splenic vascular tumors are uncommon and are more typically encountered as benign incidental findings. By contrast, splenic angiosarcoma may present acutely and dramatically and typically pursues a very aggressive clinical course. Vascular tumors in the spleen may show conventional endothelial, specialized endothelial (sinusoidal/littoral cell) or lymphatic differentiation and there is morphologic overlap between some of the currently defined diagnostic categories, within which benign, intermediate, and malignant subsets are recognized. The greatest problem in trying to better define and analyze these tumors is the availability of only relatively small case numbers. This overview describes the diagnosis and differential diagnosis of splenic vascular tumors as presently understood.

The proapoptotic activity of the “BH3-only” molecule BAD can be differentially regulated by survival factor signaling. Bad -deficient mice lacking both BAD long and BAD short proteins proved viable, and most cell types appeared to develop normally. BAD did not exclusively account for cell death after withdrawal of survival factors, but it was an intermediate for epidermal growth factor- or insulin-like growth factor I-countered apoptosis, consistent with a “sensitizing” BH3-only molecule. Lymphocytes developed normally with no premalignant hyperplasia, but they displayed subtle abnormalities in proliferation and IgG production. Despite the minimal phenotype, Bad -deficient mice progressed, with aging, to diffuse large B cell lymphoma of germinal center origin. Exposure of Bad -null mice to sublethal γ-irradiation resulted in an increased incidence of pre-T cell and pro-/pre-B cell lymphoblastic leukemia/lymphoma. Thus, proapoptotic BAD suppresses tumorigenesis in the lymphocyte lineage.

Langerhans cell histiocytosis (LCH) is a neoplastic proliferation of Langerhans cells that occurs in a range of nodal and extranodal sites. Scattered reports of LCH within the thymus exist, typically among children within the setting of multifocal, multisystem disease. Rare cases of isolated LCH involving the thymus have occurred in adult patients with myasthenia gravis. We report a case of unifocal LCH involving the thymus in a middle-aged woman with a history of a resected leiomyosarcoma but no evidence of myasthenia gravis. Computed tomographic scans revealed an anterior mediastinal mass, which was excised and measured 9.0 cm. Histologic and immunophenotypic findings (CD1a, S100, and Fascin positive and CD68 negative) were consistent with LCH. To our knowledge, this is the first example of LCH occurring in a patient with a history of soft tissue sarcoma and one of the rare reported examples of LCH presenting as a large isolated lesion in the thymus of a nonmyasthenic adult.

FIP1L1-PDGFRalpha causes hypereosinophilic syndrome (HES) and is inhibited by the tyrosine kinase inhibitor imatinib (Gleevec). Imatinib is a potent inhibitor of ABL, ARG, PDGFRalpha, PDGFRbeta, and KIT and induces durable hematologic responses in HES patients. However, we observed relapse with resistance to imatinib as consequence of a T674I mutation in FIP1L1-PDGFRalpha, analogous to the imatinib-resistant T315I mutation in BCR-ABL. We developed a murine bone marrow transplant model of FIP1L1-PDGFRalpha-induced myeloproliferative disease to evaluate the efficacy of PKC412, an alternative inhibitor of PDGFRalpha, for the treatment of HES. PKC412 is effective for treatment of FIP1L1-PDGFRalpha-induced disease and of imatinib-induced resistance due to the T674I mutation. Our data establish PKC412 as molecularly targeted therapy for HES and other diseases expressing activated PDGFRalpha and demonstrate the potential of alternative kinase inhibitors to overcome resistance in target tyrosine kinases. ispartof: Cancer cell vol:3 issue:5 pages:459-69 ispartof: location:United States status: published

The identification of antigens associated with tumor destruction is a major goal of cancer immunology. Vaccination with irradiated tumor cells engineered to secrete granulocyte–macrophage colony stimulating factor generates potent, specific, and long-lasting antitumor immunity through improved tumor antigen presentation by dendritic cells and macrophages. A phase I clinical trial of this immunization strategy in patients with disseminated melanoma revealed the consistent induction in distant metastases of dense T and B cell infiltrates that effectuated substantial tumor necrosis and fibrosis. To delineate the target antigens of this vaccine-stimulated tumor destruction, we screened a melanoma cDNA expression library with postimmunization sera from a long-term responding patient (K030). High-titer IgG antibodies recognized melanoma inhibitor of apoptosis protein (ML-IAP), a caspase antagonist containing a single baculoviral IAP repeat and a COOH-terminal RING domain. Although K030 harbored antibodies to ML-IAP at the time of study entry, multiple courses of vaccination over 4 years increased antibody titers and elicited isotype switching. Moreover, lymphocyte infiltrates in necrotic metastases included CD4 + and CD8 + T cells specific for ML-IAP, as revealed by proliferation, tetramer, enzyme-linked immunospot, and cytotoxicity analysis. Whereas melanoma cells in densely infiltrated lesions showed strong ML-IAP expression by immunohistochemistry, lethal disease progression was associated with the loss of ML-IAP staining and the absence of lymphocyte infiltrates. These findings demonstrate that ML-IAP can serve as a target for immune-mediated tumor destruction, but that antigen-loss variants can accomplish immune escape.

The zebrafish is an attractive model organism for studying cancer development because of its genetic accessibility. Here we describe the induction of clonally derived T cell acute lymphoblastic leukemia in transgenic zebrafish expressing mouse c- myc under control of the zebrafish Rag2 promoter. Visualization of leukemic cells expressing a chimeric transgene encoding Myc fused to green fluorescent protein (GFP) revealed that leukemias arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into skeletal muscle and abdominal organs. Leukemic cells homed back to the thymus in irradiated fish transplanted with GFP-labeled leukemic lymphoblasts. This transgenic model provides a platform for drug screens and for genetic screens aimed at identifying mutations that suppress or enhance c- myc – induced carcinogenesis.

ABSTRACT: Anaplastic large-cell lymphoma (ALCL) provides an excellent example of how molecular insights into tumor pathogenesis are influencing and improving tumor classification. ALCL was described initially as a subtype of T-cell/null-cell lymphoma characterized by unusual tumor cell morphology and the expression of CD30. However, it was soon recognized that a subset of ALCLs contained chromosomal translocations involving anaplastic lymphoma kinase (ALK), a novel receptor tyrosine kinase gene. These rearrangements create chimeric genes encoding self-associating, constitutively active ALK fusion proteins that activate a number of downstream effectors, including phospholipase C-gamma, phosphoinositol 3'-kinase, RAS, and signal transducer and activator of transcription proteins, all of which seem potentially important in cellular transformation. Not all tumors classified as ALCLs have ALK rearrangements and, conversely, ALK rearrangements occur in lymphomas of widely varying morphology. Hence, only molecular markers can reliably identify ALK+ ALCL. The importance of doing so is reflected by clinical studies suggesting that ALK+ ALCLs have a significantly better prognosis than other aggressive peripheral T-cell or B-cell lymphomas, including ALK− ALCLs. The unique molecular pathogenesis of ALK+ ALCL is likely to lead to novel therapeutic approaches directed at specific inhibition of ALK or downstream effectors.

We examined the diagnostic utility of fine-needle aspiration (FNA) in the immunosuppressed patient with particular attention to the value of in situ hybridization for Epstein-Barr virus encoded RNA (EBER), a test frequently performed on tissue sections in this setting. Six patients with adenopathy were identified: three with prior solid organ transplantation, two with prior bone marrow transplantation, and one with human immunodeficiency virus. Cytologic findings in involved tissues were positive for non-Hodgkin's lymphoma in one case, suspicious or atypical in three cases, negative/reactive in one case, and nondiagnostic in one case due to insufficient material. Two aspirates had a polymorphous lymphoid appearance; three showed predominantly monomorphous lymphoid morphology. Retrospectively, 4/4 FNA cell blocks were positive for EBER in a large proportion of atypical lymphoid cells. Together with the cytologic findings, EBER detection allows for more specific classification of these FNA samples as EBV-associated lymphoid proliferations.

Geminin interacts with a DNA replication initiation factor, Cdt1p, to suppress initiation of DNA replication in a Xenopus egg extract based cell-free system, leading to the expectation that the protein acts as an inhibitor of cell proliferation. Immunohistochemistry and immunoblotting for geminin, however, reveals that the protein is expressed specifically in proliferating lymphocytes and epithelial cells. This pattern is in contrast to the expression of a bona fide cell cycle inhibitor like p21/WAF1 that is specifically expressed in quiescent cells. Geminin is widely expressed in several malignancies and the number of geminin-expressing cells is directly proportional to the cell proliferation index as measured by Ki-67 expression. Therefore, instead of being a suppressor of cell proliferation, geminin expression is positively correlated with cell proliferation. Consistent with this observation, transient overexpression of wild-type geminin in cancer cells in culture did not produce a cell cycle block. A point mutation in the destruction box of geminin, however, results in a protein that is stabilized in G(1) and capable of arresting cells at the G(1)-S transition.

Constitutive activation of tyrosine kinases, such as the BCR/ABL fusion associated with t(9;22)(q34;q22), is a hallmark of chronic myeloid leukemia (CML) syndromes in humans. Expression of BCR/ABL is both necessary and sufficient to cause a chronic myeloproliferative syndrome in murine bone marrow transplantation models, and absolutely depends on kinase activity. Progression of CML to acute leukemia (blast crisis) in humans has been associated with acquisition of secondary chromosomal translocations, including the t(7;11)(p15;p15) resulting in the NUP98/HOXA9 fusion protein. We demonstrate that BCR/ABL cooperates with NUP98/HOXA9 to cause blast crisis in a murine model. The phenotype depends both on expression of BCR/ABL and NUP98/HOXA9, but tumors retain sensitivity to the ABL inhibitor STI571 in vitro and in vivo . This paradigm is applicable to other constitutively activated tyrosine kinases such as TEL/PDGFβR. These experiments document cooperative effects between constitutively activated tyrosine kinases, which confer proliferative and survival properties to hematopoietic cells, with mutations that impair differentiation, such as the NUP98/HOXA9, giving rise to the acute myeloid leukemia (AML) phenotype. Furthermore, these data indicate that despite acquisition of additional mutations, CML blast crisis cells retain their dependence on BCR/ABL for proliferation and survival.

Proliferative states such as chronic inflammation, ischemic diseases, and cancer are often accompanied by intense angiogenesis, a highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, and maturation. Aspirin-triggered lipoxins (ATLs), the 15R enantiomeric counterparts of lipoxins (LXs), are endogenous mediators generated during multicellular responses that display potent immunomodulatory actions. Herein, we report a novel action for the ATL stable analog 15-epi-16-(para-fluoro)-phenoxy-lipoxin A(4) (denoted ATL-1), which proved to be a potent inhibitor of angiogenesis. This ATL inhibited endothelial cell proliferation in the 1 to 10 nM range by approximately 50% in cells stimulated with either vascular endothelial growth factor (VEGF) at 3 ng/ml or leukotriene D(4) at 10 nM. In addition, ATL-1 (in a 10-100 nM range) inhibited VEGF (3 ng/ml)-induced endothelial cell chemotaxis. In a granuloma in vivo model of inflammatory angiogenesis, ATL-1 treatment (10 microg/mouse) reduced by approximately 50% the angiogenic phenotype, as assessed by both vascular casting and fluorescence. Together, these results identify a novel and potent previously unappreciated action of aspirin-triggered 15-epi-LX.

Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy in adults, is curable in less than 50% of patients. Prognostic models based on pre-treatment characteristics, such as the International Prognostic Index (IPI), are currently used to predict outcome in DLBCL. However, clinical outcome models identify neither the molecular basis of clinical heterogeneity, nor specific therapeutic targets. We analyzed the expression of 6,817 genes in diagnostic tumor specimens from DLBCL patients who received cyclophosphamide, adriamycin, vincristine and prednisone (CHOP)-based chemotherapy, and applied a supervised learning prediction method to identify cured versus fatal or refractory disease. The algorithm classified two categories of pa- tients with very different five-year overall survival rates (70% versus 12%). The model also ef- fectively delineated patients within specific IPI risk categories who were likely to be cured or to die of their disease. Genes implicated in DLBCL outcome included some that regulate responses to B-cell-receptor signaling, critical serine/threonine phosphorylation pathways and apoptosis. Our data indicate that supervised learning classification techniques can predict outcome in DLBCL and identify rational targets for intervention. © 2002 Nature Publishing Group http://medicine.nature.com

Inflammatory pseudotumors (IPTs) of the lymph node and spleen are an uncommon, benign cause of lymphadenopathy and/or splenomegaly that often bear striking clinicopathologic similarities to the inflammatory myofibroblastic tumors (IMTs) found in soft tissues. These tumors have classically been grouped together under the umbrella category of "inflammatory pseudotumor." Recent evidence shows that IMTs are in fact neoplastic processes that often harbor balanced chromosomal translocations involving the ALK kinase gene. These translocations result in expression of ALK kinase in IMTs as assessed by immunohistochemical studies. However, the relationship between IMT and IPT of the lymph node and spleen is uncertain. To determine if ALK tyrosine kinase expression is also present in IPT, 13 cases of IPT (9 involving lymph nodes, 4 splenic lesions) were examined for the presence of ALK tyrosine kinase by immunohistochemical staining on paraffin-embedded tissue. In addition, in situ hybridization studies for Epstein-Barr virus--encoded RNAs (EBER) and immunoperoxidase studies for human herpesvirus-8 (HHV8)--specific proteins were performed. All cases had clinical, morphologic, and immunophenotypic findings typical of IPT and had varying proportions of fibroblastic and inflammatory components. Age ranged from 11 to 75 (median, 40) years; 8 subjects were male, and 5 were female. None of the cases (0 of 13) had positive staining for ALK kinase or HHV8, and in 1 a lymph node (1 of 13) was focally positive for EBV (EBER) by in situ hybridization. The absence of ALK kinase as detected by immunohistochemical studies in IPT of the lymph node and spleen suggests that this entity is biologically distinct from the histologically similar IMT.

Peptides containing sequences derived from the new NH2 terminus of the seven-transmembrane domain thrombin receptor after thrombin cleavage can activate platelets directly. We recently demonstrated that such peptides are readily cleaved and inactivated by plasma, serum, and endothelial cell-associated aminopeptidase M. The rapid degradation and inactivation of the peptides makes it difficult to assess dose-response relationships precisely or to conduct long term incubations with cells in the presence of plasma or serum. To overcome these problems, we first substituted D-serine for the NH2-terminal L-serine in an active peptide ligand (SFLLRNPNDKY). The D-serine derivative resisted degradation in plasma, but it is less than 1% as active as the L-serine peptide. Substituting a racemic mixture of the beta-amino acid isoserine for serine in a related peptide ligand (SFLLRN) produced an active peptide ((iso-S)FLLRN) that is approximately 15-20% as potent as SFLLRN as judged by platelet aggregation. To assess the stability of the peptides in plasma, SFLLRN (1 mM) was incubated with 50% plasma for various periods of time; after 15 min, 65% of the peptide remained intact, and after 2 h only 4% remained intact. Loss of aggregating activity paralleled the loss of intact peptide. In contrast, even after 2 h of incubation with plasma, 83% of the (iso-S)FLLRN remained intact and the aggregating activity was essentially unchanged. Qualitative differences in the patterns of platelet aggregation produced by the peptides were also observed. Thus, the distinct pattern of aggregation followed by rapid disaggregation observed with submaximal aggregating doses of SFLLRN was less evident with (iso-S)FLLRN, and inhibition of aminopeptidase M by amastatin enhanced aggregation in platelet-rich plasma induced by SFLLRN but not (iso-S)FLLRN. The (iso-S)FLLRN peptide should permit improved analysis of the effects of constant levels of peptide-induced thrombin receptor stimulation in the presence of plasma or serum, or when testing the effects of the peptide on cells that contain surface-associated aminopeptidase M.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). Despite aggressive chemoimmunotherapy, ∼40% of patients die of their disease. Molecular differences in the tumor genome are likely to be major contributors to the heterogeneity of clinical response. Several investigators have identified molecular subtypes, including a “cell-of-origin” (COO) signature with activated B-cell-like (ABC) and germinal center B-cell-like (GCB) sub-types (Alizadeh, et al. Nature 2000), and a “consensus clustering” (CC) signature with oxidative phosphorylation, B-cell receptor (BCR)/proliferation, and host response sub-types (Monti, et al. 2005). Phosphoinositide-3 kinases (PI3Ks) are key cellular signaling proteins that act as a central node, relaying signals from cell surface receptors to downstream mediators such as AKT. PI3K-δ and PI3K-γ isoforms are preferentially expressed in normal and malignant leukocytes where they are critical for cell differentiation, migration, and proliferation. IPI-145 is a potent, oral PI3K-δ,γ inhibitor that has shown clinical activity in the Phase 1 trial (IPI-145-02) in patients with advanced hematologic malignancies (ClinicalTrials.gov NCT01476657). Activation of the PI3K pathway is an important component of normal BCR signaling and has been implicated in the pathogenesis of DLBCL. To further explore the role of PI3K signaling in DLBCL, a panel of more than 10 DLBCL cell lines of varying molecular profile was treated with IPI-145. PI3K-δ and PI3K-γ were expressed at varying levels across the DLBCL cell lines, without evidence of a correlation with molecular subtype. In a cellular growth inhibition assay, 5 cell lines including 4 GCB (SU-DHL-4, SU-DHL-6, OCI-LY-8, and WSU-DLCL-2) and 1 ABC (Ri-1) subtype were sensitive to IPI-145 treatment in the micromolar (μM) range or below, and 5 cell lines (OCI-LY-3, OCI-LY-7, Pfeiffer, Toledo and U2932) were insensitive to IPI-145 (IC50 >25μM). There was no evidence of a correlation between IPI-145 sensitivity and COO or CC molecular profile in this panel. IPI-145 sensitivity did correlate with evidence of PI3K pathway inhibition as measured by reduction in phospho (p)-AKT. To characterize the kinetics of pathway modulation, phosphorylation of AKT, PRAS40, and S6 was examined following a time-course of IPI-145 treatment in selected cell lines. P-AKT and p-PRAS40 were modulated by 30 minutes, whereas modulation of p-S6 was not detected until after 8 hours. Upon BCR stimulation via antibody-induced crosslinking, some cell lines exhibited enhanced AKT phosphorylation, which was inhibited with IPI-145. The GCB cell line OCI-LY-8 was moderately sensitive to IPI-145 without BCR crosslinking (low μM range) and exhibited enhanced sensitivity to IPI-145 with BCR crosslinking (sub-μM range). These results suggest that intact BCR pathway signaling contributes to IPI-145 sensitivity in DLBCL cell lines. Current efforts are focusing on identification of biomarkers that define IPI-145 responsive DLBCL subsets. IPI-145 activity was also explored in combination with ibrutinib, an irreversible inhibitor of Bruton agammaglobulinemia tyrosine kinase (BTK). Interestingly, in the setting of BCR crosslinking, OCI-LY-8 cells exhibited a robust increase in p-AKT which was completely inhibited by IPI-145 but only partially inhibited by ibrutinib. In other cell lines, such as SU-DHL-4, robust inhibition of p-BTK was observed with ibrutinib treatment but not with IPI-145. These findings suggest a potential mechanistic rationale for combination of PI3K-δ,γ and BTK inhibition. Supporting this hypothesis, a combination effect was observed in a cellular growth inhibition assay with IPI-145 plus ibrutinib in the SU-DHL-4 cell line and in the OCI-LY-8 cell line with BCR crosslinking. Studies are ongoing to explore this combination in additional DLBCL cell lines as well as in in vivo DLBCL models. Taken together, these findings suggest that a subset of DLBCL patients might benefit fromPI3K-δ,γ inhibition. Additional work is necessary to determine how to prospectively identify IPI-145-sensitive DLBCL. In addition, preliminary evidence suggests that there may be opportunities to improve targeted therapy options for DLBCL patients with the combination of IPI-145 and ibrutinib. Disclosures: Campbell: Infinity Pharmaceuticals, Inc.: Employment. Thompson:Infinity Pharmaceuticals, Inc.: Employment. Villegas:Infinity Pharmaceuticals, Inc.: Employment. Proctor:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment. Stern:Infinity Pharmaceuticals, Inc.: Employment.

Phosphoinositide-3 kinases (PI3Ks) are key cellular signaling proteins that act as a central node for relaying signals from cell surface receptors to downstream mediators, such as AKT. The PI3K-δ and PI3K-γ isoforms are preferentially expressed in normal and malignant leukocytes where they play critical roles in cell differentiation, migration, and proliferation. Constitutive activity of the PI3K pathway is common in T-cell acute lymphoblastic leukemia (T-ALL) and frequently involves the deletion of PTEN, the phosphatase that negatively regulates the PI3K pathway. An important role for the PI3K-δ and PI3K-γ isoforms has been demonstrated in the Pten-deleted genetically engineered murine model of T-ALL in conjunction with PI3K-δ and/or PI3K-γ gene knock outs (Subramanian et al, Cancer Cell, 2012). IPI-145 is a potent inhibitor of PI3K-δ and PI3K-γ currently being studied in a Phase 1 trial (IPI-145-02) in patients with advanced hematologic malignancies, including T-ALL (ClinicalTrials.gov NCT01476657). We performed in vitro studies to address the sensitivity of human and murine T-ALL cell lines to IPI-145 and to additional PI3K inhibitors with defined isoform selectivity. The human T-ALL cells examined were from cell lines that lack PTEN protein expression (Loucy, MOLT-4, CCRF-CEM, CEM/C2, p12 Ichikawa, and Karpas-45) and cell lines that express PTEN protein (MOLT13 and MOLT16). In addition, two murine cell lines derived from a Pten-deleted model of T-ALL (LPN049 and LPN236) were studied. The expression levels of class I PI3K isoforms were determined by western blotting and quantitative RT-PCR, which revealed varying levels of protein and RNA expression across the cell lines. In vitro treatment of human T-ALL cells with IPI-145 resulted in variable degrees of growth inhibition, with the PTEN-deficient Loucy cell line demonstrating the greatest sensitivity with an IC50 of 245 nM. In the cell lines tested, growth inhibition to IPI-145 was only seen in PTEN-deficient human cell lines, whereas all PTEN-expressing human T-ALL cell lines were resistant to IPI-145 (IC50 > 10 uM). However, not all PTEN-deficient human T-ALL cells demonstrated sensitivity to IPI-145 (e.g., CEM/C2), indicating that loss of PTEN does not confer sensitivity to PI3K inhibition in all settings. Inhibition of phospho-AKT (pAKT) correlated with growth inhibition, with an IC50 of 286 nM in the Loucy cell line. Studies to evaluate the mechanism of growth inhibition revealed that IPI-145 treatment resulted in apoptosis of sensitive cells as measured by 7-AAD and Annexin V staining. Cell lines derived from the Pten-deleted murine T-ALL model were also sensitive to IPI-145 with IC50s in the 300-600 nM range as measured by MTT assay. In addition, IPI-145 led to apoptosis, as measured by cleaved Caspase 3 and 7-AAD/Annexin V. Interestingly, Pten-deleted murine T-ALL cell lines showed down-regulation of pAKT and c-MYC expression with IPI-145 in a dose responsive manner that corresponded with increasing activated Caspase-3 expression. In NOTCH1-expressing murine T-ALL cell lines, down regulation of NOTCH1 and activated NOTCH1 was also observed in parallel with c-MYC down-regulation. To explore further the individual contributions of the varying PI3K isoforms on T-ALL cell growth, the effect of IPI-145 on tumor cell growth was compared with PI3K-δ, PI3K-δ,γ, and PI3K-β selective compounds in the Loucy PTEN-deficient T-ALL cell line. These experiments support an anti-leukemic effect for both PI3K-δ and PI3K-γ inhibition and indicate that the greatest effect is seen with combined PI3K-δ and PI3K-γ inhibition. A role for PI3K-β in T-ALL cell survival was not observed. Evaluation of the in vivo activity of IPI-145 on Loucy xenografts, as well as PTEN-expressing MOLT-13 xenografts, is ongoing. Together, these data provide a strong rationale for combined targeted inhibition of PI3K-δ and PI3K-γ in T-ALL. Disclosures: Huang: Infinity Pharmaceuticals, Inc.: Research Funding. Proctor:Infinity Pharmaceuticals, Inc.: Employment. Yang:Infinity Pharmaceuticals, Inc.: Research Funding. Gao:Infinity Pharmaceuticals, Inc.: Research Funding. Zhang:Infinity Pharmaceuticals, Inc.: Research Funding. Huang:Infinity Pharmaceuticals, Inc.: Research Funding. Changelian:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. You:Infinity Pharmaceuticals, Inc.: Research Funding.

The role of inhibitor of kappa Bα (IKBA) mutations in lymphoid malignancies with constitutive NF-κB signaling remains to be defined. We recently characterized the molecular signatures of primary mediastinal large B-cell lymphoma (MLBCL) and 3 subtypes of diffuse large B-cell lymphoma (DLBCL).[1][1

Abstract 1441 Introduction: Hedgehog (Hh) signaling is essential in many developmental pathways and a growing body of evidence supports a role for aberrant activation of the Hh pathway in a variety of hematological malignancies including Acute Myeloid Leukemia (AML). Primary CD34+ myeloid blasts and cell lines preferentially demonstrate active Hh signaling suggesting that the Hh pathway may be involved in leukemic stem cell maintenance and survival (Bai et al. Leukemia. 2008;22(1):226-8). In addition, disruption of Hh signaling by targeted agents in this setting results in cell death and confers sensitivity to Ara-C in chemoresistant CD34+ leukemic cell lines (Kobune et al. Cancer Science. 2009;100(5):948-55). These findings provide a rationale for targeted disruption of the Hh pathway in AML and imply that the identification of Hh signaling in AML could correlate with disease that is refractory to standard induction therapy. Methods: To extend these previous findings, the presence of Hh signaling was assessed by the identification of nuclear GLI1 by immunofluorescence in tissue microarrays (TMA) prepared from a large cohort of primary human AMLs and the data correlated with clinical outcome. Presentation bone marrow trephine samples from patients diagnosed with AML between 1994 and 2005 were retrieved from the histopathology archives at Manchester Royal Infirmary. These biopsy samples were routinely processed, formalin-fixed, paraffin-embedded, and EDTA-decalcified. All material used for preparation of the TMA was residual diagnostic tissue, anonymized and consented for its use in research. Each TMA core contained at least 20% leukemic blasts and was representative of the whole biopsy sample. A specific and highly sensitive human GLI1 immunofluorescence assay was developed to visualize nuclear GLI1in four micron thick TMA sections. Human embryonic palatal mesenchymal (HEPM) cells stimulated with SHH and treated with the Hh antagonist saridegib (IPI-926) were used as staining controls to demonstrate assay specificity. To determine the cellular context of GLI1 staining in AML, a GLI1/CD34 double immunoflourescence assay was also developed. All samples were scanned in using the TissueGnostics TissueFAXS 2.0 system. The Cy5 channel was used to detect nuclear GLI1 and the Texas Red channel was used to detect CD34 when double staining was employed. A nuclear algorithm was developed to analyze the GLI1 staining using the TissueQuest version 2.0 software. Results: Of the 160 patient samples stained, 71 samples were evaluable after filtering on the basis of the presence of adequate tissue cores on the TMA and a full set of clinical follow-up data. All patients (36 male, 34 female, 1 unknown; age range 17 to 83 years at diagnosis with a median age of 53 years) received intensive chemotherapy according to standard UK MRC AML protocols and had follow-up data for up to 5124 days. Nuclear GLI1 as a function of total marrow cells showed a wide range of expression (0.0–32.0%) with a mean of 4.8% and a median of 3.1%. In a subset of cases with CD34 positive blasts, nuclear GLI1 co-expression could be readily identified as coexpressed in the CD34 positive cells. The degree of GLI1 expression did not correlate with AML subtype or cytogenetics. Correlation of GLI1 positivity with clinical features was performed and while no association with overall survival was observed in the total population, there was a significant decrease in overall survival in patients with secondary AML that had GLI1 levels above the median (HR = 0.18; p value = 0.0112). In addition, there was a significant correlation between GLI1 either above or below the median and remission status (i.e. complete remission, partial remission or refractory disease) with higher GLI1 levels observed in patients with refractory disease (p value = 0.045) and a more significant correlation with GLI1 values as a continuous variable and remission status (p value = 0.026). Conclusions: These data suggest that high levels of GLI1 predict for poor remission status in patients with AML and poor overall survival in patients with secondary AML. The presence of high levels of nuclear GLI1 in AMLs that are refractory to chemotherapy supports a role for Hh signaling in chemoresistance, particularly in patients with secondary AML, and provides a rationale for targeted inhibition of the Hh pathway in selected AML patients. Disclosures: Campbell: Infinity Pharmaceuticals, Inc.: Employment. Quigley:Infinity Pharmaceuticals, Inc.: Employment. Keilty:Infinity Pharmaceuticals, Inc.: Employment. Kelly:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. Read:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment.

Abstract 1534 + equal contributions Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate and infrequent somatic mutations of TP53 and genes encoding cell cycle pathway components. Despite recent advances in the molecular understanding of DLBCL pathogenesis, clinical models such as the International Prognostic Index (IPI) are still used to identify high-risk patients. By integrating high-resolution high-density SNP array data with transcriptional profiles and performing pathway analysis in 180 primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary DLBCLs with single copy loss of 17p13.1 (TP53/RPL26/KDM6B) often had CNAs of an additional p53 modifier – 9p21.3 (CDKN2A/ARF), 19q13.42 (BCL2L12), 12q15 (MDM2) or 1q23.3 (MDM4/RFWD2). CNAs of the respective p53 modifiers, CDKN2A (ARF, 9p21.3), MDM2 (12q15) and MDM4/RFWD2 (1q23.3), occurred in largely separate groups of tumors. DLBCLs with CNAs of p53 pathway members frequently exhibited concurrent alterations of additional cell cycle components such as CCND3 (6p21.32), CDK6 (7q22.1), CDK2/CDK4 (12q15) and/or RB1 (13q14.2) or RBL2 (16q12.2). When the primary DLBCLs were clustered in the space of CNAs that perturb p53 pathway and cell cycle components, 66% of tumors had multiple alterations (“complex” pattern) whereas the remaining 34% of tumors lacked these lesions (“clean” signature). Tumors with “complex” alterations of p53 pathway and cell cycle components also had more total CNAs and more frequent TP53 mutations, highlighting the association between p53 deficiency, cell cycle deregulation and increased genomic instability in DLBCL. The patterns of “complex” vs. “clean” CNAs of p53 pathway and cell cycle components and the association between the “complex” signature and total CNAs were confirmed in an independent series of primary DLBCLs. To further characterize DLBCLs with “complex” vs. “clean” CNA patterns, we performed gene set enrichment analyses with publicly available series of p53 target genes and a RB-deficiency gene set which included multiple E2F targets. The p53 target transcripts were significantly less abundant in “complex” DLBCLs, directly linking their genetic signature of p53 deficiency with decreased p53 activity. Furthermore, the RB-deficiency gene set was significantly enriched in “complex” DLBCLs suggesting that these tumors had increased E2F-mediated cell cycle progression. Consistent with these observations, DLBCLs with “complex” CNA patterns also had significantly higher proliferation indices as determined by Ki67 immunostaining. We next assessed the prognostic significance of the “complex” CNA pattern in the subset of patients who were treated with R-CHOP (rituxan, cyclophosphamide, adriamycin, oncovin, prednisone) and had long-term follow-up. R-CHOP treated patients with “complex” CNA patterns had a 5-year overall survival of only 62%; in contrast, those with “clean” CNA signatures had an 100% overall survival (p =.001). The association between CN complexity and outcome was independent of transcriptionally defined categories and additive to the clinical IPI risk model. Taken together, these data provide a structural basis for deregulated cell cycle, increased cellular proliferation and unfavorable outcome in DLBCL and suggest targeted treatment strategies. Disclosures: No relevant conflicts of interest to declare.

Neuroblastoma is a developmental tumor that arises in the peripheral sympathetic nervous system and accounts for 10% of all cancer-related deaths in children. The anaplastic lymphoma kinase (ALK) gene is mutationally activated in a subset of primary neuroblastomas, including those with MYCN gene amplification, suggesting pathogenic cooperation. Because the mechanism underlying this cooperation is unclear, we generated a novel transgenic zebrafish model that overexpresses human MYCN and activated ALK in the peripheral sympathetic nervous system to analyze their interaction. The expression of MYCN in this model induces neuroblastomas in the inter-renal gland, the zebrafish analogue of the adrenal medulla, which is the site of origin observed in approximately half of childhood neuroblastomas. Furthermore, the tumors resemble human neuroblastomas histologically, immunohistochemically and ultrastructurally. Concomitant expression of activated ALK with MYCN in this model profoundly accelerates the onset of neuroblastoma and markedly increases disease penetrance. Detailed in vivo analyses show that MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts. Taken together, these findings provide a mechanism for the synergistic interplay of MYCN with activated ALK in neuroblastoma pathogenesis and demonstrate the utility of the zebrafish model in understanding human disease processes that may aid the development of improved therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4252. doi:1538-7445.AM2012-4252

Abstract 1405 Despite improvements in the treatment of AML, high-risk disease including 8p11 myeloproliferative syndrome/stem cell leukemia remains largely refractory to current therapy, and is mostly fatal. Identification of effective therapeutic targets by using candidate gene approaches has been limited by the number and variety of genetic defects associated with AML. Based on the results of a genome-wide functional screen in AML using a retroviral library of short hairpin RNAs (shRNAs), we discovered that hepatocyte growth factor (HGF) is aberrantly expressed in about 40% of patients with AML, leading to activation of its receptor MET. Autocrine activation of MET is required for survival of AML cells, as genetic and pharmacologic inhibition of HGF or MET induces apoptosis and inhibits cell growth. However, AML cells treated for more than one week with the MET kinase inhibitor crizotinib develop resistance as a result of 13-fold upregulation of HGF expression, leading to restoration of MET pathway activity, and emphasizing autocrine HGF production as the basis for MET signaling addiction. Using simple thermodynamic models, we find that ligand-mediated receptor activation due to increased ligand expression effectively antagonizes the effects of targeted kinase inhibitors, causing complete restoration of aberrant signaling activity. Here we show that this mechanism of resistance can be overcome by combining MET kinase inhibition with depletion of HGF or targeting of pathways required for compensatory HGF upregulation. In the case of KG-1 cells derived from a patient with 8p11 myeloproliferative syndrome AML with chromosomal translocation mediated activation of FGFR1, we find that signaling downstream of the FGFR is required for the upregulation of HGF that occurs in response to MET kinase inhibition. Sustained MET inhibition due to the blockade of compensatory HGF upregulation leads to durable logarithmic cell kill as a result of combined treatment with the MET kinase inhibitor crizotinib and FGFR kinase inhibitor PD173074. In addition, we find that aberrant expression of HGF is highly correlated with expression of FGF2 (Pearson's correlation = 0.79, p < 0.001), with 10% of all patients exhibiting co-expression of HGF and FGF2. This co-expression leads to autocrine co-activation of MET and FGFR1, which can be effectively targeted using combined treatment with MET and FGFR kinase inhibitors. In all, these findings suggest a therapeutic strategy for patients with co-activation of MET and FGFR, including patients with 8p11 myeloproliferative syndrome AML, establish a paradigm of effective therapeutic targeting of aberrant HGF/MET signaling in AML, and emphasize the importance of targeting compensatory ligand-dependent receptor tyrosine kinase activation, as required for the development of rational combination therapy. Disclosures: Christensen: Pfizer: Employment. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding.

Neuroblastoma is the most common extracranial solid tumor in children and originates during development of the peripheral sympathetic nervous system. Advanced-stage disease and a poor outcome are associated with amplification of the MYCN oncogene, often in combination with mutational activation or amplification of another oncogene ALK (anaplastic lymphoma kinase), suggesting that they cooperate in neuroblastoma pathogenesis. To investigate this possibility, we generated a transgenic zebrafish model of neuroblastoma in which human MYCN is expressed under the control of the dopamine-beta-hydroxylase promoter, and show that the resultant tumors recapitulate childhood neuroblastomas histologically, immunohistochemically, and ultrastructurally. Surprisingly, the tumors arise from a subpopulation of neuroblasts that migrate into the adrenal analogue (interrenal gland) in the zebrafish after organogenesis is complete. Coexpression of activated ALK with MYCN markedly increased the frequency of neuroblastoma and accelerated the time of onset, providing conclusive evidence for synergistic interplay between these two oncogenes in neuroblastoma pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4296. doi:10.1158/1538-7445.AM2011-4296

The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences in thrombogenicity between surfaces, and may provide a mechanism for purposefully passivating platelet-reactive artificial surfaces.

A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin-induced activation of HUVECs and platelets may be partially mediated by an alternative mechanism(s) or receptor(s).

Abstract 317 The JAK2V617F mutation is the most common somatic mutation in BCR-ABL negative myeloproliferative neoplasms (MPN) and small molecule JAK2 kinase inhibitors are currently being evaluated in clinical trials in MPN patients. We recently reported a Jak2V617F conditional knockin mouse MPN model in which expression of Jak2V617F was under the control of the endogenous Jak2 promoter and the phenotype closely recapitulated the features of human polycythemia vera (PV) (Mullally and Lane et al, Cancer Cell 2010). In this model the MPN phenotype developed as a result of expansion and erythroid skewing of committed myeloid progenitor populations, while the hematopoietic stem cell (HSC) compartment (enriched within LineagelowSca1+cKithigh, LSK cells) was not expanded. In contrast to the expanded progenitor population, only LSK cells had the unique capacity to initiate the MPN. Notably, treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. In light of these findings, we wished to further characterize MPN-initiating LSK cells in this model, with a view to gaining understanding of the differential molecular circuitry of JAK2V617F mutant and normal HSC. The LSK compartment, although enriched for HSC, may be further defined by immunophenotype into long-term HSC (LT-HSC, LSKCD150+CD48-), short-term HSC (ST–HSC, LSKCD150-CD48-), or multipotent progenitor cells (MPP, LSKCD150-CD48+). Gene expression analysis of total LSK cells revealed enrichment for markers of myeloid progenitor differentiation in Jak2V617F LSK cells compared to Jak2 wild-type (WT) controls, although there were no quantitative differences in the LSK sub-populations between the two groups. We therefore sought to evaluate whether the MPN-initiating cell population was contained within the LT-HSC compartment. Employing single cell sorting multi-parameter flow cytometry and limiting dilution bone marrow transplantation assays, we provide further functional characterization of the MPN-initiating population in this model. We demonstrate that Jak2V617F LT-HSCs are highly enriched for MPN-initiating activity in vivo and are able to maintain the MPN through multiple rounds of serial transplantation for periods greater than one year. The MPN phenotype remains stable during this time without evolution to myelofibrosis or leukemia. These findings provide important biological insights into MPN initiation and maintenance and have implications for the development of curative therapeutic strategies for MPN patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 472 We and others recently identified locus rearrangements involving the type I cytokine receptor subunit CRLF2 (also known as thymic stromal lymphopoietin receptor (TSLPR)) in 5–7% of all adult and pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL). CRLF2 rearrangement places full-length CRLF2 under alternate transcriptional control, and can either result from an intrachromosomal CRLF2-P2RY8 deletion or from a CRLF2-IGH translocation. Prognosis for CRLF2-rearranged B-ALL is particularly poor, suggesting that a sizable fraction of relapsed and ultimately fatal B-ALL harbor CRLF2 rearrangements. Approximately 50% of CRLF2-rearranged B-ALL harbor mutations in JAK2 (and rarely JAK1) that cluster around JAK2 Arg683. A separate 15% have a CRLF2 F232C mutation that promotes constitutive homodimerization and signaling. In the remaining cases, the driver of CRLF2 signaling is not known. To identify additional genetic alterations that contribute to CRLF2-mediated leukemogenesis, we performed next-generation sequencing on 3 CRLF2-rearranged B-ALL specimens, including one with CRLF2 F232C (#536). Exome libraries were assembled from bone marrow specimens with greater than 90% B-ALL involvement and from paired remission bone marrows. Transcriptome sequencing was also performed on cDNA isolated from the involved marrow of patient #536 to: 1) confirm the findings from exome sequencing, 2) focus on transcribed genes and 3) identify possible RNA editing events. Sequencing utilized the SOLiD (Sequencing by Oligonucleotide Ligation and Detection; Applied BioSystems) platform, with a target recovery of greater than 40 million (transcriptome) and 25 million (exome) uniquely mapping reads per sample. Mutations were considered high confidence if a minimum of 3 individual reads identified the same mutation within the involved specimen and the mutation was not identified in any reads from the remission specimen (minimum 10 reads at that base-pair). Paired exome sequencing of germline and involved marrow specimens identified 129, 297 and 630 high-confidence, non-synonymous, somatic mutations in the three samples. Of the total 1056 mutations, 1023 (96.9%) were missense and the remaining 33 (3.1%) were nonsense mutations. The expected mutation in CRLF2 F232C (#536) was recovered, as was a novel JAK2 mutation from a sample previously thought to be JAK2 wild-type (#002). Comparison between transcriptome and exome sequence markedly reduced the number of potential “driver” alterations, i.e., nonsynonymous coding mutations expressed in the tumor but not present in the remission exome. For example, from the 129 somatic alterations in the involved exome from #536, only 15 were recovered from the involved transcriptome. Sequence alterations affected genes known to be involved in histone modification, oxygen metabolism, and steroid responsiveness. To our knowledge, none of the identified mutations have previously been described. One mutation in a gene involved in E2F transcription was observed in two of three cases. In addition, 2,428 coding sequence mutations were identified from the involved marrow transcriptome of #536 that were not present in the involved marrow exome, possibly indicating a high rate of RNA editing. Additional sequence analysis and molecular epidemiology of the novel sequence alterations in CRLF2-rearranged and —unrearranged B-ALL is underway. Disclosures: No relevant conflicts of interest to declare.

Abstract 1042 Despite improvements in the treatment of acute myeloid leukemia (AML), high risk disease such as complex aberrant karyotype AML remains largely refractory to current therapy, and is mostly fatal. Identification of effective therapeutic targets by using candidate gene approaches has been limited by the number and variety of genetic defects associated with AML. Thus, we carried out a genome-wide functional screen in complex karyotype AML using a retroviral library of short hairpin RNAs (shRNAs), and discovered that shRNA-mediated depletion of hepatocyte growth factor (HGF) specifically inhibits growth of AML cells but not a panel of lymphoid cancer cells. HGF was to found to be aberrantly expressed in about 15% of patients with AML, including most patients with complex karyotype disease. In contrast to normal CD34+ cells that express MET (but not HGF), 5 of 7 cell lines derived from patients with complex karyotype AML exhibited aberrant expression of HGF that was associated with autocrine activation of its receptor MET. Depletion of HGF or MET using multiple independent shRNAs profoundly reduced proliferation and induced cell death in AML cells lines that express HGF but not those that lack HGF expression. Inhibition of MET using the tyrosine kinase inhibitor (SU11274) or HGF using neutralizing anti-HGF antibody (R&D Systems) also inhibited growth and induced apoptosis in AML cell lines dependent on HGF/MET activation but not those that lack HGF expression. Thus, aberrant HGF expression causes autocrine MET activation and oncogene dependence in a subset of patients with AML, confers sensitivity to HGF/MET inhibition, and provides a novel therapeutic target for this otherwise lethal disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 635 Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease with infrequent alterations of multiple apoptotic, developmental and signaling pathways. Inactivating somatic mutations of the p53 tumor suppressor are uncommon in DLBCL, prompting speculation regarding alternative mechanisms of modulating p53 activity in this disease. As part of a comprehensive analysis of genetic alterations in DLBCL, we recently integrated high-resolution copy number data and transcription profiles in a series of 81 newly diagnosed previously untreated DLBCLs. Copy number (CN) alterations were evaluated using Affymetrix SNP Array 6.0 data on over 1.8 million genomic loci (906,000 SNP probe sets and 946,000 copy number probes) and a data-processing pipeline incorporating the normalization, calibration, and segmentation of the CN profiles and their analysis by the GISTIC algorithm. The genes within the peak and region of each GISTIC-identified alteration were tested for association with the corresponding transcripts' expression and significance values were corrected for multiple hypothesis testing by the false discovery rate (FDR) procedure, with the correction accounting for both the multiple genes within each peak and the multiple peaks. The signature for a given copy number alteration was then defined as the set of within-peak and -region transcripts with FDR q-values < 0.25. The alteration signatures thus defined were then tested for pathway/gene set enrichment based on the hypergeometric distribution, using a compendium of gene sets from the MSigDB repository (Broad Institute). This analysis highlighted a genome-wide pattern whereby: i) individual pathways are targeted by multiple alterations at different loci (with different genes of the same pathway located within distinct CN alteration peaks or regions); and ii) members of multiple pathways are targeted by the same alteration (with genes co-located within a single CN alteration belonging to different pathways). The most highly significant enriched pathways (FDR < .05) included apoptotic, p53 signaling, and ARF pathways and identified multiple modulators and effectors of normal p53 activity. These include: deletion of the MDM2-inhibitor, ARF; amplification of the p53 E3 ligases, MDM2 and COP1; deletion of p53 itself; deletion of the ribosomal protein that enhances p53 translation following DNA damage, RPL26; and deletion of p53 effectors including BNIP3L and DFFB. Of note, the deletion of 17p13.1 encompasses both p53 and its positive modulator, RPL26. In all cases, the observed alterations in p53 pathway components would decrease functionally active p53 protein and p53 apoptotic effectors in primary DLBCLs. Taken together, these data identify alternative genetic mechanisms for reducing p53 activity in DLBCL and highlight the value of an integrated comprehensive analysis of genetic alterations in this disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 746 Classical Hodgkin Lymphoma (cHL) Reed-Sternberg (RS) cells have crippling mutations of their rearranged Ig genes and lack B-cell receptor-mediated survival signals. As a consequence, cHLs rely on alternative survival and proliferation pathways including AP-1. Although cHL RS cells express high levels of the AP-1 components, cJun and JunB, and depend on AP-1-mediated proliferation signals, the mechanism of constitutive AP-1 activation in cHL remains undefined. In a screen for genetic abnormalities in cHL, we integrated microRNA (miR) expression profiles with high resolution copy number data and identified single copy loss of the miR-15a/16-1 locus (chromosome 13q14) and decreased miR-15a/16 expression in 75% of examined HL cell lines. We isolated primary Hodgkin RS cells using laser-capture microdissection and confirmed frequent single copy loss of the miR-15a/16-1 locus by quantitative PCR and associated decreased miR-15a/16 abundance by RT-PCR in 10/23 (43%) of primary tumors. Using retroviral vectors containing miR-15a and miR-16, we reconstituted miR-15a/16 expression in HL cell lines with single copy loss of chromosome 13q14. In these HL lines, miR-15a/16 reconstitution was associated with cell cycle arrest, significantly decreased cellular proliferation and induction of apoptosis (88% apoptotic cells at 48 hours). In addition, miR-15a/16 reconstitution resulted in significant downregulation of multiple survival pathway components and known miR-15a/16 targets including panAKT, BCL2 and cyclin D1. Using a stand-alone miRanda algorithm with direct sequence entry, we also identified a candidate miR-15a/16 binding site in the AP-1 component, cJun. In multiple HL cell lines, miR-15a/16 reconstitution decreased the expression of cJun and additional known AP-1 targets such as galectin-1. Therefore, miR-15a/16-1 deletion is a frequent genetic abnormality in HL cell lines and primary tumors, a candidate mechanism of AP-1 activation in HL and a modulator of multiple critical survival pathways in this disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 4314 Background: Emerging evidence demonstrates that endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of being recruited to sites of ischemic injury and contribute to neovascularization. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Methods and Results: We previously identified that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) can preferentially differentiate into EPCs and possess high angiogenic activity under ischemic conditions compared to megakaryocyte-erythrocyte progenitors (MEPs), hematopoietic stem cells (HSCs), and common lymphoid progenitors (CLPs). Herein, we identify that a TGF-β1-responsive Kruppel-like Factor, KLF10, is robustly expressed in EPCs derived from CMPs and GMPs, compared to progenitors lacking EPC markers. KLF10–/– mice have marked defects in circulating EPCs (–23.6% vs. WT, P Background: Collectively, these observations identify that TGF-β1 signaling and KLF10 are part of a key signaling pathway that regulates EPC differentiation from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. Disclosures: No relevant conflicts of interest to declare.

Peptides derived from the recently identified thrombin receptor were tested for their ability to induce platelet aggregation in platelet-rich plasma. The 14 amino acid peptide identified as the new N-terminus after thrombin cleavage (T-14) and an 11 amino acid peptide (T-11) lacking the 3 C-terminal amino acids of T-14 were studied. Both induced platelet aggregation at micromolar concentrations, with T-11 about twice as potent as T-14. Induction of platelet aggregation by these two peptides showed an unusual pH dependence, being more potent at pH 7.2 than at pH 8.1; thrombin-induced aggregation showed a reverse pH dependence. Proton NMR studies of T-11 demonstrated that the chemical shift of the C-alpha proton of the N-terminal serine had a pH dependence that mirrored the aggregation potency. Acetylating the N-terminus of T-11 resulted in loss of aggregating activity, and this peptide did not show the pH-dependence change in chemical shift. The T-14 and T-11 peptides lost aggregating activity when incubated in plasma due to cleavage of the N-terminal serine by an enzyme identified as aminopeptidase M based on its pattern of inhibition and the ability of purified aminopeptidase M (EC3.4.11.2) to cleave the T-11 peptide. Endothelial cell aminopeptidase M was also able to cleave T-11. Inhibiting aminopeptidase M with amastatin enhanced aggregation induced by T-11 but not thrombin. These studies suggest that ionization of the N-terminus of the T-11 and T-14 peptides may be important in initiating platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Background Defects in the development or activation of T cells result in immunodeficiency associated with severe infections early in life. T-cell activation requires Ca 2+ influx through Ca 2+ -release activated Ca 2+ (CRAC) channels encoded by the gene ORAI1 . Objective Investigation of the genetic causes and the clinical phenotype of immunodeficiency in patients with impaired Ca 2+ influx and CRAC channel function. Methods DNA sequence analysis for mutations in the genes ORAI1 , ORAI2 , ORAI3 , and stromal interaction molecule (STIM) 1 and 2, as well as mRNA and protein expression analysis of ORAI1 in immunodeficient patients. Immunohistochemical analysis of ORAI1 tissue distribution in healthy human donors. Results We identified mutations in ORAI1 in patients from 2 unrelated families. One patient is homozygous for a frameshift nonsense mutation in ORAI1 (ORAI1-A88SfsX25), and a second patient is compound heterozygous for 2 missense mutations in ORAI1 (ORAI1-A103E/L194P). All 3 mutations abolish ORAI1 expression and impair Ca 2+ influx and CRAC channel function. The clinical syndrome associated with ORAI1 deficiency is characterized by immunodeficiency with a defect in the function but not in the development of lymphocytes, congenital myopathy, and anhydrotic ectodermal dysplasia with a defect in dental enamel calcification. In contrast with the limited clinical phenotype, we found ORAI1 protein expression in a wide variety of cell types and organs. Conclusion Ca 2+ influx through ORAI1 is crucial for lymphocyte function in vivo . Despite almost ubiquitous ORAI1 expression, the channel has a nonredundant role in only a few cell types judging from the limited clinical phenotype in ORAI1-deficient patients.

Abstract 910 Despite modern intensive therapy, 25% of children with T-ALL will develop treatment-resistant disease, which carries a dismal prognosis. However, no clinical or biologic features have been found to predict prognosis robustly enough to be incorporated into current clinical protocols. The inability to accurately identify patients at high risk of treatment failure at the time of diagnosis is a major impediment to further improvements in outcome in T-ALL, as patients at high risk of treatment failure, who would probably benefit from the introduction of novel therapeutic agents, cannot be differentiated from those who might be cured with reduced-intensity regimens. In an effort to identify genomic alterations present at the time of diagnosis that would predict response to therapy, we performed array comparative genomic hybridization (CGH) on DNA extracted from diagnostic specimens from 47 children with T-ALL treated on either Children's Oncology Group Protocol 9404 or DFCI ALL Consortium Protocol 00-001, which are very similar regimens. The samples analyzed included all of the specimens available from cases in which therapy failed, comprising 9 induction failure and 13 relapse cases, and a control group of 25 long-term event-free survivors. The absence of deletions of genomic DNA at the TCRg, TCRb, and TCRa/TCRd loci at the time of diagnosis, indicating that V(D)J recombination had not occurred, was associated with induction failure, as was deletion of the CDKN2A tumor suppressor locus. The absence of TCRg and TCRb deletions were also associated with inferior event-free and overall survival. The lack of T cell receptor rearrangements in these cases indicates that PCR-based minimal residual disease testing may not be useful in these high-risk cases. The most robust marker of treatment failure identified, lack of homozygous TCRg rearrangement, strongly predicted induction failure (75% vs. 8%,P = 0.0002), inferior event-free survival (P = 0.0009) and inferior overall survival (P < 0.0001) in these patients. Our findings were validated using quantitative DNA PCR, an assay that is readily suited to clinical applications. Analysis of gene expression data demonstrated that the group of cases identified by lack of TCR rearrangement showed significant biologic overlap with the high-risk “early T-cell precursor” subtype of T-ALL recently identified using gene expression profiling. Taken together, these data indicate that a subset of T-ALL patients with a very poor response to contemporary therapy can be accurately identified at the time of diagnosis, raising the possibility that alternative therapies can be administered early in treatment to this high group of patients in order to improve their outcome. Disclosures: No relevant conflicts of interest to declare.

Abstract 947 Large hemizygous chromosomal deletions are among the most common molecular abnormalities in cancer, but the identification of critical haploinsufficiency disease genes within the deleted regions has been difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a well-studied chromosomal deletion syndrome characterized by a consistent clinical phenotype with macrocytic anemia and thrombocytosis. We have previously shown that while hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not account for the thrombocytosis. Evaluation of the effects of all protein coding genes in the CDR on hematopoietic differentiation showed no genes other than RPS14 altered the ratio of megakaryocytic to erythroid cells, either alone or in combination with RPS14. We therefore examined the 5q- syndrome CDR for non-coding RNAs and identified a microRNA, miR-145, which targets Fli-1, a transcriptional factor that regulates megakaryocyte development. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 in CD34+ cells decreases megakaryocyte production, while inhibition of miR-145 or overexpression of Fli-1 has the reciprocal effect. These findings have been validated in vivo using transgenic mice. Moreover, the combined loss of miR-145 and RPS14 cooperate to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate for the first time that coordinate deletion of a microRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome. Disclosures: No relevant conflicts of interest to declare.

Abstract 96 EBV-associated post-transplant lymphoproliferative disorders (PTLD) are potentially fatal EBV-driven B cell proliferations that develop in immunocompromised solid organ or bone marrow allograft recipients. Proliferation of EBV-infected B cells in PTLD is maintained by expression of EBV latent genes such as latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance. Management of early PTLD lesions is currently based on reduction or withdrawal of immunosuppression which increases the risk of graft rejection. For these reasons, alternative treatments that specifically boost host anti-EBV immune responses are needed. To identify novel T-cell inhibitory molecules induced by EBV and expressed in PTLDs, we compared the transcription profiles of a series of EBV-transformed lymphoblastoid B-cell lines (LCLs) and diffuse large B-cell lymphoma (DLBCL) cell lines. The LCLs overexpressed galectin-1 (Gal-1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4+ Th1 and CD8+ cytotoxic T cells. Gal-1 protein expression was also uniformly high in LCLs and low or undetectable in DLBCL lines by western blotting. Immunohistochemical staining of primary tumors confirmed abundant Gal-1 expression in PTLD B-cells, whereas DLBCLs were negative. To elucidate the mechanisms responsible for Gal-1 overexpression in EBV-associated PTLD, we performed transcriptional reporter assays using cotransfected FLAG-tagged LMP1 and/or LMP2A and a luciferase reporter vector driven by Gal-1 promoter. LMP2A and LMP1 each increased Gal-1-driven luciferase expression 3- to 6-fold, respectively, and the combination of LMP2A and LMP1 was synergistic. In complementary experiments, siRNA-mediated depletion of LMP2A decreased Gal-1 protein abundance in EBV-transformed lymphoblastoid cells. The mechanism of LMP1 and LMP2A-induced Gal-1 expression in LCLs was both AP-1 and PI3K-dependent; the PI3K selective inhibitor, LY294002, markedly inhibited Gal-1 expression in these cells. Given the tolerogenic and immunosuppressive function of Gal-1 and the EBV-driven overexpression of the lectin in LCLs and primary PTLDs, we postulated that Gal-1 might be a targetable immune escape mechanism in EBV-associated malignancies. For these reasons, we assessed the neutralizing activity of a recently developed series of anti-Gal1 monoclonal antibodies. After confirming that these reagents selectively identified both recombinant and native Gal1, we asked whether certain Gal-1 monoclonal antibodies neutralized recombinant Gal-1-mediated apoptosis of EBV-specific polyclonal, largely CD8+, T-cells. Preincubation of human recombinant Gal-1 (hrGal-1) with Gal-1 monoclonal antibodies, but not with isotype-matched controls, selectively inhibited hrGal-1 induced apoptosis of EBV-specific CD8+ T-cells. Taken together, these studies indicate that EBV-transformed LCLs and primary EBV-associated PTLDs express Gal-1 in a LMP-1 and LMP2A-dependent manner. Given the acknowledged role of Gal-1 in mediating an immunopriviledged tumor microenvironment, the lectin likely inhibits the host anti-tumor response in these tumors. Antibody-mediated neutralization of secreted Gal-1 may represent a novel immunotherapeutic strategy in PTLD and other Gal1-expressing tumors. Disclosures: No relevant conflicts of interest to declare.