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Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a 5-day course of granulocyte colony-stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor plerixafor is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was coadministered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization achieved by the VLA4 inhibitor and CXCR2 agonist combination in mice compared with currently approved HSPC mobilization methods, the combination represents an exciting potential strategy for clinical development in the future.

Purpose Epidermal growth factor receptor (EGFR) mutations confer sensitivity to EGFR tyrosine kinase inhibitors in patients with advanced non–small-cell lung cancer (NSCLC). There are limited and conflicting reports on the frequency of EGFR mutations in Latinos. Patients and Methods Samples from 642 patients with NSCLC from seven institutions in the United States and Latin America were assessed for EGFR mutations (exons 18 to 21) at Clinical Laboratory Improvement Amendments-certified central laboratories. Results EGFR mutation analysis was successfully performed in 480 (75%) of 642 patients; 90 (19%) were Latinos, 318 (66%) were non-Latino whites, 35 (7%) were non-Latino Asians, 30 (6%) were non-Latino blacks, and seven (2%) were of other races or ethnicities. EGFR mutations were found in 21 (23%) of 90 Latinos with varying frequencies according to the country of origin; Latinos from Peru (37%), followed by the United States (23%), Mexico (18%), Venezuela (10%), and Bolivia (8%). In never-smoker Latinos and Latinos with adenocarcinoma histology, EGFR mutation frequencies were 38% and 30%, respectively. There was a significant difference in the frequency of EGFR mutations among the different racial and ethnic subgroups analyzed (P < .001), with non-Latino Asians having the highest frequency (57%) followed by Latinos (23%), non-Latino whites (19%), and non-Latino blacks (10%). There was no difference between Latinos (23%) and non-Latinos (22%; P = .78) and Latinos and non-Latino whites (P = .37). Patients from Peru had an overall higher frequency of mutations (37%) than all other Latinos (17%), but this difference only exhibited a trend toward significance (P = .058). Conclusion There was no significant difference between the frequency of EGFR mutations in NSCLC in Latinos and non-Latinos.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. A great majority of GISTs is driven by pathological activation of KIT or platelet-derived growth factor receptor-α (PDGFRA), two closely related receptor tyrosine kinases. However, other genetic changes including gain-of-function BRAF mutations and loss of succinate dehydrogenase (SDH) complex activity have been identified in the subsets of KIT-, PDGFRA-wild type tumors. Genetic mutations affecting KIT, PDGFRA, BRAF and SDH complex functions are believed to be mutually exclusive events. Recently, KRAS codon 12 and 13 mutations were reported in a small subset of KIT or PDGFRA mutant GISTs. Moreover, in in vitro experiments, KIT mutants with concurrent KRAS mutation showed resistance to imatinib, a receptor tyrosine kinase inhibitor used in GIST treatment. The aim of this study was to evaluate a large cohort of GISTs to define frequency and clinical significance of KRAS mutations in this type of cancer. A well-characterized cohort of 514 GISTs was screened for KRAS mutations using Sanger sequencing (n = 450) and pyrosequencing (n = 64). In all, 350 gastric, 100 intestinal and 64 primary disseminated GISTs were analyzed. No KRAS mutations were found. In GIST, KRAS mutations are extremely rare if they exist (

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive and largely incurable hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). Using RNA interference screening, we identified the E-box transcription factor TCF4 as a master regulator of the BPDCN oncogenic program. TCF4 served as a faithful diagnostic marker of BPDCN, and its downregulation caused the loss of the BPDCN-specific gene expression program and apoptosis. High-throughput drug screening revealed that bromodomain and extra-terminal domain inhibitors (BETi’s) induced BPDCN apoptosis, which was attributable to disruption of a BPDCN-specific transcriptional network controlled by TCF4-dependent super-enhancers. BETi’s retarded the growth of BPDCN xenografts, supporting their clinical evaluation in this recalcitrant malignancy.

We have analyzed the role of the REL family members in Hodgkin lymphoma (HL). shRNA targeting of each REL member showed that HL was uniquely dependent on relB, in contrast to several other B-cell lymphomas. In addition, relA and c-rel shRNA expression also decreased HL cell viability. In exploring relB activation further, we found stable NF-κB inducing kinase (NIK) protein in several HL cell lines and that NIK shRNA also affected HL cell line viability. More importantly, 49 of 50 HL patient biopsies showed stable NIK protein, indicating that NIK and the noncanonical pathway are very prevalent in HL. Lastly, we have used a NIK inhibitor that reduced HL but not other B-cell lymphoma cell viability. These data show that HL is uniquely dependent on relB and that the noncanonical pathway can be a therapeutic target for HL. Furthermore, these results show that multiple REL family members participate in the maintenance of a HL phenotype.

Analysis of the Polyp Prevention Trial showed an association between an isorhamnetin-rich diet and a reduced risk of advanced adenoma recurrence; however, the mechanism behind the chemoprotective effects of isorhamnetin remains unclear. Here, we show that isorhamnetin prevents colorectal tumorigenesis of FVB/N mice treated with the chemical carcinogen azoxymethane and subsequently exposed to colonic irritant dextran sodium sulfate (DSS). Dietary isorhamnetin decreased mortality, tumor number, and tumor burden by 62%, 35%, and 59%, respectively. MRI, histopathology, and immunohistochemical analysis revealed that dietary isorhamnetin resolved the DSS-induced inflammatory response faster than the control diet. Isorhamnetin inhibited AOM/DSS–induced oncogenic c-Src activation and β-catenin nuclear translocation, while promoting the expression of C-terminal Src kinase (CSK), a negative regulator of Src family of tyrosine kinases. Similarly, in HT-29 colon cancer cells, isorhamnetin inhibited oncogenic Src activity and β-catenin nuclear translocation by inducing expression of csk, as verified by RNA interference knockdown of csk. Our observations suggest the chemoprotective effects of isorhamnetin in colon cancer are linked to its anti-inflammatory activities and its inhibition of oncogenic Src activity and consequential loss of nuclear β-catenin, activities that are dependent on CSK expression. Cancer Res; 73(17); 5473–84. ©2013 AACR.

Defective lymphocyte apoptosis results in chronic lymphadenopathy and/or splenomegaly associated with autoimmune phenomena. The prototype for human apoptosis disorders is the autoimmune lymphoproliferative syndrome (ALPS), which is caused by mutations in the FAS apoptotic pathway. Recently, patients with an ALPS-like disease called RAS-associated autoimmune leukoproliferative disorder, in which somatic mutations in NRAS or KRAS are found, also were described. Despite this progress, many patients with ALPS-like disease remain undefined genetically. We identified a homozygous, loss-of-function mutation in PRKCD (PKCδ) in a patient who presented with chronic lymphadenopathy, splenomegaly, autoantibodies, elevated immunoglobulins and natural killer dysfunction associated with chronic, low-grade Epstein-Barr virus infection. This mutation markedly decreased protein expression and resulted in ex vivo B-cell hyperproliferation, a phenotype similar to that of the PKCδ knockout mouse. Lymph nodes showed intense follicular hyperplasia, also mirroring the mouse model. Immunophenotyping of circulating lymphocytes demonstrated expansion of CD5+CD20+ B cells. Knockdown of PKCδ in normal mononuclear cells recapitulated the B-cell hyperproliferative phenotype in vitro. Reconstitution of PKCδ in patient-derived EBV-transformed B-cell lines partially restored phorbol-12-myristate-13-acetate–induced cell death. In summary, homozygous PRKCD mutation results in B-cell hyperproliferation and defective apoptosis with consequent lymphocyte accumulation and autoantibody production in humans, and disrupts natural killer cell function.

Purpose: Multiple myeloma is a usually incurable malignancy of plasma cells. New therapies are urgently needed for multiple myeloma. Adoptive transfer of chimeric antigen receptor (CAR)–expressing T cells is a promising new therapy for hematologic malignancies, but an ideal target antigen for CAR-expressing T-cell therapies for multiple myeloma has not been identified. B-cell maturation antigen (BCMA) is a protein that has been reported to be selectively expressed by B-lineage cells including multiple myeloma cells. Our goal was to determine if BCMA is a suitable target for CAR-expressing T cells. Experimental Design: We conducted an assessment of BCMA expression in normal human tissues and multiple myeloma cells by flow cytometry, quantitative PCR, and immunohistochemistry. We designed and tested novel anti-BCMA CARs. Results: BCMA had a restricted RNA expression pattern. Except for expression in plasma cells, BCMA protein was not detected in normal human tissues. BCMA was not detected on primary human CD34+ hematopoietic cells. We detected uniform BCMA cell-surface expression on primary multiple myeloma cells from five of five patients. We designed the first anti-BCMA CARs to be reported and we transduced T cells with lentiviral vectors encoding these CARs. The CARs gave T cells the ability to specifically recognize BCMA. The anti-BCMA-CAR–transduced T cells exhibited BCMA-specific functions including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. Importantly, anti-BCMA-CAR–transduced T cells recognized and killed primary multiple myeloma cells. Conclusions: BCMA is a suitable target for CAR-expressing T cells, and adoptive transfer of anti-BCMA-CAR–expressing T cells is a promising new strategy for treating multiple myeloma. Clin Cancer Res; 19(8); 2048–60. ©2013 AACR.

Abstract 937 Multiple myeloma (MM) is a usually incurable malignancy of plasma cells. New therapies are urgently needed for MM. Development of immunotherapies targeting antigens expressed by MM cells could improve the outcomes of patients with multiple myeloma (MM). Chimeric antigen receptors (CARs) are fusion proteins containing antigen-recognition moieties and T-cell activation domains. T cells genetically modified to express CARs can specifically recognize tumor antigens, and CAR-expressing T cells have potent in vivo activity against some types of lymphoma and leukemia. Unfortunately, development of CAR-expressing T-cell therapies for MM has been hindered because most proteins expressed on MM cells are also expressed on essential normal cells. We report here our work developing CARs that target B-cell maturation antigen (BCMA). BCMA is a protein that has been reported to be selectively expressed by B-lineage cells including MM cells. We hypothesized that BCMA would be a suitable target for CAR-expressing T cells. We evaluated BCMA expression in all major human organs, and we designed and tested the first anti-BCMA CARs. We assessed BCMA RNA expression in all major human organs by quantitative PCR. Although BCMA RNA expression was mainly limited to hematologic tissues, low levels of BCMA RNA were detected in gastrointestinal organs, trachea, and testes. Next, we assessed BCMA protein expression in samples of 33 normal human organs by immunohistochemistry (IHC). BCMA-expressing plasma cells were detected in the lamina propria of several gastrointestinal organs by IHC, but BCMA protein was not detected in other gastrointestinal tissues. BCMA-expressing gastrointestinal plasma cells were probably the source of the low levels of BCMA RNA detected in normal gastrointestinal organs. Except for plasma cells, BCMA expression was not detected in any normal human tissues by IHC. Furthermore, BCMA was not detected on primary human CD34+hematopoietic cells by flow cytometry. In contrast, we detected uniform BCMA cell-surface expression by either flow cytometry or IHC on primary MM cells from 5 of 5 patients. Because BCMA is expressed by MM cells but not by normal essential tissues, we reasoned that BCMA would be an appropriate target for CAR-expressing T cells. We designed anti-BCMA CARs that contained antigen receptor domains derived from anti-BCMA monoclonal antibodies. The CARs also contained the signaling moiety of the CD28 molecule and the signaling domains of the CD3-zeta molecule. DNA encoding these CARs was ligated into a self-inactivating lentiviral vector, and human T cells were transduced with the anti-BCMA CARs. After transduction, anti-BCMA CAR expression was routinely detected on more that 60% of the transduced T cells by flow cytometry. Notably, we were able to successfully culture and transduce T cells from heavily treated patients with advanced MM. T cells expressing anti-BCMA CARs produced IFN-gamma, IL-2, and tumor necrosis factor in a BCMA-specific manner. T cell degranulation, which is required for perforin-mediated cytotoxicity, is associated with upregulation of CD107a. Anti-BCMA-CAR-transduced T cells upregulated CD107a when stimulated with BCMA-expressing target cells but not when stimulated with BCMA-negative target cells. Anti-BCMA-CAR-transduced T cells proliferated in a BCMA-specific manner. Anti-BCMA-CAR-transduced T cells killed BCMA-expressing multiple myeloma cells lines but not BCMA-negative cell lines. Importantly, anti-BCMA-CAR-transduced T cells produced interferon-gamma when cultured with BCMA-expressing primary MM cells but not when cultured with BCMA-negative peripheral blood mononuclear cells. Anti-BCMA-CAR-transduced T cells killed 66% of autologous primary MM cells in a BCMA-specific manner at a T cell to MM cell ratio of 7:1 in a 4-hour cytotoxicity assay. In summary, BCMA is expressed on MM cells of many patients, and BCMA is not expressed by normal essential tissues; therefore, BCMA is a suitable target for CAR-expressing T cells. We constructed and tested the first reported anti-BCMA CARs, and we showed that these T cells exhibited BCMA-specific functions including killing of primary MM cells. Adoptive transfer of anti-BCMA-CAR-transduced T cells is a promising new strategy for treating MM. These findings are the first steps toward clinical trials of anti-BCMA CAR-expressing T cells. Disclosures: Kochenderfer: National Cancer Institute: Inventor on patent application by National Cancer Institute., Inventor on patent application by National Cancer Institute. Patents & Royalties.

The absence of curative therapies for recurrent forms of metastatic cancer has prompted a vigorous search for novel treatment strategies. Cancer immunotherapy uses the host immune system, either directly or indirectly, to combat cancer. The more successful cancer immunotherapies are antibody-based. In this study we hypothesized that a chemical antibody (chembody) with an intact Fc region can be constructed by conjugating tumor cell specific peptidomimetic agents (ligands) to the Fab region of human IgG (hIgG). The ligand of interest, RGD, binds effectively to αVβ3 integrin expressed on tumor cells. The resultant chembody will have the ability to opsonize the tumor cells and elicit host immune response against cancer cells by recruiting the immune effector cells. In this study, we (1) conjugate naïve hIgG with the ligand, (2) verify ligand conjugation, and (3) test the efficacy of the chembody. The 2-iminothiolane conjugation method was employed to conjugate the ligand to naïve hIgG. The conjugation was verified by performing Western blot analysis, MALDI-MS assay, and cell binding assay. The Western blot and Coomassie blue staining showed that conjugation was successful while the MALDI-MS assay showed a 1:3 conjugation ratio. Colorimetric cytotoxicity assay will be used to test the efficacy of the chembody. We expect chembodies with intact Fc region to be tumor specific, and actively recruit immune cells to the tumor site and have low immunogenicity. This novel individualized medicine approach in cancer immunotherapy can be very effective in eliminating the remaining cells responsible for relapse after chemotherapy or radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5634.