Lan Pham

Development of intermediate-grade (mantle cell) and low-grade (small lymphocytic and marginal zone) human non-Hodgkin's lymphomas xenotransplanted in severe combined immunodeficiency mouse models

We have used severe combined immunodeficiency (SCID) (c.b.-17, ICR/SCID) mice to develop xenotransplantation (XT) models for human intermediate-and-low-grade non-Hodgkin’s lymphomas (NHL). In the past, SCID mice have provided a variety of useful XT models for human hematopoietic neoplasms that primarily involve the acute leukemias and some nonhematopoietic tumors, but only rare reports exist on use of the SCID mouse model in the study of primary tumor cells from NHL. Intermediate-grade and low-grade NHL are the most common lymphomas seen in adults. There is no effective therapy for those types of NHL, and they have not been established in an animal model to date. The lack of an animal model has hampered studies that can evaluate the disease process in vivo as well as the definition of therapeutic parameters involved in treatment. We report in this study that primary patient samples of NHL ( intermediate grade and low grade) have been successfully established in SCID mice after XT. NHL include intermediate-grade (mantle cell lymphoma) and low-grade (eg, small lymphocytic lymphoma/chronic lymphocytic lymphoma and marginal zone lymphoma) forms. Studies have been directed toward creating appropriate conditions for the optimal grafting of these NHL in SCID mice so that the disease process in humans could be accurately simulated. These studies indicate that development of XT-human lymphoma cells in SCID mice appear to be linked to their biologic and/or clinical behavior, transplanted lymphoma cell number, and age, as well as to the natural killer cell status of the SCID mouse recipients. Evidence has also shown that NHL cells can exhibit homing or trafficking patterns in SCID recipients that resemble those observed in patients with gastrointestinal lymphomatous involvement (particularly that of mantle cell lymphoma). Our studies also indicate that artefactual influences, such as the outgrowth of Epstein-Barr virus-associated lymphoblastoid lesions, are rare occurrences in the human NHL/SCID models that we have established.

Advances in Molecular Pathway-Directed Cancer Systems Imaging and Therapy

Molecular nuclear imaging agents enable the comprehensive characterization of therapeutic intervention and can be used in patient selection, pharmacokinetic, dosage-finding, and proof-of-concept studies. The effort in image-guided cell therapy and theranostic approaches in parallel with instrumentation development would be more comprehensive in the outcome assessment of patient response to treatment. To extend the threshold of being able to provide personalized therapy for patients, the integration of imaging findings to genomic and proteomic systems profiling is essential to validate targeted pathways. n nThe Food and Drug Administration (FDA) permits radiopharmaceuticals produced under well-controlled conditions in central commercial facilities to be distributed to local clinics where they are administered. In addition, radiopharmaceutical production process must adhere to Current Good Manufacturing Practice (CGMP) compliance to ensure the quality of drug product that meets acceptance criteria. The CGMP compliance covers manufacturing process and facility, quality guidelines, and personnel training. Y.-T. Chi et al. reported the design of CGMP production for 18F- and 68Ga-radiopharmaceuticals. The Pharmaceutical Inspection Convention and Pharmaceutical Inspection Cooperation Scheme (jointly referred to as PIC/S) are two international instruments between countries and pharmaceutical inspection authorities, which provide together an active and constructive cooperation in the field of Good Manufacturing Practice (GMP). PIC/S mission is to lead the international development, implementation, and maintenance of harmonized CGMP standards and quality systems of inspectorates in the field of medicinal products. They reviewed FDA and PIC/S guidelines for the synthesis of radiopharmaceuticals. Two examples, 68Gallium-[1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid]-D-Phe1,Tyr3-octreotate (68Ga-DOTATATE) and 18F-fluorodeoxyglucose (18F-FDG), were manufactured under CGMP process. They have reviewed acceptance criteria for these clinic useful radiopharmaceuticals. n nRadiopharmaceutical chemistry requires intricate handling of radioactive materials, fast reaction times, ease of synthesis, and reproducible results. In the preclinical setting, radiopharmaceuticals are typically synthesized manually. Such applications use in vitro and small animal models to validate the agent and require low levels of radioactivity. The use of manual synthesis for clinical imaging, however, is challenging for multiple reasons: (1) clinical agents must meet strict sterility and pyrogenicity requirements which are validated from batch to batch; (2) batch-to-batch reproducibility is required to demonstrate suitable radiochemical yield, radiochemical purity, and other quality control analyses; (3) synthesis time must be fast when dealing with radionuclides with a short half-life; (4) clinical studies require multiple patient doses and would expose radiochemists to much higher levels of radioactivity; and (5) production cost and availability of the technology may limit the viability of the agent in routine clinical practice. I.-H. Shih et al. reported the manufacturing of a cGMP grade of [18F]fluoropropoxytryptophan (18F-FTP) to assess tryptophan transporters. PET imaging studies were performed with 18F-FTP and 18F-FDG in prostate and small cell lung tumor-bearing animal models. They have reported that 18F-FTP could be synthesized with high radiochemical yield. They conclude that 18F-FTP may provide potential applications in differential diagnosis and prediction of early treatment response for carcinoids. n nMolecular imaging science has been focused on imaging guidance in the areas of targeting epigenetic abnormalities and disease microenvironment in overcoming resistance in diseases. Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. Y.-J. Hsieh et al. reported the rational design of a triple reporter gene for multimodality molecular imaging. They reported that an optimized triple fusion reporter constructed with DsRedm-fl-ttksr39 was developed and validated for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities. Their findings may facilitate different fields of biomedical research and applications. n nThe use of image-guided technologies to select patient for personalized therapy and to monitor therapeutic outcomes is the focus of this special issue. S. H.-H. Yeh et al. reported the evaluation of inhibitory effect of recreational drugs on dopaminergic terminal neuron by 18F-FDOPA and whole-body autoradiography. They used 18F-FDOPA PET imaging and autoradiographic techniques toassess the impact of recreational drugs such as ketamine, cocaine, and methamphetamine on dopamine neurons in peripheral organs. They have demonstrated that both dynamic 18F-FDOPA PET and autoradiography were useful crossing-validating tools to evaluate the alteration of dopaminergic neurons in peripheral tissues. D. Smith et al. reported the application of patched targeting peptides for imaging and treatment of hedgehog positive breast tumors. Hedgehog (Hh) signaling is involved in breast cancer growth and metastasis and high tumor Sonic Hedgehog (SHh) expression is correlated with poor prognosis in invasive ductal carcinoma. Peptides which bind the PTCH receptor have recently been reported to have a growth inhibitory effect in tumors with activated Hh signaling. These peptides may be used as molecular imaging probes to monitor changes in Hh expression after chemotherapy. Their studies showed that peptides which bind the SHh docking site in PTCH-1 correlate with PTCH-1 expression and can be used to image PTCH-1 in vivo. They conclude that radiolabeled peptides may enable examining the activity of the Hh signaling pathway and evaluating response to anticancer therapies. n nIn summary, this special issue covers advances in molecular imaging in drug manufacturing under CGMP environment, preclinical drug discovery, image-guided drug response, and target validation using PET/SPECT hybrid with CT. n n nDavid J. Yang n nLan Pham n nMei-Hsiu Liao n nFan-Lin Kong n nHiroji Uemura n nYen-Yu Ian Shih

Abstract 4391: Overcoming primary ibrutinib resistance in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a rare and incurable subtype of B-cell lymphoma. In a phase II study of ibrutinib in MCL patients, most of the patients responded and had long durable remissions; however, 22.7% of patients were considered to be non-responsive to ibrutinib, and an additional 22/110 patients displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment, with both responses classified as primary ibrutinib resistant. Therefore, understanding the mechanisms mediating primary ibrutinib resistance may identify new prognostic and predictive markers and potential therapeutic targets. Bruton9s tyrosine kinase (BTK) plays an important role in B-cell development, activation, and differentiation. Upon B-cell receptor (BCR) activation, BTK is phosphorylated and activated by SYK. Phosphoinositide 3-kinase (PI3K) is recruited to the BCR, and the induction of these signaling activities leads to the downstream activation of multiple effector proteins, including nuclear factor-kB (NF-kB), AKT, RAS, mTOR and mitogen-activated protein kinase (MAPK). BTK and PI3K have been shown to function independently to mediate BCR signaling, suggesting that PI3K signaling activities may underlie ibrutinib resistance independently of BTK. PI3K has also been associated with primary ibrutinib resistance. To further elucidate the mechanisms underlying ibrutinib resistance, we conferred primary ibrutinib resistance using both MCL cell lines and MCL-bearing patient-derived xenograft (PDX) mouse models and used whole exome sequencing (WES) and reverse phase protein analysis (RPPA) to identify any genetic and expression changes associated with primary ibrutinib resistance. WES did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. RPPA analysis showed a significant increase in the PI3K/AKT/mTOR/MCL-1 compensatory pathway component levels in ibrutinib-resistant cell lines when compared with their parental cells as well as ibrutinib-resistant PDXs. To determine whether inhibiting these pathways would overcome primary ibrutinib resistance, we tested various therapeutic combinations targeting these pathways. Ibrutinib plus the PI3K inhibitor idelalisib, the AKT inhibitor ACP-319, the mTOR inhibitors AZD8055 or BEZ235 as well as the proteasome inhibitor carfilzomib inhibited tumor growth in vitro and in vivo in the PDX mouse models, demonstrating that targeting these alternative pathways may overcome ibrutinib resistance. This work strongly suggests that targeting the PI3K/AKT/mTOR/MCL-1 compensatory pathway successfully inhibits the viability of ibrutinib-resistant MCL tumor cells both in vitro and in vivo and identified potential therapies that can be used to treat ibrutinib-resistant patients in clinical practice. Citation Format: Leo Zhang, Lan Pham, Hui Zhang, Jingmeng Xie, Wenjing Tao, Taylor Bell, Zhihong Chen, Krystle Nomie, Bingliang Fang, Michael Wang. Overcoming primary ibrutinib resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4391.

Abstract B18: B-cell lymphoma patient-derived xenograft models: The road to personalized therapy

Lymphoma is the most common hematological malignancy, and B-cell lymphoma accounts for 85% of all lymphomas. The current overall cure rate for B-cell lymphoma is estimated at ~30%, even with the development and application of novel therapies, and the majority of patients relapse after treatment due to the development of drug resistance. The evaluation of novel drug targets using established B-cell lymphoma cell lines is limited by the inexact correlation between responsiveness observed in the cell line versus the patient sample. However, patient-derived xenograft (PDX) mouse models have been shown to recapitulate the diversity of growth, metastasis, and histopathology of the original tumor, overcoming the limitations of cell lines. Furthermore, recent studies have indicated that PDXs can recapitulate treatment responses of the parental tumor and can successfully choose a therapeutic target and regimen for the patient. We previously established a SCID-hu in vivo human primary mantle cell lymphoma (MCL) PDX model, the first human primary MCL animal model for biological and therapeutic research, and investigated the disease biology and potential novel human MCL therapies using this model. In this MCL PDX model, the engraftment and growth of patient MCL cells were dependent on the human bone marrow microenvironment supplied by an implanted human fetal bone chip. Our clinical information and reports show that numerous B-cell lymphoma subtypes involve bone marrow; therefore, we expanded our PDX model to include various B-cell lymphomas to study the clonal evolution of tumors and to evaluate novel therapies for the treatments of these diseases. PDX models (n=20) were established for multiple different types of B-cell lymphoma, including MCL (n=12), Burkitt9s lymphoma (n=1), marginal zone lymphoma (n=2), follicular lymphoma (n=2), chronic lymphocytic leukemia (n=1) and diffuse large B-cell lymphoma (n=2). The engraftment rate was high at 95%, the tumor xenografts rapidly grew at 3-4 weeks/generation, and 68% of the tumor xenografts were passaged for multiple generations, even up to 14 generations. Further demonstrating the utility of this model to recapitulate the characteristics of the original patient tumor, the tumor xenograft cells migrated to the lymph nodes, spleen, bone marrow, and gastrointestinal tract of the host mice, mimicking the disease progression observed in humans, and HE Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B18.

Abstract 306: Development of EC-DG as a molecular theranostic personalized medicine

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnPurpose: D-glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. We have then synthesized Tc-99m- ethylenedicysteine-glucosamine (EC-DG). We found Tc-99m-EC-DG was involved in cell proliferation in lung, breast and head and neck cultures and could assess breast cancer treatment outcome in vivo by planar scintigraphy. Tc-99m-EC-DG is a safe imaging agent in lung cancer patients. This study was amied to (1) assess a novel response to unlabeled rhenium-EC-DG (Re-EC-DG) involving the translation regulation of hypoxia inducible factor (HIF)-1alpha expression in lymphoma cells, and, (2) evaluate feasibility of using EC-DG for theranostic approaches in cancers.nnMethods: For theranostic assessment studies, we synthesized cold Re-EC-DG. Re-EC-DG was synthesized via a two-step synthesis. The first step was to synthesize Re-EC by reacting rheniumoxo trichloride with EC. The second step was to react Re-EC with D-glucosamine tetraacetate, followed by de-acetylation. Twelve types of DLBCL cells were incubated with Re-EC-DG at various concentrations (0-10 mM) and TUNEL assays were used to determine cell apoptosis. To ascertain the mechanism of the anticancer properties for Re-EC-DG, DLBCL-LY10 cells were treated with Re-EC-DG (0-5 mM) for 48 hrs. Immunoblotting were then performed on nuclear extracts with 50 µg. For radiotheranostic assessment studies, 13762 breast tumor-bearing rats were imaged with In-111-EC-DG and tumor/muscle ratios were determined at 0.5-24 hrs. Radiation absorbed dose was estimated for the use of Y-90-EC-DG.nnResults: There was a dose response relationship of Re-EC-DG inhibition in DLBCL cells. Extensive apoptosis was observed at 24 hrs in lymphoma cell cultures. Re-EC-DG showed significant tumorcidal activity compared to normal B-lymphocyte activity at doses >0.17 µmol. Re-EC-DG caused a decreased expression of HIF-1alpha under normoxic conditions in DLBCL-LY10 cells. Tumor-to-muscle ratios for In-111-EC-DG were 5.43±0.45 to 7.80±0.05 whereas In-111-EC had 3.24±0.32 to 4.64±0.16 at 0.5-24 hrs. Radiation exposure of In-111-EC-DG to whole body, blood-forming organs, gonads, and effective dose equivalent for a single dose at 5 mCi was below the limits of 3 rad annually and 5 rad total. The absorbed dose in all other organs was below the limits of 5 rad annually and 15 rad total.nnConclusion: EC-DG is a useful molecular theranostic compound. In-111-EC-DG has favorable dosimetry, providing a potential use of Y-90-EC-DG to treat cancers. Re-EC-DG inhibits HIF-1alpha expression and is an attractive anti-proliferation compound.nnCitation 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 306. doi:10.1158/1538-7445.AM2011-306