Miriam R. Anver

PET/CT imaging of renal cell carcinoma with (18)F-VM4-037: a phase II pilot study.

BackgroundCarbonic anhydrase IX (CA-IX) is a potential imaging biomarker of clear cell renal cell carcinoma (ccRCC). Here, we report the results of a phase II clinical trial of a small molecule radiotracer targeting CA-IX (18F-VM4-037) in ccRCC.MethodsBetween October 2012 and May 2013, 11 patients with kidney masses underwent 18F-VM4-037 PET/CT prior to surgery. Dynamic imaging was performed for the first 45xa0min post injection and whole-body imaging was obtained at 60xa0min post injection. Tumors were surgically excised or biopsied within 4xa0weeks of imaging.ResultsAll patients tolerated the radiotracer well with no adverse events. Ten of the 11 patients had histologically confirmed malignancy. One patient had a Bosniak Type 3 cyst with no tumor found at surgery. Two patients had extrarenal disease and 9 had tumors only in the kidney. Primary ccRCC lesions were difficult to visualize on PET alone due to high uptake of the tracer in the adjacent normal kidney parenchyma, however when viewed in conjunction with CT, the tumors were easily localized. Metastatic lesions were clearly visible on PET. Mean SUV for primary kidney lesions was 2.55 in all patients; in patients with histologically confirmed ccRCC, the mean SUV was 3.16. The time-activity curves (TAC) are consistent with reversible ligand binding with peak activity concentration at 8xa0min post injection followed by washout. Distribution Volume Ratio (DVR) of the lesions was measured using the Logan graphical analysis method. The mean DVR value across the 9 kidney lesions was 5.2xa0±xa02.8, (range 0.68–10.34).Conclusion18F-VM4-037 is a well-tolerated PET agent that allows same day imaging of CA-IX expression. The agent demonstrated moderate signal uptake in primary tumors and excellent visualization of CA-IX positive metastases. While the evaluation of primary ccRCC lesions is challenging due to high background activity in the normal kidney parenchyma, 18F-VM4-037 may be most useful in the evaluation of metastatic ccRCC lesions.

Mass Spectrometry–Based Metabolomics Identifies Longitudinal Urinary Metabolite Profiles Predictive of Radiation-Induced Cancer

Nonlethal exposure to ionizing radiation (IR) is a public concern due to its known carcinogenic effects. Although latency periods for IR-induced neoplasms are relatively long, the ability to detect cancer as early as possible is highly advantageous for effective therapeutic intervention. Therefore, we hypothesized that metabolites in the urine from mice exposed to total body radiation (TBI) would predict for the presence of cancer before a palpable mass was detected. In this study, we exposed mice to 0 or 5.4 Gy TBI, collected urine samples periodically over 1 year, and assayed urine metabolites by using mass spectrometry. Longitudinal data analysis within the first year post-TBI revealed that cancers, including hematopoietic, solid, and benign neoplasms, could be distinguished by unique urinary signatures as early as 3 months post-TBI. Furthermore, a distinction among different types of malignancies could be clearly delineated as early as 3 months post-TBI for hematopoietic neoplasms, 6 months for solid neoplasms, and by 1 year for benign neoplasms. Moreover, the feature profile for radiation-exposed mice 6 months post-TBI was found to be similar to nonirradiated control mice at 18 months, suggesting that TBI accelerates aging. These results demonstrate that urine feature profiles following TBI can identify cancers in mice prior to macroscopic detection, with important implications for the early diagnosis and treatment.

Combination of 13-Cis Retinoic Acid and Lovastatin: Marked Antitumor Potential In Vivo in a Pheochromocytoma Allograft Model in Female Athymic Nude Mice

Currently, there are no reliably effective therapeutic options for metastatic pheochromocytoma (PCC) and paraganglioma. Moreover, there are no therapies that may prevent the onset or progression of tumors in patients with succinate dehydrogenase type B mutations, which are associated with very aggressive tumors. Therefore, we tested the approved and well-tolerated drugs lovastatin and 13-cis-retinoic acid (13cRA) in vitro in an aggressive PCC mouse cell line, mouse tumor tissue-derived (MTT) cells, and in vivo in a PCC allograft nude mouse model, in therapeutically relevant doses. Treatment was started 24 hours before sc tumor cell injection and continued for 30 more days. Tumor sizes were measured from outside by caliper and sizes of viable tumor mass by bioluminescence imaging. Lovastatin showed antiproliferative effects in vitro and led to significantly smaller tumor sizes in vivo compared with vehicle treatment. 13cRA promoted tumor cell growth in vitro and led to significantly larger viable tumor mass and significantly faster increase of viable tumor mass in vivo over time compared with vehicle, lovastatin, and combination treatment. However, when combined with lovastatin, 13cRA enhanced the antiproliferative effect of lovastatin in vivo. The combination-treated mice showed slowest tumor growth of all groups with significantly slower tumor growth compared with the vehicle-treated mice and significantly smaller tumor sizes. Moreover, the combination-treated group displayed the smallest size of viable tumor mass and the slowest increase in viable tumor mass over time of all groups, with a significant difference compared with the vehicle- and 13cRA-treated group. The combination-treated tumors showed highest extent of necrosis, lowest median microvessel density and highest expression of α-smooth muscle actin. The combination of high microvessel density and low α-smooth muscle actin is a predictor of poor prognosis in other tumor entities. Therefore, this drug combination may be a well-tolerated novel therapeutic or preventive option for malignant PCC.

Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies

Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM.

Gadd45a acts as a modifier locus for lymphoblastic lymphoma.

Gadd45a−/− and p53−/− mice and cells derived from them share similar phenotypes, most notably genomic instability. However, p53−/− mice rapidly develop a variety of neoplasms, while Gadd45a−/− mice do not. The two proteins are involved in a regulatory feedback loop, whereby each can increase the expression or activity of the other, suggesting that common phenotypes might result from similar molecular mechanisms. Mice lacking both genes were generated to address this issue. Gadd45a−/−p53−/− mice developed tumors with a latency similar to that of tumor-prone p53−/− mice. However, while p53−/− mice developed a variety of tumor types, nearly all Gadd45a−/−p53−/− mice developed lymphoblastic lymphoma (LBL), often accompanied by mediastinal masses as is common in human patients with this tumor type. Deletion of Gadd45a in leukemia/lymphoma-prone AKR mice decreased the latency for LBL. These results indicate that Gadd45a may act as modifier locus for T-cell LBL, whereby deletion of Gadd45a enhances development of this tumor type in susceptible mice. Gadd45a is localized to 1p31.1, and 1p abnormalities have been described in T-cell lymphomas. Related human tumor samples did not show Gadd45a deletion or mutation, although changes in expression could not be ruled out.

Interleukin-27 Is a Potential Rescue Therapy for Acute Severe Colitis Through Interleukin-10–Dependent, T-Cell–Independent Attenuation of Colonic Mucosal Innate Immune Responses

Background: If treatment with intravenous steroids fail, inflammatory bowel disease patients with acute severe colitis face systemic anti–tumor necrosis factor biologic rescue therapy or colectomy. Interleukin (IL)-27 is a cytokine with an immunosuppressive role in adaptive immune responses. However, the IL-27 receptor complex is also expressed on innate immune cells, and there is evidence that IL-27 can impact the function of innate cell subsets, although this particular functionality in vivo is not understood. Our aim was to define the efficacy of IL-27 in acute severe colitis and characterize novel IL-27–driven mechanisms of immunosuppression in the colonic mucosa. Methods: We assessed oral delivery of Lactococcus lactis expressing an IL-27 hyperkine on the innate immune response in vivo in a genetically intact, noninfective, acute murine colitis model induced by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid in SJL/J mice. Results: IL-27 attenuates acute severe colitis through the reduction of colonic mucosal neutrophil infiltrate associated with a decreased CXC chemokine gradient. This suppression was T cell independent and IL-10 dependent, initially featuring enhanced mucosal IL-10. IL-27 was associated with a reduction in colonic proinflammatory cytokines and induced a multifocal, strong, positive nuclear expression of phosphorylated STAT-1 in mucosal epithelial cells. Conclusion: We have defined novel mechanisms of IL-27 immunosuppression toward colonic innate immune responses in vivo. Mucosal delivery of IL-27 has translational potential as a novel therapeutic for inflammatory bowel disease, and it is a future mucosal directed rescue therapy in acute severe inflammatory bowel disease.

Cancer Incidence in C3H Mice Protected from Lethal Total-Body Radiation after Amifostine

Amifostine is a potent antioxidant that protects against ionizing radiation effects. In this study, we evaluated the effect of Amifostine administered before total-body irradiation (TBI), at a drug dose that protects against TBI lethality, for potential protection against radiation-induced late effects such as a shortened lifespan and cancer. Three groups of mice were studied: 0 Gy control; 10.8 Gy TBI with Amifostine pretreatment; and 5.4 Gy TBI alone. Animals were monitored for their entire lifespan. The median survival times for mice receiving 0, 5.4 or 10.8 Gy TBI were 706, 460 and 491 days, respectively. Median survival of both irradiated groups was significantly shorter compared to nonirradiated mice (P < 0.0001). Cancer incidence (hematopoietic and solid tumors) was similar between the irradiated groups and was significantly greater than for the 0 Gy controls. The ratio of hematopoietic-to-solid tumors differed among the groups, with the 5.4 Gy group having a higher incidence of hematopoietic neoplasms compared to the 10.8 Gy/Amifostine group (1.8-fold). Solid tumor incidence was greater in the 10.8 Gy/Amifostine group (1.6-fold). There are few mouse lifespan studies for agents that protect against radiation-induced lethality. Mice treated with 10.8 Gy/Amifostine yielded a lower incidence of hematopoietic neoplasms and higher incidence of solid neoplasms. In conclusion, mice protected from lethal TBI have a shortened lifespan, due in large part to cancer induction after exposure compared to nonexposed controls. Amifostine treatment did protect against radiation-induced hematopoietic tumors, while protection against solid neoplasms was significant but incomplete.

Abstract POSTER-TECH-1102: Caspase 8 cooperates with IKKβ to protect ovarian cancer cells from necroptosis

Ovarian cancer is a leading cause of cancer death in women, and identification of factors determining clinical outcome is essential to enable pathway-directed therapeutic advances. We recently showed that NF-κB signaling is constitutively active in a subset of ovarian cancer patients. Over-expression of the critical IκBα-regulatory kinase IKKβ, and its defined NF-κB gene signature, correlated with poor overall survival in that population. In order to identify cooperating pathways engaging in compensatory signaling in NF-κB-dependent ovarian cancer cells, genome-wide RNA interference was used as a loss-of-function genetic screen for key regulators of cell survival in response to IKKβ inhibition. The pro-apoptotic protease CASPASE 8 was identified. Ovarian cancer cells became more sensitive to killing with IKKb inhibitor when CASPASE 8 was depleted by shRNA. In agreement, co-expression of CASPASE 8 and NF-κB gene signature was observed in two large cohorts of primary ovarian cancer patient samples, and was largely confined to immune-related subtypes. In vitro, TNFα stimulation of Ovcar3 cells required CASPASE 8 for apoptotic cell death induced by IKKβ or cIAP1 inhibition, suggesting that both enzymatic activity of CASPASE 8 and its association with TNFR-induced cIAP protein complexes are mechanistically involved. However, CASPASE 8 depletion combined with IKKβ inhibition and cIAP1 depletion produced additional, CASPASE 8-independent cell death, in a RIP1-dependent manner. The data presented herein demonstrate our novel finding that Caspase 8 signals in conjunction with IKKβ to maintain viability of a subtype ovarian cancer cells, especially in the presence of TNFα stimulation. Ovarian cancer is known to be a heterogeneous disease, prone to developing resistance to chemotherapies designed primarily to induce apoptosis. The NF-kB pathway is one means by which tumors survive. Our findings point towards a new avenue for treatment of this disease, namely that cancers may be therapeutically susceptible to inducers of necroptosis, in addition to IKKβ blockade. The current data link our in vitro findings to primary patient samples, underscoring the relevance of our findings to future clinical application. Therefore, dual therapy with an IKKβ inhibitor and apoptosis- or necroptosis-inducing treatments may add together in molecularly selected ovarian cancer patient subsets. Citation Format: Lidia Hernandez, Anne M. Noonan, Ethan Sagher, Holger Kohlhammer, George Wright, L. Tiffany Reed, Patricia S. Steeg, Miriam Anver, David D. Bowtell, Christina M. Annunziata. Caspase 8 cooperates with IKKβ to protect ovarian cancer cells from necroptosis [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1102.

Abstract 1251: Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnNADPH oxidase 5 (Nox5), one of the seven members of the NADPH oxidase gene family that generate reactive oxygen species (ROS), has been implicated in physiological and pathophysiological processes. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas; expression in most non-malignant tissues was weak to modest. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms regulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 melanoma cells by generating stable Nox5-overexpressing clones and knockdown clones. Overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that Nox5 expression in human melanoma UACC-257 cells could contribute to cell proliferation, invasion, amoeboid morphology, and the ability to grow in the absence of serum or growth factors in part due to the generation of high local concentrations of extracellular ROS that modulate multiple signaling networks that regulate Hif-1α, p27Kip1, and the cadherin switch.nnCitation Format: Smitha Antony, Yongzhong Wu, Guojian Jiang, Jennifer L. Meitzler, Han Liu, Agnes Juhasz, Jiamo Lu, Miriam R. Anver, Krishnendu K. Roy, James H. Doroshow. Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2015-1251