Cure of Micrometastatic B-Cell Lymphoma in a SCID Mouse Model Using 213Bi-Anti-CD20 Monoclonal Antibody.

We studied the feasibility of using the α-emitting 213Bi-anti-CD20 therapy with direct bioluminescent tracking of micrometastatic human B-cell lymphoma in a SCID mouse model. Methods: A highly lethal SCID mouse model of minimal-tumor-burden disseminated non-Hodgkin lymphoma (NHL) was established using human Raji lymphoma cells transfected to express the luciferase reporter. In vitro and in vivo radioimmunotherapy experiments were conducted. Single- and multiple-dose regimens were explored, and results with 213Bi-rituximab were compared with various controls, including no treatment, free 213Bi radiometal, unlabeled rituximab, and 213Bi-labeled anti-HER2/neu (non-CD20-specific antibody). 213Bi-rituximab was also compared in vivo with the low-energy ß-emitter 131I-tositumomab and the high-energy ß-emitter 90Y-rituximab. Results: In vitro studies showed dose-dependent target-specific killing of lymphoma cells with 213Bi-rituximab. Multiple in vivo studies showed significant and specific tumor growth delays with 213Bi-rituximab versus free 213Bi, 213Bi-labeled control antibody, or unlabeled rituximab. Redosing of 213Bi-rituximab was more effective than single dosing. With a single dose of therapy given 4 d after intravenous tumor inoculation, disease in all untreated controls, and in all mice in the 925-kBq 90Y-rituximab group, progressed. With 3,700 kBq of 213Bi-rituximab, 75% of the mice survived and all but 1 survivor was cured. With 2,035 kBq of 131I-tositumomab, 75% of the mice were tumor-free by bioluminescent imaging and 62.5% survived. Conclusion: Cure of micrometastatic NHL is achieved in most animals treated 4 d after intravenous tumor inoculation using either 213Bi-rituximab or 131I-tositumomab, in contrast to the lack of cures with unlabeled rituximab or 90Y-rituximab or if there was a high tumor burden before radioimmunotherapy. α-emitter-labeled anti-CD20 antibodies are promising therapeutics for NHL, although a longer-lived α-emitter may be of greater efficacy.

Myocardial substrate and route of administration determine acute cardiac retention and lung bio-distribution of cardiosphere-derived cells.

BACKGROUND: Quantification of acute myocardial retention and lung bio-distribution of cardiosphere-derived cells (CDCs) following transplantation is important to improve engraftment. METHODS AND RESULTS: We studied acute(1 hour) cardiac/lung retention in 4 groups (n = 25) of rats (normal--NL, acute ischemia-reperfusion--AI-RM, acute permanent ligation-PL, and chronic infarct by ischemia-reperfusion--CI-R) using intra-myocardial delivery, 1 group using intracoronary delivery (acute ischemia-reperfusion, AI-RC, n = 5) and 1 group using intravenous delivery (acute ischemia-reperfusion, AI-RV, n = 5) of CDCs by PET. Cardiac retention was similar in the NL, AI-RM, CI-R, and A-IRC groups (13.6% ± 2.3% vs. 12.0% ± 3.9% vs. 9.9 ± 2.8 vs. 15.4% ± 5.5%; P = NS), but higher in PL animals (22.9% ± 5.2%; P < .05). Low cardiac retention was associated with significantly higher lung activity in NL and AI-RM groups (43.3% ± 5.6% and 39.9% ± 9.3%), compared to PL (28.5% ± 5.9%), CI-R (20.2% ± 9.3%), and A-IRC (19.9% ± 5.6%) animals (P < .05 vs. AI-RM and NL). Lung activity was highest following intravenous CDC delivery (55.1% ± 9.3%, P < .001) and was associated with very low cardiac retention (0.8% ± 1.06%). Two-photon microscopy indicated that CDCs escaped to the lungs via the coronary veins following intra-myocardial injection. CONCLUSIONS: Acute cardiac retention and lung bio-distribution vary with the myocardial substrate and injection route. Intra-myocardially injected CDCs escape into the lungs via coronary veins, an effect that is more pronounced in perfused myocardium.

CDC91L1 (PIG-U) is a newly discovered oncogene in human bladder cancer.

Genomic amplification at 20q11-13 is a common event in human cancers. We isolated a germline translocation breakpoint at 20q11 from a bladder cancer patient. We identified CDC91L1, the gene encoding CDC91L1 (also called phosphatidylinositol glycan class U (PIG-U), a transamidase complex unit in the glycosylphosphatidylinositol (GPI) anchoring pathway), as the only gene whose expression was affected by the translocation. CDC91L1 was amplified and overexpressed in about one-third of bladder cancer cell lines and primary tumors, as well as in oncogenic uroepithelial cells transformed with human papillomavirus (HPV) E7. Forced overexpression of CDC91L1 malignantly transformed NIH3T3 cells in vitro and in vivo. Overexpression of CDC91L1 also resulted in upregulation of the urokinase receptor (uPAR), a GPI-anchored protein, and in turn increased STAT-3 phosphorylation in bladder cancer cells. Our findings suggest that CDC91L1 is an oncogene in bladder cancer, and implicate the GPI anchoring system as a potential oncogenic pathway and therapeutic target in human cancers.

Specificity of the anti-glycolytic activity of 3-bromopyruvate confirmed by FDG uptake in a rat model of breast cancer.

PURPOSE: To evaluate the anti-glycolytic effects of 3-BrPA on rats bearing RMT mammary tumors, by determining FDG uptake after intravenous administration of the therapeutic dose. MATERIALS AND METHODS: Sixteen rats bearing RMT tumors were treated either with 15 mM 3-BrPA in 2.5 ml of PBS or with 2.5 ml of PBS. After treatment, all rats received FDG and were sacrificed 1 h later. RESULTS: 3-BrPA treatment significantly decreased FDG uptake in tumors by 77% (p = 0.002). FDG uptake did not significantly decrease in normal tissues after treatment. CONCLUSION: Our study showed that 3-BrPA exhibits a strong anti-glycolytic effect on RMT cells implanted in rats.

Targeting of VX2 rabbit liver tumor by selective delivery of 3-bromopyruvate: a biodistribution and survival study.

The aim of this study was to determine the biodistribution and tumor targeting ability of (14)C-labeled 3-bromopyruvate ([(14)C]3-BrPA) after i.a. and i.v. delivery in the VX2 rabbit model. In addition, we evaluated the effects of [(14)C]3-BrPA on tumor and healthy tissue glucose metabolism by determining (18)F-deoxyglucose (FDG) uptake. Last, we determined the survival benefit of i.a. administered 3-BrPA. In total, 60 rabbits with VX2 liver tumor received either 1.75 mM [(14)C]3-BrPA i.a., 1.75 mM [(14)C]3-BrPA i.v., 20 mM [(14)C]3-BrPA i.v., or 25 ml of phosphate-buffered saline (PBS). All rabbits (with the exception of the 20 mM i.v. group) received FDG 1 h before sacrifice. Next, we compared survival of animals treated with i.a. administered 1.75 mM [(14)C]3-BrPA in 25 ml of PBS (n = 22) with controls (n = 10). After i.a. infusion, tumor uptake of [(14)C]3-BrPA was 1.8 +/- 0.2% percentage of injected dose per gram of tissue (%ID/g), whereas other tissues showed minimal uptake. After i.v. infusion (1.75 mM), tumor uptake of [(14)C]3-BrPA was 0.03 +/- 0.01% ID/g. After i.a. administration of [(14)C]3-BrPA, tumor uptake of FDG was 26 times lower than in controls. After i.v. administration of [(14)C]3-BrPA, there was no significant difference in tumor FDG uptake. Survival analysis showed that rabbits treated with 1.75 mM 3-BrPA survived longer (55 days) than controls (18.6 days). Intra-arterially delivered 3-BrPA has a favorable biodistribution profile, combining a high tumor uptake resulting in blockage of FDG uptake with no effects on healthy tissue. The local control of the liver tumor by 3-BrPA resulted in a significant survival benefit.

In vitro evaluation of radioprotective and radiosensitizing effects of rituximab.

UNLABELLED: Clinical radioimmunotherapies with anti-CD20 monoclonal antibodies involve administering a predose of unlabeled anti-CD20 antibodies to favorably alter the biodistribution profile of the subsequently administered radiolabeled antibodies and mediate antitumor effects. Prior in vitro data suggested that unlabeled anti-CD20 monoclonal antibodies radiosensitize lymphoma cells as well. We assessed the antiproliferative and possible radiosensitizing capabilities of an anti-CD20 monoclonal antibody, rituximab. METHODS: Luciferase-transfected (via a lentivirus vector) CD20+ human Raji lymphoma cells in log-phase growth were incubated with or without rituximab (20 microg/mL) for either 1 or 24 h before external-beam radiation exposure. Cell counts were measured with a luciferase assay at 24-h intervals. Subsets of these cells were also analyzed for cell cycle status by flow cytometry. RESULTS: Rituximab pretreatment and irradiation were found to significantly inhibit tumor cell growth compared with irradiation alone (by a factor of 0.40 at 1 Gy [P < 0.01]). One hour of rituximab pretreatment modestly radiosensitized tumor cells at a radiation dose of 1 Gy (by a factor of 1.03 compared with the results for nonirradiated cells). At higher radiation doses (2 and 12 Gy), 1 h of rituximab pretreatment paradoxically radioprotected tumor cells by factors of 0.25 (P < 0.01) and 0.54 (P < 0.05), respectively. Rituximab predosing for 24 h was found to be radiosensitizing at a radiation dose of 4 Gy (by a factor of 2.84 [P < 0.01]) but radioprotective at radiation doses of 1, 8, and 12 Gy (by factors of 0.10 [P < 0.01], 2.50 [P < 0.01], and 2.07 [P < 0.05], respectively). These results correlated with retardation of the cell cycle at 6 d after rituximab administration, as determined by flow cytometry. CONCLUSION: Rituximab demonstrated a direct tumor antiproliferative effect in the absence of radiation. At lower levels of radiation exposure, rituximab radiosensitized Raji lymphoma cells. At higher doses of radiation, rituximab paradoxically protected tumor cells against ionizing radiation, possibly through effects on the cell cycle. These radiobiologic effects of rituximab should be carefully considered in the design of radioimmunotherapeutic trials.

Comparison of uptake of multiple clinical radiotracers into brown adipose tissue under cold-stimulated and nonstimulated conditions.

UNLABELLED: Our objective was to determine whether multiple clinically useful radiotracers accumulate in brown adipose tissue (BAT) and to assess their uptake in rats kept at room temperature or exposed to a cold environment. METHODS: The following radiotracers were injected intravenously into groups of 6 female Wistar rats: (201)Tl-chloride (TlCl), (123)I-metaiodobenzylguanidine (MIBG), (99m)Tc-sestamibi (MIBI), (18)F- or (3)H-FDG, (3)H-l-methionine, and (3)H-thymidine. BAT-stimulated animals were maintained at 4 degrees C for 4 h before tracer injection, whereas control animals were kept at approximately 22.5 degrees C. The animals were sacrificed at 20-60 min after tracer injection, and BAT, major organs, and blood were extracted, weighed, and measured for radioactivity. The localization of uncoupling protein-1, glucose transporter-1, and norepinephrine transporter was evaluated with immunohistochemical staining in both groups. RESULTS: We determined the percentage injected dose (%ID) per gram of each radiotracer in interscapular BAT, normalized to blood %ID/g. In control animals, this uptake ratio (+/-SD) was 8.44 +/- 3.39 for (201)TlCl, 9.77 +/- 6.06 for (123)I-MIBG, 37.30 +/- 14.42 for (99m)Tc-MIBI, 5.47 +/- 4.44 for (18)F- or (3)H-FDG, 1.93 +/- 0.87 for (3)H-l-methionine, and 1.22 +/- 0.74 for (3)H-thymidine. Compared with uptake at room temperature, uptake after exposure to cold increased 26.4-fold (P < 0.01) for (18)F- or (3)H-FDG and increased significantly (P < 0.05) for (201)Tl (2.04-fold), (123)I-MIBG (3.25-fold), and (3)H-l-methionine (3.11-fold). Immunohistochemical staining revealed increased glucose transporter-1 and norepinephrine transporter expression in BAT cell membranes and blood vessels after exposure to cold, whereas uncoupling protein-1 was expressed in the cytoplasm under both control and cold-stimulated conditions. CONCLUSION: BAT uptake of (18)F- or (3)H-FDG, (123)I-MIBG, and (3)H-l-methionine was significantly increased over the control state by exposure to cold. Increased uptake of (201)TlCl relative to blood in cold-stimulated BAT suggests that blood flow in BAT is increased by exposure to cold. The greater increased uptake with (18)F- or (3)H-FDG, (123)I-MIBG, and (3)H-l-methionine, and the immunohistostaining findings, suggest that other factors in addition to blood flow (e.g., increased metabolism, increased transport, or metabolic trapping of the tracers) are involved in cold-stimulated BAT activation. Knowledge that high uptake in BAT may possibly be observed on clinical scans using several radiotracers, especially after patients are exposed to the cold, may lead to more accurate interpretation of clinical studies.

Effect of nicotine and ephedrine on the accumulation of 18F-FDG in brown adipose tissue.

UNLABELLED: This study evaluated the effect of various beta-adrenergic agonists on (18)F-FDG uptake in brown adipose tissue (BAT) in rats using ex vivo biodistribution studies. METHODS: Caffeine (10 mg/kg of body weight, n = 4), ephedrine (5 mg/kg of body weight, n = 4), nicotine (0.8 mg/kg of body weight, n = 9), or a mixture of nicotine and ephedrine (0.8 mg/kg of body weight and 5 mg/kg of body weight, respectively, n = 9) was injected into the peritoneal cavity of female Lewis rats 30 min before intravenous (18)F-FDG injection. One hour after injection of (18)F-FDG, the animals were sacrificed, and BAT, other major organs, and blood were extracted. The biodistribution results were compared with body temperature data. RESULTS: In the rats injected with nicotine or ephedrine, the mean uptake of (18)F-FDG, in percentage injected dose (%ID)/(g of interscapular BAT) x (kg of body weight), was significantly increased (7.9-fold for nicotine and 3.7-fold for ephedrine), compared to the control rats. Nicotine had the strongest effect on (18)F-FDG uptake in BAT. Caffeine increased BAT uptake slightly, but this increase did not reach statistical significance. The combination of nicotine and ephedrine increased the uptake 12.0-fold, compared with control rats; more than either nicotine or ephedrine alone. Uptake of (18)F-FDG in most other major organs did not change significantly. The effect of nicotine was blocked by prior injection of beta-adrenergic antagonists. A transient decrease in body temperature was observed in the nicotine-injected group, and this effect was canceled by prior injection of beta-adrenergic antagonists. No significant change in baseline temperature was seen before or after beta-adrenergic agonist injection. CONCLUSION: Nicotine caused a greater increase in (18)F-FDG uptake in BAT than did other interventions, and the effect was increased when nicotine was combined with ephedrine. The effect of nicotine was completely blocked by prior injection of beta-adrenergic antagonists, indicating that beta-adrenergic agonists increase the metabolism of BAT. These preclinical data suggest that patients should avoid nicotine and ephedrine before undergoing (18)F-FDG PET to minimize (18)F-FDG uptake in BAT.

Effects of pegfilgrastim on normal biodistribution of 18F-FDG: preclinical and clinical studies.

UNLABELLED: The purpose of this study was to evaluate the effects of pegfilgrastim, a long-acting granulocyte colony-stimulating factor, on the normal biodistribution of (18)F-FDG in an animal model and in humans. METHODS: Two groups of 12 rats received a single subcutaneous injection of either normal saline or pegfilgrastim. One, 7, 14, and 21 d after injection, biodistribution studies were performed 1 h after (18)F-FDG injection. Sixteen breast cancer patients underwent baseline (18)F-FDG PET/CT and, approximately 1 wk after receiving 1 dose of docetaxel and adjunctive pegfilgrastim, follow-up (18)F-FDG PET/CT (scan 2). Standardized uptake values corrected for lean body mass (SUL) were determined for several normal organs before and after therapy. RESULTS: In rats, bone marrow (18)F-FDG uptake (standardized uptake value) was higher in the pegfilgrastim group 1 d after injection (mean +/- SD, 8.3 +/- 4.1 vs. 2.5 +/- 0.2, P < 0.05), whereas (18)F-FDG uptake in blood was lower (0.41 +/- 0.06 vs. 0.49 +/- 0.01, P < 0.05). In patients, mean SUL was higher in bone marrow (4.49 +/- 1.50 vs. 1.33 +/- 0.22, P < 0.0001), spleen (3.29 +/- 0.83 vs. 1.23 +/- 0.23, P < 0.0001), and liver (1.45 +/- 0.25 vs. 1.31 +/- 0.23, P = 0.01) but lower in brain (4.18 +/- 0.76 vs. 5.14 +/- 1.44, P < 0.01) on scan 2 than on the baseline scan. CONCLUSION: In both the animal model and humans, pegfilgrastim markedly increased bone marrow uptake of (18)F-FDG and reduced (18)F-FDG uptake in some normal tissues. These profound alterations in (18)F-FDG biodistribution induced by pegfilgrastim must be considered when one is evaluating quantitative (18)F-FDG PET scans for tumor response to therapy.

Stunning and its effect on 3H-FDG uptake and key gene expression in breast cancer cells undergoing chemotherapy.

UNLABELLED: Shortly after chemotherapy, relatively little is known about the expression of key genes and proteins involved in glycolysis. Doxorubicin (DOX) and 5-fluorouracil (5FU) are two commonly used chemotherapy agents that work through differing pathways. Glucose transporter-1 (Glut-1) and hexokinase II (HKII) proteins are highly expressed in many breast carcinomas, but their status while undergoing DOX or 5FU chemotherapy has not been systematically evaluated. METHODS: We evaluated, in vitro, the messenger RNA (mRNA) and protein levels of Glut-1 and HKII in MCF-7, a breast adenocarcinoma cell line, and (3)H-FDG uptake, both in untreated conditions and during treatment with either DOX or 5FU for 24 h. Six time points were evaluated: untreated at time 0; treated for 1 h; treated for 24 h; and 1, 2, and 3 d after chemotherapy. We analyzed tumor cell Glut-1 and HKII mRNA expression with real-time polymerase chain reaction and Western blotting, (3)H-FDG uptake per cell, and cell viability with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. RESULTS: DOX was more effective than 5FU in killing the cancer cells under our study conditions. In untreated MCF-7 cells, the mRNA levels of HKII were typically higher than those of Glut-1, and (3)H-FDG uptake was strongly related to Glut-1 mRNA expression (R(2) = 0.85). Once treated with either drug, (3)H-FDG uptake declined initially, the mRNA ratio was reversed, and Glut-1 mRNA levels were higher than HKII levels. This was verified in the protein assay. With DOX treatment, the cells showed increased Glut-1 mRNA and decreased HKII mRNA for the duration of active treatment; these levels returned to those seen in the untreated cells once the treatment was stopped for 24 h. However, HKII protein levels remained somewhat low. No correlation was seen between (3)H-FDG uptake and HKII mRNA in DOX- and 5FU-treated cells (R(2) = 0.14 and 0.0038, respectively). CONCLUSION: After DOX or 5FU therapy, the relationship between (3)H-FDG uptake and viable cell number can become disjointed, with transient declines in (3)H-FDG uptake in excess of the decline in cell number despite increased Glut-1 mRNA levels. This transient "stunning" has potential implications for (3)H-FDG PET, especially soon after treatment is initiated. However, (3)H-FDG remains a generally valid marker of viable cell number after cancer chemotherapy.

In vitro evaluation of adenosine 5'-monophosphate as an imaging agent of tumor metabolism.

UNLABELLED: Adenosine appears to play an important role in tumor growth and metastasis. Synthesized (11)C-adenosine 5'-monophosphate (AMP) has recently been reported as a potential tumor-imaging radiotracer. METHODS: A variety of human tumor cell lines (SKOV-3, SCC-15, U251, U87, Raji, and Daudi) were incubated with 3.7 kBq (0.1 microCi) of [2-(3)H]AMP ((3)H-AMP), [5,6-(3)H]FDG ((3)H-FDG), or [2-(3)H]adenosine ((3)H-adenosine) in low-physiologic-glucose serum-free medium. Selected cells were exposed to caffeine, dipyridamole, adenosine 5'-(alpha,beta-methylene)diphosphate (APCP), or unlabeled adenosine before exposure to the radiotracer. R-phycoerythrin-conjugated mouse antihuman monoclonal antibody to human CD73 was used for immunophenotyping. High-performance liquid chromatography was used to characterize the intracellular metabolites of (3)H-AMP after intracellular uptake. RESULTS: Intracellular uptake of (3)H-AMP was significant-10 to 100 times the uptake of (3)H-FDG, depending on the particular tumor cell line. Preexposure of SKOV-3 cells to caffeine, a competitive inhibitor of adenosine receptors, did not affect cellular uptake of (3)H-AMP. However, preexposure of SKOV-3 cells to dipyridamole, an equilibrative nucleoside transporter inhibitor; APCP, a CD73 (ecto-5'-nucleotidase) inhibitor; or cold adenosine significantly inhibited cellular uptake of (3)H-AMP. SKOV-3 uptake of (3)H-adenosine was inhibited by dipyridamole but not APCP. U251 uptake of (3)H-AMP was significantly inhibited by dipyridamole and APCP. U87 uptake of (3)H-AMP was only partially inhibited by dipyridamole and APCP. However, Raji and Daudi cells had significantly lower uptake of (3)H-AMP than of (3)H-FDG but had significantly increased uptake of (3)H-adenosine, which was inhibited by dipyridamole. Raji and Daudi cells were negative, but the SKOV-3 cells positive, for CD73 cell-surface expression. (3)H-Adenosine metabolites were persistently retained after influx into the cell, predominantly as (3)H-adenosine triphosphate and (3)H-adenosine diphosphate. CONCLUSION: Cancer cell lines evaluated in vitro had significantly elevated uptake of radiolabeled AMP, on the order of 10- to 100-fold, in comparison to radiolabeled FDG. The mechanism of intracellular uptake depends predominantly on equilibrative nucleoside transporters after conversion of AMP to adenosine by CD73 in SKOV-3, SCC-15, and U251 cells. Intracellular retention is due to phosphorylation to adenosine triphosphate and adenosine diphosphate. Raji and Daudi cells have low uptake of radiolabeled AMP because of a lack of CD73 expression. This in vitro evaluation using (3)H-AMP with tumor cell lines supports the potential of (11)C-AMP for use in targeting the nucleoside transport pathway in PET imaging of tumors.

High-throughput molecular analysis of urine sediment for the detection of bladder cancer by high-density single-nucleotide polymorphism array.

The detection of urothelial malignancies remains challenging. The majority of patients diagnosed with bladder cancer require life-long surveillance for disease recurrence. Monitoring strategies rely predominantly on invasive endoscopic techniques, which are inconvenient and uncomfortable. Multiple in vitro diagnostic technologies have been developed to supplant the contemporary standard of care. The U.S. Food and Drug Administration has approved several assays, but [because of inferior performance characteristics (low sensitivity and specificity)] these tests have not made a significant impact on practice, to date. We sought to develop a test for bladder cancer with better performance characterization detection based on a novel molecular approach. Matched urine and peripheral blood lymphocyte samples were obtained before surgery from 31 patients with bladder cancer (10 pTa, 4 pT1, and 17 pT2>or). DNA from these samples was subjected to allelic imbalance analysis using HuSNP chips and was validated in parallel with microsatellite analysis for loss of heterozygosity and microsatellite instability. Peripheral blood lymphocyte and urine DNA obtained from 14 individuals without clinical evidence of genitourinary malignancy served as controls. Thirty-one of 31 (100%) urine DNA samples from patients with bladder tumors were found to have 24 or more single-nucleotide polymorphism (SNP) DNA alterations. In general, SNP alterations were more common in urine samples from pT2>or tumors than pTa or pT1 tumors. SNP alterations were not identified in nine normal control subjects and in four of five patients with hematuria. These data support the noninvasive HuSNP chip assay in urine DNA as a valuable tool for the detection of bladder cancer (on a high-throughput-automated platform).

Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer.

The serum of cancer patients often harbors increased free DNA levels, which can potentially be used for cancer detection. Because genetic and epigenetic alterations of the adenomatous polyposis coli (APC) gene are common events in gastrointestinal tumor development, we sought to investigate the frequency and level of aberrant APC promoter methylation in primary tumors and paired preoperative serum or plasma samples of lung cancer patients by semiquantitative methylation-specific fluorogenic real-time PCR. We detected methylation of APC in 95 of 99 (96%) primary lung cancer tissues. Forty-two of 89 (47%) available serum and/or plasma samples from these cases carried detectable amounts of methylated APC promoter DNA. In contrast, no methylated APC promoter DNA was detected in serum samples from 50 healthy controls. A highly elevated APC methylation level in lung cancer tissue was the only independent factor predicting inferior survival in this cohort (P = 0.015). APC methylation analysis appears to be promising as a prognostic factor in primary lung cancer and as a noninvasive tumor marker in plasma and/or serum DNA.

Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung.

Frequent losses of chromosome 19p have recently been observed in sporadic lung adenocarcinomas, targeting the location of a critical tumor suppressor gene. Here we performed fine mapping of the short arm of chromosome 19 and found that the LKB1/STK11 gene mapped in the minimal-deleted region. Because germ-line mutations at LKB1/STK11 result in the Peutz-Jeghers syndrome and an increased risk of cancer, we performed a detailed genetic screen of the LKB1/STK11 gene in lung tumors. We detected a high frequency of somatic alterations (mainly nonsense mutations) in primary lung adenocarcinomas and in lung cancer cell lines. Thus, our findings demonstrate for the first time that LKB1/STK11 inactivation is a very common event and may be integrally involved in the development of sporadic lung adenocarcinoma.

Interaction and colocalization of PGP9.5 with JAB1 and p27(Kip1).

PGP9.5 (UCH-L1) is a member of the ubiquitin C-terminal hydrolase (UCH) family of proteins that is expressed in neuronal tissues. Our previous studies have shown that PGP9.5 was highly expressed in primary lung cancers and lung cancer cell lines. Additionally, the frequency of PGP9.5 over expression increases with tumor stage, indicating that PGP9.5 may play a role in lung cancer tumorigenesis. We used the yeast two-hybrid system to identify proteins that interact with PGP9.5. We show that PGP9.5 interacts with at least three proteins, one of which is JAB1, a Jun activation domain binding protein that can bind to p27(Kip1) and is involved in the cytoplasmic transportation of p27(Kip1) for its degradation. We also show that PGP9.5 is associated with JAB1 in vitro and in vivo; and that both proteins can be a part of a heteromeric complex containing p27(Kip1) in the nucleus in lung cancer cells. Furthermore, under serum-restimulation, nuclear translocation of both PGP9.5 and JAB1 coincides with a reduced level of p27(Kip1) in the nucleus. In contrast, when cells are contact inhibited, both PGP9.5 and JAB1 became more perinuclear and cytoplasmic in localization while p27(Kip1) was present only in the nucleus. Therefore, PGP9.5 may contribute to p27(Kip1) degradation via its interaction and nuclear translocation with JAB1.

Intense (18)F-FDG uptake in brown fat can be reduced pharmacologically.

UNLABELLED: Physiologic (18)F-FDG uptake in areas of supraclavicular fat in humans ("USA-Fat") has recently been recognized as (18)F-FDG uptake in apparent brown adipose tissue (BAT) using fused PET/CT technology. In this study, we evaluated (18)F-FDG uptake in BAT of rats to determine whether pharmacologic or physiologic interventions affect the uptake, knowing that BAT has a high density of adrenergic innervation. METHODS: Seven- to 8-wk-old female Lewis rats receiving intravenous (18)F-FDG injections were examined under various conditions to evaluate (18)F-FDG biodistribution into interscapular BAT and major organs. In series 1, rats were given ketamine-based anesthesia or were exposed to cold (4 degrees C for 4 h) to determine whether these interventions increased (18)F-FDG uptake in BAT. In series 2, anesthetized rats (ketamine-based anesthesia) were given propranolol, reserpine, or diazepam intraperitoneally before (18)F-FDG injection to determine whether the drug reduced (18)F-FDG uptake in BAT. The control and treated groups in series 2 were also evaluated with (18)F-FDG PET/CT imaging. RESULTS: In series 1, anesthesia or exposure to cold increased (18)F-FDG uptake in BAT to levels 14-fold and 4.9-fold, respectively, greater than the control nonstimulated values. BAT uptake was high, comparable to that in the brain. In series 2, (18)F-FDG uptake in BAT was significantly decreased to less than 30% of the control level after propranolol or reserpine (P < 0.05). Diazepam did not significantly decrease (18)F-FDG uptake in BAT. (18)F-FDG PET/CT findings reflected these biodistribution data: The control and diazepam groups exhibited intense (18)F-FDG uptake in BAT, whereas the propranolol and reserpine groups showed only faint to mild (18)F-FDG uptake in BAT. Among several organs whose (18)F-FDG uptake was affected after predosing drugs, the heart exhibited considerable decreases in tracer uptake with propranolol or reserpine. CONCLUSION: This rodent study demonstrated that BAT can exhibit high (18)F-FDG uptake under stimulated conditions including exposure to cold and that propranolol or reserpine treatment can remarkably reduce the high (18)F-FDG uptake in BAT. The effect of these drugs on (18)F-FDG uptake in human BAT, as well as on tracer accumulation in other organs, should carefully be evaluated clinically to minimize the USA-Fat artifact.

Systemic administration of 3-bromopyruvate in treating disseminated aggressive lymphoma.

The Warburg hypothesis states that aggressive cancers obtain much of their adenosine triphosphate (ATP) by metabolizing glucose directly to lactic acid. As a result of its high tumor selectivity, 3-bromopyruvic acid (3-BrPA), a well-known inhibitor of energy metabolism, has been proposed as a specific anticancer agent. We investigated the effect of 3-BrPA in a mouse model of aggressive metastatic lymphoma. Epstein-Barr-virus-infected human Raji lymphoma cells with lentivirally transfected green fluorescent protein and luciferase were incubated with RPMI/fetal bovine serum, and various concentrations of 3-BrPA were used to determine the LD50 in vitro. In total, 18 severely combined immunodeficient mice were injected with 1 million human Raji lymphoma cells via the tail vein. Using bioluminescent imaging, tumor growth was measured daily for 12 days to determine the tumor burden. At day 0 (start of treatment), the mice were randomized. Six mice received 10 mg/kg 3-BrPA i.p. daily for 7 days, 6 mice received 1 treatment at day 0, and 6 mice received the control buffer. Tumor growth was assessed daily from day 0 until day 7 using bioluminescent imaging. All data were normalized to acquisition time (luminescence/second; L/s). Body weight was measured daily to determine the toxicity of 3-BrPA. The LD50 for Raji lymphoma cells exposed to 3-BrPA in vitro was 11 µM with an extremely steep dose response curve. At day 0, tumor activity medians in the group with daily treatment was 2131 L/s (244-12,725), with a 1-day dose of 3095 L/s (523-9650) and in the nontreated control group, 2997 L/s (1521-6911). In mice treated with a daily dose of 10 mg/kg 3-BrPa for 7 days, a significant reduction in tumor activity was found during the whole treatment period compared with the control mice (P = 0.0043 at day 7). In mice with a single treatment at day 0, growth delay was only evident at day 2 (P = 0.0152 at day 2) but not for the rest of the observation period. The only manifestation of toxicity of the daily administration of 10 mg/kg 3-BrPA was a reduction in body weight. Body weight at day 0 was 17.22 g ± 0.84 g in the treatment group and 17.58 g ± 0.86 g in the control group. Body weight at day +6 was 15.02 g ± 2.04 g in the treated group and 19.4 g ± 0.63 g in the control group. 3-BrPA demonstrated a significant positive tumor response both in vitro and in vivo. This, to our knowledge, is the first report of the use of 3-BrPA in a systemic tumor model. Based on these data, 3-BrPA holds promise for treatment of systemic metastatic cancers.

CT Hounsfield units of brown adipose tissue increase with activation: preclinical and clinical studies.

UNLABELLED: Brown adipose tissue (BAT) densities assessed as CT Hounsfield units (HUs) were evaluated in a rodent model and in patients to determine whether HUs changed in relation to BAT activity. METHODS: Serial (18)F-FDG PET/CT was performed on rats under both room temperature control conditions and after 4 h of cold-stimulation, which is known to activate BAT. The maximum standardized uptake values and CT HUs of BAT were measured, and tissues were examined in the laboratory. Image records from cancer patients who underwent PET/CT were reviewed, and 23 patients were identified who displayed both high and low (18)F-FDG uptake into BAT on serial (18)F-FDG PET/CT scans. The maximum standardized uptake values and CT HUs of BAT were compared in these scans. RESULTS: The mean (+/-SD) CT HUs of cold-activated BAT (-12.4 +/- 22.4) were significantly higher than those (-27.9 +/- 9.6) of the controls in the rat model. The CT HUs of BAT (-71.6 +/- 18.0) in the patients with high (18)F-FDG uptake were significantly higher than those (-104.4 +/- 16.8) of the patients with low (18)F-FDG uptake . A decrease in relative lipid content is seen in activated BAT in rats on histology. CONCLUSION: The CT HUs of BAT increased in activated conditions in both animals and patients, likely because of lipid consumption by activated BAT.

Induction of thyroid gene expression and radioiodine uptake in melanoma cells: novel therapeutic implications.

Both the MAP kinase and PI3K/Akt pathways play an important role in the pathogenesis of melanoma. We conducted the present study to test the hypothesis that targeting the two pathways to potently induce cell inhibition accompanied with thyroid iodide-handling gene expression for adjunct radioiodine ablation could be a novel effective therapeutic strategy for melanoma. We used specific shRNA approaches and inhibitors to individually or dually suppress the MAP kinase and PI3K/Akt pathways and examined the effects on a variety of molecular and cellular responses of melanoma cells that harbored activating genetic alterations in the two pathways. Suppression of the MAP kinase and PI3K/Akt pathways showed potent anti-melanoma cell effects, including the inhibition of cell proliferation, transformation and invasion, induction of G(0)/G(1) cell cycle arrest and, when the two pathways were dually suppressed, cell apoptosis. Remarkably, suppression of the two pathways, particularly simultaneous suppression of them, also induced expression of genes that are normally expressed in the thyroid gland, such as the genes for sodium/iodide symporter and thyroid-stimulating hormone receptor. Melanoma cells were consequently conferred the ability to take up radioiodide. We conclude that dually targeting the MAP kinase and PI3K/Akt pathways for potent cell inhibition coupled with induction of thyroid gene expression for adjunct radioiodine ablation therapy may prove to be a novel and effective therapeutic strategy for melanoma.

Noninvasive quantification and optimization of acute cell retention by in vivo positron emission tomography after intramyocardial cardiac-derived stem cell delivery.

OBJECTIVES: The aim of this study was to quantify acute myocardial retention of cardiac-derived stem cells (CDCs) and evaluate different delivery methods with positron emission tomography (PET). BACKGROUND: Success of stem cell transplantation for cardiac regeneration is partially limited by low retention/engraftment of the delivered cells. A clinically applicable method for accurate quantification of cell retention would enable optimization of cell delivery. METHODS: The CDCs were derived from syngeneic, male Wistar Kyoto (WK) rats labeled with [(18)F]-fluoro-deoxy-glucose ((18)FDG) and injected intramyocardially into the ischemic region of female WK rats after permanent left coronary artery ligation. The effects of fibrin glue (FG), bradycardia (adenosine), and cardiac arrest were examined. Imaging with (18)FDG PET was performed for quantification of cell retention. Quantitative polymerase chain reaction (PCR) for the male-specific SRY gene was performed to validate the PET results. RESULTS: Myocardial retention of cells suspended in phosphate-buffered saline 1 h after delivery was 17.6 +/- 11.5% by PCR and 17.8 +/- 7.3% by PET. When CDCs were injected immediately after induction of cardiac arrest, retention was increased to 75.6 +/- 18.6%. Adenosine slowed the ventricular rate and doubled CDC retention (35.4 +/- 5.3%). A similar increase in CDC retention was observed after epicardial application of FG at the injection site (37.5 +/- 8.2%). The PCR revealed a significant increase in 3-week cell engraftment in the FG animals (22.1 +/- 18.6% and 5.3 +/- 3.1%, for FG and phosphate-buffered saline, respectively). CONCLUSIONS: In vivo PET permits accurate measurement of CDC retention early after intramyocardial delivery. Sealing injection sites with FG or lowering ventricular rate by adenosine might be clinically translatable methods for improving stem cell engraftment in a beating heart.