Monitoring Therapy with MEK Inhibitor U0126 in a Novel Wilms Tumor Model in Wt1 Knockout Igf2 Transgenic Mice Using 18F-FDG PET with Dual-Contrast Enhanced CT and MRI: Early Metabolic Response Without Inhibition of Tumor Growth

Purpose

The understanding of the role of genetic alterations in Wilms tumor development could be greatly advanced using a genetically engineered mouse models that can replicate the development and progression of this disease in human patients and can be monitored using non-invasive structural and molecular imaging optimized for renal tumors.

Procedures

Repetitive dual-contrast computed tomography (CT; intravenous and intraperitoneal contrast), T2-weighted magnetic resonance imaging (MRI), and delayed 2-deoxy-2-[¹⁸F]fluoro-d-glucose (¹⁸F-FDG) positron emission tomography (PET) were utilized for characterization of Igf2 biallelic expression/Wt1 knockout mouse model of Wilms tumor. For CT imaging, Ioversol 678 mg/ml in 200 μl was administered i.p. followed by 100 μl injected intravenously at 20 and 15 min prior to imaging, respectively. Static PET imaging studies were acquired at 1, 2, and 3 h after i.v. administration of ¹⁸F-FDG (400 μCi). Coronal and sagittal T1-weighted images (T_E/T_R 8.5/620 ms) were acquired before and immediately after i.v. injection of 0.4 ml/kg gadopentetate dimeglumine followed by T2-weighted images (T_E/T_R 60/300 ms). Tumor tissue samples were characterized by histopathology and immunohistochemistry for Glut1, FASN, Ki67, and CD34. In addition, six Wt1-Igf2 mice were treated with a mitogen-activated protein kinase (MEK) inhibitor U0126 (50 μmol/kg i.p.) every 4 days for 6 weeks. ¹⁸F-FDG PET/CT imaging was repeated at different days after initiation of therapy with U0126. The percent change of initial tumor volume and SUV was compared to non-treated historic control animals.

Results

Overall, the best tumor-to-adjacent kidney contrast as well as soft tissue contrast for other abdominal organs was achieved using T2-weighted MRI. Delayed ¹⁸F-FDG PET (3-h post ¹⁸F-FDG administration) and dual-contrast CT (intravenous and intraperitoneal contrast) provided a more accurate anatomic and metabolic characterization of Wilms tumors in Wt1-Igf2 mice during early development and progression of renal tumors. Over the 8-month period, 46 Wt1-Igf2 mice and 8 littermate control mice were studied. Renal tumors were identified in 54.3 % of Wt1-Igf2 mice between post-natal 50–100 days. In 35.6 % of Wt1-Igf2 mice, tumors were localized in the right kidney; in 24 %, in the left kidney, while 40.4 % of Wt1-Igf2 mice had bilateral kidney tumors. Metastatic lesions were identified in 15.4 % of Wt1-Igf2 mice. Increased levels of Glut1 and IGF1R expression, high Ki67 labeling index, and a dense network of CD34+ microvessels in renal tumors was consistent with increased ¹⁸F-FDG accumulation. Treatment with a MEK 1/2 inhibitor U0126 did not cause the inhibition of tumor growth as compared to untreated animals. However, after the first three to four doses (~2 weeks of treatment), a decrease in ¹⁸F-FDG SUV was observed, as compared to pre-treatment levels (p?<?0.05, paired Student t test), which constitutes a metabolic response. Six weeks later, despite continuing therapy, the ¹⁸F-FDG SUV increased again to previous levels.

Conclusions

The optimized dual contrast PET/CT imaging with early post i.v. and i.p. contrast CT and 3 h delayed PET imaging after ¹⁸F-FDG administration provides a sensitive and reliable method for detecting early tumor lesions in this endogenous mouse model of Wilms tumor and for monitoring their growth in response to targeted therapies. Therapy with MEK inhibitor U0126 produces only a transient inhibition of tumor glycolytic activity but does not inhibit tumor growth, which is due to continuing IGF2-induced signaling from IGF1R through the PI3K-AKT-mTOR pathway.     

A Unique Interaction of IVS-I-1 (G>A) (HBA2: c.95+1G>A) with Hb Adana (HBA2: c.179G>A) Presenting as Transfusion-Dependent α-Thalassemia

Abstract

Nondeletional α-globin mutations are known to cause more serious clinical effects than deletional ones. A rare IVS-I-1 (G>A) (HBA2: c.95+1G>A) donor splice site mutation interferes with normal splicing of pre mRNA and results in activation of a cryptic splice site as well as a frameshift mutation. Hb Adana [HBA2: c.179G>A (or HBA1)] is a highly unstable variant hemoglobin (Hb) resulting from a mutation at codon 59 on the HBA2 or HBA1 gene, recognized to cause severe α-thalassemia (α-thal) syndromes. We report a unique case of compound heterozygosity for these two mutations in a 9-year-old boy who presented with a Hb level of 5.3?g/dL and hepatomegaly at the age of 15?months. He required regular blood transfusions in view of a Hb level of <7.0?g/dL and failure to thrive. He had thalassemic red cell indices and peripheral blood film. The Hb electrophoresis only showed a raised Hb F level (3.3%) and a pre run peak but the Hb H inclusion test was negative. His father had thalassemic red cell indices but a normal Hb level. His mother had almost normal Hb levels and red cell indices. Hb Adana involving the HBA2 gene was detected by mutiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in the proband and his father. DNA sequencing of the HBA2 gene confirmed the IVS-I-1 mutation in the proband and his mother. This case highlighted the unique interaction of the IVS-I-1 mutation with Hb Adana in a young Malay boy presenting with transfusion-dependent α-thal.     

Imaging of <Emphasis Type="Italic">Sleeping Beauty</Emphasis>-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography

Purpose

We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells.

Procedures

We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19⁺ artificial antigen-presenting cells.

Results

After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19⁺ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2′-deoxy-2′-[¹⁸F]fluoro-5-ethyl-1-β-d-arabinofuranosyl-uracil ([¹⁸F]FEAU).

Conclusion

This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

     

Blocking catabolism with eniluracil enhances PET studies of 5-[18F]fluorouracil pharmacokinetics.

Noninvasive methods for measuring the pharmacokinetics of chemotherapeutic drugs such as 5-fluorouracil (FU) are needed for individualized optimization of treatment regimens. PET imaging of [18F]FU (PET/[18F]FU) is potentially useful in this context, but PET/[18F]FU is severely hampered by low tumor uptake of radiolabel and rapid catabolism of FU in vivo. Pretreatment with eniluracil (5-ethynyluracil) prevents catabolism of FU. Hypothesizing that suppression of catabolism would enhance PET/[18F]FU, we examined the effects of eniluracil on the short-term pharmacokinetics of the radiotracer. METHODS: Anesthetized rats bearing a subcutaneous rat colorectal tumor were given eniluracil or placebo and injected intravenously 1 h later with [18F]FU or [3H]FU. In the 18F studies, dynamic PET image sequences were obtained 0-2 h after injection. Tumors were excised and frozen at 2 h and then analyzed for labeled metabolites by high-performance liquid chromatography. Biodistribution of radiolabel was determined by direct tissue assay. RESULTS: Eniluracil improved tumor visualization in PET images. With eniluracil, tumor standardized uptake values ([activity/g]/[injected activity/g body weight]) increased from 0.72 +/- 0.06 (mean +/- SEM; n = 6) to 1.57 +/- 0.20 (n = 12; P < 0.01), and tumor uptake increased by factors of 2 or more relative to plasma (P < 0.05) and bone, liver, and kidney (P < 0.01). Without eniluracil (n = 5), 57% +/- 4% of recovered radiolabel in tumor at 2 h was on catabolites, with the rest divided among FU (2% +/- 1%), anabolites of FU (38% +/- 7%), and unidentified peaks (4% +/- 2%). With eniluracil (n = 8), catabolites, FU, and anabolites comprised 2% +/- 1%, 41% +/- 5%, and 57% +/- 4%, respectively, of the recovered radiolabel in tumors. CONCLUSION: Eniluracil increased tumor accumulation of 18F relative to host tissues and fundamentally changed the biochemical significance of that accumulation. With catabolism suppressed, tumor radioactivity reflected the therapeutically relevant aspect of FU pharmacokinetics--namely, uptake and anabolic activation of the drug. With this approach, it may be feasible to measure the transport and anabolism of [18F]FU in tumors by kinetic modeling and PET. Such information may be useful in predicting and increasing tumor response to FU.     

Brain trace elements in Pick's disease

The trace element content of the brain of two patients with Pick's disease examined postmortem was studied using instrumental neutron activation analysis. Results showed significant increases in chlorine, iron, manganese, sodium, and phosphorus and significant decreases in chromium, cesium, rubidium, and selenium and in the mean freeze-dried to wet-weight ratio for patients with Pick's disease compared with control patients. Brain zinc content was not elevated in the two patients, a finding that fails to support the hypothesis that elevated zinc levels play a role in the pathogenesis of Pick's disease.     

A human-derived reporter gene for noninvasive imaging in humans: mitochondrial thymidine kinase type 2.

A human-derived intrinsically nonimmunogenic reporter gene was tested for PET imaging of different molecular-genetic processes for potential clinical use. METHODS: The human mitochondrial thymidine kinase type 2 (hTK2) reporter gene truncated at the N terminus (DeltahTK2), alone or fused with green fluorescent protein (GFP), was used for preclinical evaluation in a mouse model. The levels of enzymatic activity of DeltahTK2 and DeltahTK2 GFP proteins were assessed using radiotracer accumulation and prodrug activation assays in vitro and in subcutaneous tumors grown from the corresponding cell lines in nude mice. Kinetic analyses of (124)I-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-iodouracil (FIAU), (18)F-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-ethyluracil (FEAU), or (18)F-9-(4-(18)F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) uptake in tumors and biodistribution studies were performed. RESULTS: DeltahTK2 was successfully expressed in the cytoplasm of transduced cells. A new anti-hTK2 monoclonal antibody 8G2 was developed. The levels of FIAU and FEAU accumulation in cells expressing DeltahTK2 and DeltahTK2 GFP were at least 10-fold higher than in wild-type cells in vitro and about 6 times higher in vivo. We determined that FEAU is a more specific reporter substrate for DeltahTK2 than FIAU, whereas FHBG is not phosphorylated by this enzyme. In addition, we showed that DeltahTK2 transduced cells can be eliminated by treatment with d-arabinofuranosyl-cytosine. CONCLUSION: We have tested a human-derived reporter gene that is likely to be nonimmunogenic and potentially allows for long-term monitoring of different molecular-genetic processes by nuclear imaging techniques in humans. Using (124)I-FIAU, (18)F-FIAU, or (18)F-FEAU, it should be possible to image DeltahTK2 reporter gene expression with PET in preclinical and clinical studies.     

Antihyperglycemic Effect of Trigonella Foenum-Graecum (Fenugreek) Seed Extract in Alloxan-Induced Diabetic Rats and Its Use in Diabetes Mellitus: A Brief Qualitative Phytochemical and Acute Toxicity Test on the Extract

The effects of ethanol extract of Trigonella foenum-graecum (Fenugreek) seeds on the blood glucose levels in alloxan-induced diabetic rats at different doses (2g/kg, 1g/kg, 0.5g/kg and 0.1g/kg) were studied. The hypoglycemic effect of extract was compared with that of the standard antidiabetic drug (glimepiride, 4mg/kg) single dose. The extract showed significant activity against the diabetic state induced by alloxan but the intensity of hypoglycemic effect varied from dose to dose. The most effective dose recognized was 1g/kg but that is still lower than the standard antidiabetic drug. No acute toxicity was observed for ethanol extract of T. foenum-graecum seed when it was administered orally at high dose level (3 g/kg body weight), which is higher than effective antihyperglycemic dose, and closely observed for 24 hrs for any mortality and next 10 days for any delayed toxic effects on gross behavioral activities. Phytochemical group tests were also accomplished and presence of alkaloids, steroids and carbohydrates were recognized in the extract.     

Dietary B vitamin intakes and urinary total arsenic concentration in the Health Effects of Arsenic Longitudinal Study (HEALS) cohort, Bangladesh

Purpose  

The objective of this analysis was to evaluate the effects of dietary B vitamin intakes on creatinine-adjusted urinary total arsenic concentration among individuals participating in the Health Effects of Arsenic Longitudinal Study (HEALS) cohort in Araihazar, Bangladesh. Arsenic exposure is a major public health problem in Bangladesh, where nearly 77 million people have been chronically exposed to arsenic through the consumption of naturally contaminated groundwater. Dietary factors influencing the metabolism of ingested arsenic may potentially be important modifiers of the health effects of arsenic in this population.     

Evaluation of 2′-deoxy-2′-[18F]fluoro-5-methyl-1-β-l-arabinofuranosyluracil ([18F]-l-FMAU) as a PET imaging agent for cellular proliferation: comparison with [18F]-d-FMAU and [18F]FLT

PURPOSE: Clevudine (L: -FMAU) an un-natural analogue of thymidine, is in clinical trials for the treatment of hepatitis B virus (HBV). L: -FMAU is phosphorylated by cellular kinases such as thymidine kinase 1 and deoxycytidine kinase, and its triphosphate form inhibits HBV deoxyribonucleic acid synthesis. Thus, L: -FMAU, radiolabeled with an appropriate isotope, may be useful for positron emission tomography (PET) imaging of tumor proliferation. We evaluated [18F]-L-FMAU as a PET imaging agent in tumor-bearing mice and compared the results with those of two other radiotracers, [18F]-d-FMAU and [18F]-FLT. METHODS: Subcutaneous xenografts of the human lung cancer cell lines H441 and H3255 were established in mice. A micro-PET scanner was used to obtain images of the tumor-bearing animals with [18F]-L-FMAU, [18F]-D-FMAU, and [18F]-FLT. RESULTS: At 2 h postinjection, the tumor uptake (% ID/g) of 18F]-L: -FMAU, 18F]-D: -FMAU, and [18F]-FLT in the faster-growing H441 cells was 3.13 +/- 1.11, 7.74 +/- 1.39, and 5.10 +/- 1.45, respectively. The corresponding values for the slower-growing H3255 cells were 1.38 +/- 0.81, 4.49 +/- 1.08, and 0.57 +/- 0.33. Tumor/muscle ratios of accumulation for [18F]-L: -FMAU, [18F]-D: -FMAU, and [18F]-FLT in H441 cells were 4.15 +/- 1.82, 3.37 +/- 1.19, and 12.94 +/- 4.38, respectively, and the corresponding values in H3255 cells were 1.62 +/- 0.50, 1.96 +/- 0.74, and 1.50 +/- 0.90. CONCLUSIONS: [18F]-L: -FMAU may be a useful agent for imaging tumor proliferation in fast-growing human lung cancers by PET.     

Receptor mediated uptake of a radiolabeled contrast agent sensitive to beta-galactosidase activity

Radiolabeling of the MRI contrast agent 1-[2-(beta-galactopyranosyloxy)propyl]-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane with (111)In, and its evaluation is reported. Radiolabeling was performed in acetate buffer with 50-78% radiochemical yield. In vitro studies revealed that the asialoglycoprotein receptor-poor cell line MH1C1 has low uptake, while the receptor-rich cell lines BNL-CL2 and Hep G2 have higher uptake. In vivo, the uptake of the compound in receptor-rich organ liver was very high. Blocking the receptor in vivo, reduced liver uptake by 90% suggesting that the compound localizes in receptor-enriched tissues by binding to the asialoglycoprotein receptor.