Evaluation and comparison of 3’-[18F]-fluoro-3’-deoxy-L-thymidine (FLT) and 2-[18F]-fluoro-2-deoxyglucose (FDG)-PET to monitor early response following both cyclophosphamide and temsirolimus treatment in a mouse model of Burkitt lymphoma.
Daudi xenograft mice were treated with either cyclophosphamide or temsirolimus and imaged with FLT-PET and FDG-PET on appropriate days post therapy inititiation. Immunohistochemical (IHC) studies (H&E, TUNEL, CD20, PCNA and ki-67) and DNA flow cytometry studies were performed.
FDG tumor uptake decreased immediately after cyclophosphamide treatment while FLT-PET showed only a late and less pronounced decrease. A fast induction of apoptosis was observed together with an early accumulation of cells in the S-phase of the cell cycle, suggesting DNA repair. Temsirolimus treatment reduced both FDG and FLT tumor uptake immediately after therapy and resulted in a fast induction of apoptosis and G0-G1 phase accumulation.
FLT response was less distinct than FDG response and may be controlled by DNA repair early after cyclophosphamide. Nevertheless, FLT-PET was able to reflect decreased proliferation following temsirolimus.
FDG-PET; FLT-PET; Burkitt lymphoma; cyclophosphamide; mTOR inhibition; therapy response
PURPOSE: We report on a novel preclinical pancreatic cancer research model that uses bioluminescence imaging (BLI)-guided irradiation of orthotopic xenograft tumors, sparing of surrounding normal tissues, and quantitative, noninvasive longitudinal assessment of treatment response. MATERIALS AND METHODS: Luciferase-expressing MiaPaCa-2 pancreatic carcinoma cells were orthotopically injected in nude mice. BLI was compared to pathologic tumor volume, and photon emission was assessed over time. BLI was correlated to positron emission tomography (PET)/computed tomography (CT) to estimate tumor dimensions. BLI and cone-beam CT (CBCT) were used to compare tumor centroid location and estimate setup error. BLI and CBCT fusion was performed to guide irradiation of tumors using the small animal radiation research platform (SARRP). DNA damage was assessed by γ-H2Ax staining. BLI was used to longitudinally monitor treatment response. RESULTS: Bioluminescence predicted tumor volume (R = 0.8984) and increased linearly as a function of time up to a 10-fold increase in tumor burden. BLI correlated with PET/CT and necropsy specimen in size (P < .05). Two-dimensional BLI centroid accuracy was 3.5 mm relative to CBCT. BLI-guided irradiated pancreatic tumors stained positively for γ-H2Ax, whereas surrounding normal tissues were spared. Longitudinal assessment of irradiated tumors with BLI revealed significant tumor growth delay of 20 days relative to controls. CONCLUSIONS: We have successfully applied the SARRP to a bioluminescent, orthotopic preclinical pancreas cancer model to noninvasively: 1) allow the identification of tumor burden before therapy, 2) facilitate image-guided focal radiation therapy, and 3) allow normalization of tumor burden and longitudinal assessment of treatment response.
Positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG-PET) has increasingly been used to evaluate the efficacy of anticancer agents. We investigated the role of FDG-PET as a predictive marker for response to mammalian target of rapamycin (mTOR) inhibition in advanced solid tumor patients and in murine xenograft models.
Patients and Methods
Thirty-four rapamycin-treated patients with assessable baseline and treatment FDG-PET and computed tomography scans were analyzed from two clinical trials. Clinical response was evaluated according to Response Evaluation Criteria in Solid Tumors, and FDG-PET response was evaluated by quantitative changes and European Organisation for Research and Treatment of Cancer (EORTC) criteria. Six murine xenograft tumor models were treated with temsirolimus. Small animal FDG-PET scans were performed at baseline and during treatment. The tumors were analyzed for the expression of pAkt and GLUT1.
Fifty percent of patients with increased FDG-PET uptake and 46% with decreased uptake had progressive disease (PD). No objective response was observed. By EORTC criteria, the sensitivity of progressive metabolic disease on FDG-PET in predicting PD was 19%. Preclinical studies demonstrated similar findings, and FDG-PET response correlated with pAkt activation and plasma membrane GLUT1 expression.
FDG-PET is not predictive of proliferative response to mTOR inhibitor therapy in both clinical and preclinical studies. Our findings suggest that mTOR inhibitors suppress the formation of mTORC2 complex, resulting in the inhibition of Akt and glycolysis independent of proliferation in a subset of tumors. Changes in FDG-PET may be a pharmacodynamic marker for Akt activation during mTOR inhibitor therapy. FDG-PET may be used to identify patients with persistent Akt activation following mTOR inhibitor therapy.
Purpose: Early assessment of tumor responses to chemotherapy could enhance treatment outcomes by ensuring that, from the beginning, treatments meet the individualized needs of patients. In this study, we applied multiple modality molecular imaging techniques to pre-clinical monitoring of early tumor responses to Doxil, focusing on imaging of apoptosis.
Methods: Mice bearing UM-SCC-22B human head and neck squamous cancer tumors received either PBS or 1 to 2 doses of Doxil® (doxorubicin HCl liposome injection) (10 mg/kg/dose). Bioluminescence signals from an apoptosis-responsive reporter gene were captured for apoptosis evaluation. Tumor metabolism and proliferation were assessed by 18F-FDG and 3'-18F-fluoro-3'-deoxythymidine (18F-FLT) positron emission tomography. Diffusion-weighted magnetic resonance imaging (DW-MRI) was performed to calculate averaged apparent diffusion coefficients (ADCs) for the whole tumor volume. After imaging, tumor samples were collected for histological evaluation, including terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), anti-CD31, and Ki-67 immunostaining.
Results: Two doses of Doxil significantly inhibited tumor growth. Bioluminescence imaging (BLI) indicated apoptosis of tumor cells after just 1 dose of Doxil treatment, before apparent tumor shrinkage. 18F-FDG and 18F-FLT PET imaging identified decreased tumor metabolism and proliferation at later time points than those at which BLI indicated apoptosis. MRI measurements of ADC altered in response to Doxil, but only after tumors were treated with 2 doses. Decreased tumor proliferation and increased apoptotic cells were confirmed by changes of Ki-67 index and apoptotic ratio.
Conclusion: Our study of tumor responses to different doses of Doxil demonstrated that it is essential to combine apoptosis imaging strategies with imaging of other critical biological or pathological pathways, such as metabolism and proliferation, to improve clinical decision making in apoptosis-related diseases and interventions.
Multimodality Imaging; Doxil; Cancer Therapy; Response Monitoring; Apoptosis
Objectives: Most chemotherapy agents cause tumor cell death primarily by the induction of apoptosis. The ability to noninvasively image apoptosis in vivo could dramatically benefit pre-clinical and clinical evaluation of chemotherapeutics targeting the apoptotic pathway. This study aims to visualize the dynamics of apoptotic process with temporal bioluminescence imaging (BLI) using an apoptosis specific bioluminescence reporter gene. Methods: Both UM-SCC-22B human head and neck squamous carcinoma cells and 4T1 murine breast cancer cells were genetically modified with a caspase-3 specific cyclic firefly luciferase reporter gene (pcFluc-DEVD). Apoptosis induced by different concentrations of doxorubicin in the transfected cells was evaluated by both annexin V staining and BLI. Longitudinal BLI was performed in xenografted tumor models at different time points after doxorubicin or Doxil treatment, to evaluate apoptosis. After imaging, DNA fragmentation in apoptotic cells was assessed in frozen tumor sections using TUNEL staining. Results: Dose- and time-dependent apoptosis induced by doxorubicin in pcFluc-DEVD transfected UM-SCC-22B and 4T1 cells was visualized and quantified by BLI. Caspase-3 activation was confirmed by both caspase activity assay and GloTM luciferase assay. One dose of doxorubicin treatment induced a dramatic increase in BLI intensity as early as 24 h after treatment in 22B-pcFluc-DEVD xenografted tumors. Sustained signal increase was observed for the first 3 days and the fluorescent signal from ex vivo TUNEL staining was consistent with BLI imaging results. Long-term imaging revealed that BLI signal consistently increased and reached a maximum at around day 12 after the treatment with one dose of Doxil. Conclusions: BLI of apoptosis with pcFluc-DEVD as a reporter gene facilitates the determination of kinetics of the apoptotic process in a real-time manner, which provides a unique tool for drug development and therapy response monitoring.
apoptosis; cyclic firefly luciferase; bioluminescence imaging; doxorubicin; caspase-3.
Recent developments have established molecular imaging of mouse models with small-animal PET and bioluminescence imaging (BLI) as an important tool in cancer research. One of the disadvantages of these imaging modalities is the lack of anatomic information. We combined small-animal PET and BLI technology with small-animal CT to obtain fusion images with both molecular and anatomic information.
We used small-animal PET/CT and BLI to detect xenografts of different cell lines and metastases of a melanoma cell line (A375M-3F) that had been transduced with a lentiviral vector containing a trimodality imaging reporter gene encoding a fusion protein with Renilla luciferase, monomeric red fluorescent protein, and a mutant herpes simplex virus type 1 thymidine kinase.
Validation studies in mouse xenograft models showed a good coregistration of images from both PET and CT. Melanoma metastases were detected by 18F-FDG PET, 9-[4-18F-fluoro-3-(hydroxymethyl) butyl]guanine (18F-FHBG) PET, CT, and BLI and confirmed by ex vivo assays of Renilla luciferase and mutant thymidine kinase expression. 18F-FHBG PET/CT allowed detection and localization of lesions that were not seen on CT because of poor contrast resolution and were not seen on 18F-FDG PET because of higher background uptake relative to 18F-FHBG.
The combination of 18F-FHBG PET, small-animal CT, and BLI allows a sensitive and improved quantification of tumor burden in mice. This technique is potentially useful for the study of the biologic determinants of metastasis and for the evaluation of novel cancer treatments.
microPET; small-animal CT; bioluminescence imaging; metastasis; mouse; melanoma
Introduction and Purpose. Monitoring solid tumor growth and metastasis in small animals is important for cancer research. Noninvasive techniques make longitudinal studies possible, require fewer animals, and have greater statistical power. Such techniques include FDG positron emission tomography (FDG-PET), magnetic resonance imaging (MRI), and optical imaging, comprising bioluminescence imaging (BLI) and fluorescence imaging (FLI). This study compared the performance and usability of these methods in the context of mouse tumor studies. Methods. B16 tumor-bearing mice (n = 4 for each study) were used to compare practicality, performance for small tumor detection and tumor burden measurement. Using RETAAD mice, which develop spontaneous melanomas, we examined the performance of MRI (n = 6 mice) and FDG-PET (n = 10 mice) for tumor identification. Results. Overall, BLI and FLI were the most practical techniques tested. Both BLI and FDG-PET identified small nonpalpable tumors, whereas MRI and FLI only detected macroscopic, clinically evident tumors. FDG-PET and MRI performed well in the identification of tumors in terms of specificity, sensitivity, and positive predictive value. Conclusion. Each of the four methods has different strengths that must be understood before selecting them for use.
Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[18F]fluoro-D-glucose (FDG) and the thymidine analog, 3′-deoxy-3′-[18F] fluorothymidine (FLT).
The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects.
SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue.
Dual PI3K/mTOR inhibition using BGT226 is effective in ALK-positive anaplastic large cell lymphoma and can be monitored with both FDG-PET and FLT-PET early on in the course of therapy.
lymphoma; phosphatidylinositol-3-kinase; mammalian target of rapamycin; inhibition; positron emission tomography
Molecular subtypes of glioblastoma (GBM) with distinct alterations have been identified. There is need for reproducible, versatile preclinical models that resemble specific GBM phenotypes to facilitate preclinical testing of novel therapies. We present a cell line-based murine Proneural GBM model and characterize its response to radiation therapy.
Proneural gliomas were generated by injecting PDGF-IRES-Cre retrovirus into the subcortical white matter of adult mice that harbor floxed tumor suppressors (Pten and p53) and stop-floxed reporters. Primary cell cultures were generated from the retrovirus induced tumors and maintained in vitro for multiple passages. RNA sequencing-based expression profiling of the resulting cell lines was performed. The tumorigenic potential of the cells was assessed by intracranial injection into adult naïve mice from different strains. Tumor growth was assessed by bioluminescence imaging (BLI). BLI for tumor cells and brain slices were obtained and compared to in vivo BLI. Response to whole-brain radiation was assessed in glioma-bearing animals.
Intracranial injection of Pdgf+Pten−/−p53−/−luciferase+ glioma cells led to formation of GBM-like tumors with 100% efficiency (n=48) and tumorigenesis was retained for more than 3 generations. The cell lines specifically resembled Proneural GBM based on expression profiling by RNA-Seq. Pdgf+Pten−/−p53−/−luciferase+ cell number correlated with BLI signal. Serial BLI measured tumor growth and correlated with size and location by ex-vivo imaging. Moreover, BLI predicted tumor-related mortality with a 93% risk of death within 5 days following a BLI signal between 1×108−5×108 photons/sec/cm2. BLI signal had transient but significant response following radiotherapy, which corresponded to a modest survival benefit for radiated mice (p<0.05).
Intracranial injection of Pdgf+Pten−/−p53−/−luciferase+ cells constitutes a novel and highly reproducible model, recapitulating key features of human Proneural GBM, and can be used to evaluate tumor-growth and response to therapy.
glioma; proneural; murine; radiotherapy; cell line
Intracranial vestibular schwannoma xenografts can be successfully established and followed with bioluminescent imaging (BLI).
Transgenic and xenograft mouse models of vestibular schwannomas have been previously reported in the literature. However, none of these models replicate the intracranial location of these tumors to reflect the human disease. Additionally, traditional imaging methods (magnetic resonance imaging, computed tomography) for following tumor engraftment and growth are expensive and time consuming. BLI has been successfully used to longitudinally follow tumor treatment responses in a noninvasive manner. BLI’s lower cost and labor demands make this a more feasible approach for tumor monitoring in studies involving large numbers of mice.
Patient excised vestibular schwannomas were cultured and transduced with firefly luciferase expressing lentivirus. One million cells were stereotactically injected into the right caudate nucleus of 21 nonobese diabetic/severe combined immunodeficient mice. Schwannoma engraftment and growth was prospectively followed for 30 weeks after injection with BLI. After animal sacrifice, the presence of human tumor cells was confirmed with fluorescent in situ hybridization.
Eight (38%) of 21 mice successfully engrafted the schwannoma cells. All of these mice were generated from 4 (67%) of the 6 patient excised tumors. These 8 mice could be differentiated from the nonengrafted mice at 21 weeks. The engrafted group emitted BLI of greater than 100,000 photons/s (range, 142,478–3,106,300 photons/s; average, 618,740 photons/s), whereas the nonengrafted group were all under 100,000 photons/s (range, 0–76,010 photons/s; average, 10,737 photons/s) (p < 0.001). Fluorescent in situ hybridization analysis confirmed the presence of viable human schwannoma cells in much greater numbers in those mice with stable or growing tumors compared with those whose tumors regressed.
We have successfully established an intracranial schwannoma xenograft model that can be followed with noninvasive BLI. We hope to use this model for in vivo testing of schwannoma tumor therapies.
Acoustic neuroma; Bioluminescence; NOD/SCID mice; Vestibular schwannoma; Xenograft
The integral role of positron-emission tomography (PET) using the glucose analog tracer fluorine-18 fluorodeoxyglucose (FDG) in the staging of non-small cell lung cancer (NSCLC) is well established. Evidence is emerging for the role of PET in response assessment to neoadjuvant therapy, combined-modality therapy, and early detection of recurrence. Here, we review the current literature on these aspects of PET in the management of NSCLC. FDG-PET, particularly integrated 18F-FDG-PET/CT, scans have become a standard test in the staging of local tumor extent, mediastinal lymph node involvement, and distant metastatic disease in NSCLC. 18F-FDG-PET sensitivity is generally superior to computed tomography (CT) scans alone. Local tumor extent and T stage can be more accurately determined with FDG-PET in certain cases, especially in areas of post-obstructive atelectasis or low CT density variation. FDG-PET sensitivity is decreased in tumors <1 cm, at least in part due to respiratory motion. False-negative results can occur in areas of low tumor burden, e.g., small lymph nodes or ground-glass opacities. 18F-FDG-PET-CT nodal staging is more accurate than CT alone, as hilar and mediastinal involvement is often detected first on 18F-FDG-PET scan when CT criteria for malignant involvement are not met. 18F-FDG-PET scans have widely replaced bone scintography for assessing distant metastases, except for the brain, which still warrants dedicated brain imaging. 18F-FDG uptake has also been shown to vary between histologies, with adenocarcinomas generally being less FDG avid than squamous cell carcinomas. 18F-FDG-PET scans are useful to detect recurrences, but are currently not recommended for routine follow-up. Typically, patients are followed with chest CT scans every 3–6 months, using 18F-FDG-PET to evaluate equivocal CT findings. As high 18F-FDG uptake can occur in infectious, inflammatory, and other non-neoplastic conditions, 18F-FDG-PET-positive findings require pathological confirmation in most cases. There is increased interest in the prognostic and predictive role of FDG-PET scans. Studies show that absence of metabolic response to neoadjuvant therapy correlates with poor pathologic response, and a favorable 18F-FDG-PET response appears to be associated with improved survival. Further work is underway to identify subsets of patients that might benefit individualized management based on FDG-PET.
PET; non-small cell lung cancer; staging; response assessment; follow-up
Despite the use of radiation and chemotherapy, the prognosis for children with diffuse brainstem gliomas is extremely poor. There is a need for relevant brainstem tumor models that can be used to test new therapeutic agents and delivery systems in pre-clinical studies. We report the development of a brainstem-tumor model in rats and the application of bioluminescence imaging (BLI) for monitoring tumor growth and response to therapy as part of this model. Luciferase-modified human glioblastoma cells from five different tumor cell sources (either cell lines or serially-passaged xenografts) were implanted into the pontine tegmentum of athymic rats using an implantable guide-screw system. Tumor growth was monitored by BLI and tumor volume was calculated by three-dimensional measurements from serial histopathologic sections. To evaluate if this model would allow detection of therapeutic response, rats bearing brainstem U-87 MG or GS2 glioblastoma xenografts were treated with the DNA methylating agent temozolomide (TMZ). For each of the tumor cell sources tested, BLI monitoring revealed progressive tumor growth in all animals, and symptoms caused by tumor burden were evident 26–29 days after implantation of U-87 MG, U-251 MG, GBM6, and GBM14 cells, and 37–47 days after implantation of GS2 cells. Histopathologic analysis revealed tumor growth within the pons in all rats and BLI correlated quantitatively with tumor volume. Variable infiltration was evident among the different tumors, with GS2 tumor cells exhibiting the greatest degree of infiltration. TMZ treatment groups were included for experiments involving U-87 MG and GS2 cells, and in each case TMZ delayed tumor growth, as indicated by BLI monitoring, and significantly extended survival of animal subjects. Our results demonstrate the development of a brainstem tumor model in athymic rats, in which tumor growth and response to therapy can be accurately monitored by BLI. This model is well suited for pre-clinical testing of therapeutics that are being considered for treatment of patients with brainstem tumors.
Brainstem tumor; Animal model; Bioluminescence; Temozolomide
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.
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 (TE/TR 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 (TE/TR 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.
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.
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.
Transgenic mice; Wilms tumor; 18F-FDG; PET/CT; CT; MRI
A single dynamic PET acquisition using multiple tracers administered closely in time could provide valuable complementary information about a tumor’s status under quasi-constant conditions. This study aims to investigate the utility of dual-tracer dynamic PET imaging with 18F-Alfatide II (18F-AlF-NOTA-E[PEG4-c(RGDfk)]2) and 18F-FDG for parametric monitoring of tumor responses to therapy.
We administered doxorubicin to one group of athymic nude mice with U87MG tumors and Abraxane to another group of mice with MDA-MB-435 tumors. To monitor therapeutic responses, we performed dual-tracer dynamic imaging, in sessions that lasted 90 min, starting by injecting the mice via tail vein catheters with 18F-Alfatide II, followed 40 minutes later by 18F-FDG. To achieve signal separation of the two tracers, we fit a three-compartment reversible model to the time activity curve (TAC) of 18F-Alfatide II for the 40 min prior to 18F-FDG injection, and then extrapolated to 90 min. The 18F-FDG tumor TAC was isolated from the 90 min dual tracer tumor TAC by subtracting the fitted 18F-Alfatide II tumor TAC. With separated tumor TACs, the 18F-Alfatide II binding potential (Bp=k3/k4) and volume of distribution (VD), and 18F-FDG influx rate ((K1×k3)/(k2 + k3)) based on the Patlak method were calculated to validate the signal recovery in a comparison with 60-min single tracer imaging and to monitor therapeutic response.
The transport and binding rate parameters K1-k3 of 18F-Alfatide II, calculated from the first 40 min of dual tracer dynamic scan, as well as Bp and VD, correlated well with the parameters from the 60 min single tracer scan (R2 > 0.95). Compared with the results of single tracer PET imaging, FDG tumor uptake and influx were recovered well from dual tracer imaging. Upon doxorubicin treatment, while no significant changes in static tracer uptake values of 18F-Alfatide II or 18F-FDG were observed, both 18F-Alfatide II Bp and 18F-FDG influx from kinetic analysis in tumors showed significant decreases. For Abraxane therapy of MDA-MB-435 tumors, significant decrease was only observed with 18F-Alfatide II Bp value from kinetic analysis but not 18F-FDG influx.
The parameters fitted with compartmental modeling from the dual tracer dynamic imaging are consistent with those from single tracer imaging, substantiating the feasibility of this methodology. Even though no significant differences in tumor size were found until 5 days after doxorubicin treatment started, at day 3 there were already substantial differences in 18F-Alfatide II Bp and 18F-FDG influx rate. Dual tracer imaging can measure 18F-Alfatide II Bp value and 18F-FDG influx simultaneously to evaluate tumor angiogenesis and metabolism. Such changes are known to precede anatomical changes, and thus parametric imaging may offer the promise of early prediction of therapy response.
dual-tracer dynamic PET; parametric imaging; 18F-Alfatide II; 18F-FDG; therapy response
Noninvasive imaging technologies have the potential to enhance the monitoring and improvement of adoptive therapy with tumor-targeted T lymphocytes. We established an imaging methodology for the assessment of spatial and temporal distributions of adoptively transferred genetically modified human T cells in vivo for treatment monitoring and prediction of tumor response in a systemic prostate cancer model.
RM1 murine prostate carcinoma tumors transduced with human prostate-specific membrane antigen (hPSMA) and a Renilla luciferase reporter gene were established in SCID/beige mice. Human T lymphocytes were transduced with chimeric antigen receptors (CAR) specific for either hPSMA or human carcinoembryonic antigen (hCEA) and with a fusion reporter gene for herpes simplex virus type 1 thymidine kinase (HSV1tk) and green fluorescent protein, with or without click beetle red luciferase. The localization of adoptively transferred T cells in tumor-bearing mice was monitored with 2′-18F-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-ethyluracil (18F-FEAU) small-animal PET and bioluminescence imaging (BLI).
Cotransduction of CAR-expressing T cells with the reporter gene did not affect CAR-mediated cytotoxicity. BLI of Renilla and click beetle red luciferase expression enabled concurrent imaging of adoptively transferred T cells and systemic tumors in the same animal. hPSMA-specific T lymphocytes persisted longer than control hCEA-targeted T cells in lung hPSMA-positive tumors, as indicated by both PET and BLI. Precise quantification of T-cell distributions at tumor sites by PET revealed that delayed tumor progression was positively correlated with the levels of 18F-FEAU accumulation in tumor foci in treated animals.
Quantitative noninvasive monitoring of genetically engineered human T lymphocytes by PET provides spatial and temporal information on T-cell trafficking and persistence. PET may be useful for predicting tumor response and for guiding adoptive T-cell therapy.
PET; molecular imaging; adoptive immunotherapy; HSV1tk; 18F-FEAU
In breast cancer endocrine therapy, post-therapy Ki-67 assay of biopsy material predicts recurrence-free survival but is invasive and prone to sampling error. [18F]Fluorodeoxyglucose (FDG) positron emission tomography (PET) has shown an early agonist or ‘flare’ response to tamoxifen and estradiol, but has not been tested in response to estrogen-lowering aromatase inhibitors (AIs). We hypothesized that decreased agonistic response to AIs would result in early FDG uptake decline. We also measured early response to trastuzumab (T), another targeted agent for breast cancer with differing mechanisms of action. Our study was designed to test for an early decline in FDG uptake in response to AI or T and to examine association with Ki-67 measures of early response.
Patients with any stage of newly diagnosed or recurrent breast cancer were eligible and enrolled prior to initiation (or resumption) of AI or T therapy. FDG PET and tissue biopsy were planned before and after 2 weeks of AI or T therapy, with pretreatment archival tissue permitted. Cutoffs of ≥20% decline in standardized uptake value (SUV) as FDG PET early response and ≤5% post-treatment expression as Ki-67 early response were defined prior to analysis.
Forty-two patients enrolled, and 40 (28 AI, 12 T) completed serial FDG-PET imaging. Twenty-two patients (17 AI, 5 T) had newly diagnosed disease, and 23 (14 AI, 9 T) had metastatic disease (5 newly diagnosed). Post-treatment biopsy was performed in 25 patients (63%) and was either refused or not feasible in 15. Post-treatment biopsy yielded tumor in only 17/25 cases (14 AI, 3 T). Eleven of 14 AI patients with post-therapy tissue showed FDG PET early response, and there was 100% concordance of PET and post-therapy Ki-67 early response. For the T group, 6/12 showed an FDG PET early response, including 2/3 patients with post-therapy biopsy, all with Ki-67 >5%.
Substantial changes in FDG PET SUV occurred over 2 weeks of AI therapy and were associated with low post-therapy proliferation. SUV decline was seen in response to T, but few tissue samples were available to test association with Ki-67. Our results support further investigation of FDG PET as a biomarker for early response to AI therapy.
FDG PET; Ki-67; Breast cancer; Aromatase inhibitor; Trastuzumab; Pharmacodynamic response; Early response
18F-FDG has been used to image mouse xenograft models with small-animal PET for therapy response. However, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of 18F-FDG small-animal PET studies.
Mouse tumor xenografts were formed with B16F10 murine melanoma cells. A 7-min small-animal PET scan was performed 1 h after a 3.7- to 7.4-MBq 18F-FDG injection via the tail vein. A second small-animal PET scan was performed 6 h later after reinjection of 18F-FDG. Twenty-five sets of studies were performed. Mean injected dose per gram (%ID/g) values were calculated from tumor regions of interest. The coefficient of variation (COV) from studies performed on the same day was calculated to determine the reproducibility. Activity from the second scans performed after 6 h were adjusted by subtracting the estimated residual activity from the first 18F-FDG injection. For 7 datasets, an additional scan immediately before the second injection was performed, and residual activity from this additional delayed scan was subtracted from the activity of the second injection. COVs of both subtraction methods were compared. Blood glucose values were measured at the time of injection and used to correct the %ID/g values.
The COV for the mean %ID/g between 18F-FDG small-animal PET scans performed on the same day 6 h apart was 15.4% ± 12.6%. The delayed scan subtraction method did not produce any significant change in the COV. Blood glucose correction increased the COV. The injected dose, tumor size, and body weight did not appear to contribute to the variability of the scans.
18F-FDG small-animal PET mouse xenograft studies were reproducible with moderately low variability. Therefore, serial small-animal PET studies may be performed with reasonable accuracy to measure tumor response to therapy.
18F-FDG; small-animal PET; reproducibility; tumor xenograft; drug development
To determine whether treatment response to the Aurora B kinase inhibitor, AZD1152, could be monitored early in the course of therapy by non-invasive [18F]FDG and/or [18F]FLT PET imaging.
AZD1152-treated and control HCT116 and SW620 xenograft-bearing animals were monitored for tumor size and by [18F]FDG and [18F]FLT PET imaging. Additional studies assessed the endogenous and exogenous contributions thymidine synthesis in the two cell lines.
Both xenografts showed a significant volume-reduction to AZD1152. In contrast, [18F]FDG uptake did not demonstrate a treatment response. [18F]FLT uptake decreased to less than 20% of control values in AZD1152-treated HCT116 xenografts, whereas [18F]FLT uptake was near background levels in both treated and untreated SW620 xenografts. The EC50 for AZD1152-HQPA was ~10 nM in both SW620 and HCT116 cells; in contrast, SW620 cells were much more sensitive to Methotrexate (MTX) and 5-Fluorouracil (5FU) than HCT116 cells. Immunoblot analysis demonstrated marginally lower expression of thymidine kinase in SW620 compared to HCT116 cells. The above results suggest that SW620 xenografts have a higher dependency on the de novo pathway of thymidine utilization than HCT116 xenografts.
AZD1152 treatment showed anti-tumor efficacy in both colon cancer xenografts. Although [18F]FDG PET was inadequate in monitoring treatment-response, [18F]FLT PET was very effective in monitoring response in HCT116 xenografts, but not in SW620 xenografts. These observations suggest that de novo thymidine synthesis could be a limitation and confounding factor for [18F]FLT PET imaging and quantification of tumor proliferation, and this may apply to some clinical studies as well.
Colon cancer; HCT116; SW620; Positron-Emission Tomography; AZD1152; Methotrexate - MTX; 5-Fluorouracil – 5-FU; Fluorodeoxythymidine – FLT; Fluorodeoxyglucose – FDG
Near infrared (NIR) photoimmunotherapy (PIT) is a new type of cancer treatment based on a monoclonal antibody (mAb)-NIR phthalocyanine dye, (IR700) conjugate. In vitro cancer-specific cell death occurs during NIR light exposure in cells previously incubated with mAb-IR700 conjugates. However, documenting rapid cell death in vivo is more difficult.
A luciferase-transfected breast cancer cell (epidermal growth factor receptor+, MDA-MB-468luc cells) was produced and used for both in vitro and in vivo experiments for monitoring the cell killing effect of PIT. After validation of cytotoxicity with NIR exposure up to 8 J/cm2in vitro, we employed an orthotopic breast cancer model of bilateral MDA-MB-468luc tumors in female athymic mice, which subsequently received a panitumumab-IR700 conjugate in vivo. One side was used as a control, while the other was treated with NIR light of dose ranging from 50 to 150 J/cm2. Bioluminescence imaging (BLI) was performed before and after PIT.
Dose-dependent cell killing and regrowth was successfully monitored by the BLI signal in vitro. Although tumor sizes were unchanged, BLI signals decreased by >95% immediately after PIT in vivo when light intensity was high (>100 J/cm2), however, in mice receiving lower intensity NIR (50 J/cm2), tumors recurred with gradually increasing BLI signal.
PIT induced massive cell death of targeted tumor cells immediately after exposure of NIR light that was demonstrated with BLI in vivo.
Photoimmunotherapy; Theranostics; Cell death; Epidermal growth factor receptor; Molecular targeting; Monoclonal antibody; Bioluminescence imaging
Over the last several years epidemiological data has emerged which suggests that the anti-diabetic drug metformin (MET), an AMP-activated protein kinase (AMPK) activator, improves progression free survival in multiple cancers; more than 30 clinical trials are underway to confirm this finding. We postulated that the role of AMPK as a central cellular energy sensor would result in opposite effects on glucose uptake and proliferation, suggesting different roles for 18F-FDG and 18F-FLT in assessing its effectiveness as an anti-neoplastic agent.
Colon cancer cell lines HT29 (human) and MC26 (murine) were treated for 24 or 72hrs with a range of MET (0–10mM). Western blotting was used to study the activation of AMPK after MET treatment. Glucose uptake and cell proliferation were measured by cell retention studies with either 18F-FDG or 18F-FLT. EdU (a thymidine analogue) and Annexin-Propidium Iodine flow cytometry were performed to determine cell cycle S-phase and apoptotic changes. In vivo 18F-FDG and 18F-FLT PET images were acquired before and 24hrs after MET treatment on HT29 tumor bearing mice.
After 24hrs of MET incubation, phosphorylated AMPK increased several fold in both cell lines while total AMPK was unchanged. In cell retention studies, 18F-FDG uptake increased whereas 18F-FLT retention decreased significantly in both cell lines. Cells in S-phase decreased 36% in HT29 and 33% in MC26 cells following MET therapy. Apoptosis increased 10.5× and 5.8×, in HT29 and MC26 cells, respectively after 72hrs of incubation with MET. PET imaging showed increased 18F-FDG uptake (mean SUV: 0.71±0.03 and 1.29±0.11 pre and post MET therapy, p<0.05) and decreased 18F-FLT uptake (mean SUV: 1.18±0.05 and 0.89±0.01 pre and post MET therapy, p<0.05) in HT29 tumor bearing mice.
MET, through activation of the AMPK pathway, exerts a dose dependent increase in tumor glucose uptake while decreasing cell proliferation in human and murine colon cancer cells. Thus, changes in 18F-FDG uptake post MET treatment may be misleading; 18F-FLT imaging is a promising alternative that correlates with tumor response.
Metformin; AMPK; Tumor metabolic imaging; Proliferation imaging
Hyperthermia is used in combination with radiotherapy and/or chemotherapy in the treatment of various types of cancer. Currently, the tumor cell response to hyperthermia is determined largely based on the size reduction of tumor mass, which is insensitive.
We tested the feasibility of bioluminescent imaging (BLI) in evaluation of the tumor cell response to hyperthermia by exposing luciferase-expressing MDA-MB-231-luc human breast cancer cells to high temperature (43 °C) for 10 minutes to 2 hours. The tumor cells were the imaged and the light signal generated by the tumor cells was quantified with BLI. To validate its usefulness, the light signal intensity was comparatively analyzed with the tumor cell clonogenicity and cell viability, which were measured with classic clonogenic and MTT assays.
The light signal intensity determined by BLI was closely correlated with the absolute number of viable cells as well as the cell viability measured with the traditional MTT assay under normal culture condition. Relative to the clonogenicity of tumor cells after exposure to hyperthermia, however, BLI underestimated, while MTT assay overestimated the cell viability. Difference in the interpretation of tumor cell clonogenic ability following hyperthermia with BLI, MTT dye, and clonogenic assay may be due to the different mechanisms of the three measurements as well as the fact that hyperthermia can induce cell damage at levels of both transient and permanent.
BLI is sensitive, convenient, and potentially valuable in the evaluation and monitoring of tumor cell response to treatments including hyperthermia.
Hyperthermia; tumor cell response; bioluminescent imaging; breast cancer
AIM: To evaluate the ability of 18F-fluorodeoxyglucose positron emission and computed tomography (18F-FDG PET/CT) in restaging of hepatocellular carcinoma (HCC) after treatment.
METHODS: We reviewed a database of the diagnostic performance of 18F-FDG PET/CT scan for patients with HCC following local or regional treatment. The database consisted of 18F-FDG PET/CT information of 21 male and 4 female (age range, 27-81 years; mean age, 51.6 years) patients who had received surgical resection and/or interventional treatments and then underwent 18F-FDG PET/CT scan. All patients had received enhanced CT scan of the liver two weeks before or after the 18F-FDG PET/CT scan. Intrahepatic recurrence and/or extrahepatic metastases were confirmed by histological analysis or clinical and imaging follow-up. The accuracy of 18F-FDG PET/CT study was determined by histopathological results or by clinical and imaging follow-up.
RESULTS: 18F-FDG PET/CT was abnormal in 19 of the 25 (76.0%) patients. In detecting HCC recurrence, 18F-FDG PET/CT scored 17 true positives, 5 true negatives, 2 false positives and 1 false negative. The sensitivity, specificity and accuracy of 18F-FDG PET/CT in detecting HCC recurrence was 89.5%, 83.3% and 88%, respectively. 18F-FDG PET/CT had an impact on management of these patients by settling the problem of an unexplained increase in alpha-fetoprotein after treatment (14 patients), by monitoring response to the treatment and guiding additional regional therapy (12 patients), by identifying extrahepatic metastases (10 patients), by identifying tumor growth or thrombosis in the portal vein (6 patients), or by guiding surgical resection of extrahepatic metastases (2 patients).
CONCLUSION: Our results suggest that whole body 18F-FDG PET/CT may be useful in the early evaluation of residual, intrahepatic recurrent or extrahepatic metastatic lesions and able to provide valuable information for the management of HCC recurrence.
18F-fluorodeoxyglucose; Positron emission tomography/computed tomography; Hepatocellular carcinoma; Surgeon resection; Interventional treatment; Residual lesion; Intrahepatic recurrence; Extrahepatic metastases; Restaging
In studies of diffuse large B-cell lymphoma, positron emission tomography with [18F]fluorodeoxyglucose (FDG-PET) performed after two to four cycles of chemotherapy has demonstrated prognostic significance. However, some patients treated with immunochemotherapy experience a favorable long-term outcome despite a positive interim FDG-PET scan. To clarify the significance of interim FDG-PET scans, we prospectively studied interim FDG-positive disease within a risk-adapted sequential immunochemotherapy program.
Patients and Methods
From March 2002 to November 2006, 98 patients at Memorial Sloan-Kettering Cancer Center received induction therapy with four cycles of accelerated R-CHOP (rituximab + cyclophosphamide, doxorubicin, vincristine, and prednisone) followed by an interim FDG-PET scan. If the FDG-PET scan was negative, patients received three cycles of ICE (ifosfamide, carboplatin, and etoposide) consolidation therapy. If residual FDG-positive disease was seen, patients underwent biopsy; if the biopsy was negative, they also received three cycles of ICE. Patients with a positive biopsy received ICE followed by autologous stem-cell transplantation.
At a median follow-up of 44 months, overall and progression-free survival were 90% and 79%, respectively. Ninety-seven patients underwent interim FDG-PET scans; 59 had a negative scan, 51 of whom are progression free. Thirty-eight patients with FDG-PET–positive disease underwent repeat biopsy; 33 were negative, and 26 remain progression free after ICE consolidation therapy. Progression-free survival of interim FDG-PET–positive/biopsy-negative patients was identical to that in patients with a negative interim FDG-PET scan (P = .27).
Interim or post-treatment FDG-PET evaluation did not predict outcome with this dose-dense, sequential immunochemotherapy program. Outside of a clinical trial, we recommend biopsy confirmation of an abnormal interim FDG-PET scan before changing therapy.
PET using 18F-FDG has prognostic value when performed at the completion of initial chemotherapy in patients with diffuse large B-cell lymphoma (DLBCL). 18F-FDG PET may also be predictive of outcome when performed during the treatment course of DLBCL, but robust prospective studies and standardization of 18F-FDG PET interpretation in this setting are lacking.
In this prospective study, patients with advanced-stage DLBCL were treated with standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy, and 18F-FDG PET/CT was performed after cycle 2 or 3 and at the end of therapy. The 18F-FDG PET/CT scans were interpreted according to the International Harmonization Project for Response Criteria in Lymphoma, and the maximum standardized uptake value (SUV) of the most 18F-FDG–avid lesions was recorded.
Fifty patients were enrolled, and all underwent interim 18F-FDG PET/CT. At a median follow-up of 33.9 mo, the positive predictive value (PPV) of interim 18F-FDG PET/CT for relapse or progression was 42%, and the negative predictive value (NPV) was 77%. Interim 18F-FDG PET/CT was significantly associated with event-free survival (P = 0.017) and with progression-free survival (P = 0.04) but not with overall survival (P = 0.08). End-of-therapy 18F-FDG PET/CT had high PPV and NPV (71% and 80%, respectively) and was significantly associated with event-free survival, progression-free survival, and overall survival (P < 0.001). SUV measurements did not discriminate patients who relapsed or progressed from those who remained in remission.
When performed after 2 cycles of immunochemotherapy and interpreted according to International Harmonization Project criteria, early response assessment with PET/CT has a high NPV but low PPV in patients with advanced-stage DLBCL. Prospective trials are required to validate different criteria for the interpretation of interim 18F-FDG PET/CT and establish the role of interim 18F-FDG PET/CT in the management of patients with DLBCL.
18F-FDG; PET; PET/CT; non-Hodgkin lymphoma; prognosis
The role of [18F]fluorodeoxyglucose ([18F]FDG) PET in staging of sarcoma is well established. The aim of this preclinical study was to compare [18F]fluorothymidine ([18F]FLT) PET to [18F]FDG PET regarding early metabolic changes of sarcoma in the course of targeted cancer therapy. SCID mice bearing sarcoma A673 xenotransplants were used for investigation of tumor response after treatment with the multikinase inhibitor Sorafenib. [18F]FLT and/or [18F]FDG-PET were performed prior to and early after initiation of treatment. Tumoral uptake (% Injected Dose per gram (%ID/g) of [18F]FLT-PET was compared to [18F]FDG-PET. Results were correlated with histopathology and in vitro data including cellular uptake, cell cycle-related protein expression, cell cycle distribution and apoptosis. In vitro experiments showed that A673 cells were sensitive to Sorafenib. In vivo, tumor growth was inhibited in comparison to a 4-fold increase of the tumor volume in control mice. Using [18F]FDG as tracer, a moderate reduction in tracer uptake (n=15, mean relative %ID/g 74%, range 35%-121%, p=0.03) was observed. The decrease in %ID/g using [18F]FLT-PET was significantly higher (p=0.003). The mean relative %ID/g in [18F]FLT uptake on day + 5 was significantly reduced to 54% compared to baseline (n=15, range 24%-125%, SD=29%). The PET analysis 24 hr after therapy showed a significant reduction of the mean [18F]FLT-%ID/g (p=0.04). The reduction of %ID/g on day + 1 in [18F]FDG-PET was not statistically significant (p=0.99). In conclusion, both [18F]FDG- and [18F]FLT-PET were able to predict response to Sorafenib treatment. In contrast to [18F]FDG-PET, [18F]FLT-PET was more predictive for very early response to treatment.
Molecular imaging; sarcoma; PET; proliferation; [18F]FLT; [18F]FDG