Rebecca A. Dumont

Monitoring tumor glucose utilization by positron emission tomography for the prediction of treatment response to epidermal growth factor receptor kinase inhibitors.

Purpose: The mechanisms underlying the sensitivity of non–small cell lung cancer to epidermal growth factor receptor (EGFR) kinase inhibitors are complex, and there are no established markers to accurately predict treatment outcome in individual patients. Experimental Design: We investigated whether tumors responding to EGFR inhibitors can be identified by measuring treatment-induced changes in glucose utilization by positron emission tomography with the glucose analogue fluorodeoxyglucose (FDG-PET). We studied a panel of cell lines with a spectrum of sensitivity to EGFR kinase inhibitors. After incubation with the EGFR kinase inhibitor gefitinib for various time points, FDG uptake, glucose transport rates, and hexokinase activity were determined. FDG uptake in vivo was assessed by microPET imaging of tumor xenografts in mice. Results: In gefitinib-sensitive cell lines, there was a dramatic decrease in FDG uptake as early as 2 hours after treatment. Immunoblots showed the translocation of glucose transporters (GLUT3) from the plasma membrane to the cytosol; glucose transport rates were reduced 2.6-fold at this time. There was also a modest reduction of hexokinase activity. These metabolic alterations preceded changes in cell cycle distribution, thymidine uptake, and apoptosis. MicroPET studies showed an up to 55% decrease of tumor FDG uptake in sensitive xenografts within 48 hours. In contrast, gefitinib-resistant cells exhibited no measurable changes in FDG uptake, either in cell culture or in vivo. Conclusion: Glucose metabolic activity closely reflects response to gefitinib therapy. FDG-PET may be a valuable clinical predictor, early in the course of treatment, for therapeutic responses to EGFR kinase inhibitors.

Novel 64Cu- and 68Ga-Labeled RGD Conjugates Show Improved PET Imaging of ανβ3 Integrin Expression and Facile Radiosynthesis

PET with 18F-labeled arginine-glycine-aspartic acid (RGD) peptides can visualize and quantify ανβ3 integrin expression in patients, but radiolabeling is complex and image contrast is limited in some tumor types. The development of 68Ga-RGD peptides would be of great utility given the convenience of 68Ga production and radiolabeling, and 64Cu-RGD peptides allow for delayed imaging with potentially improved tumor-to-background ratios. Methods: We used the chelators DOTA,1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), and 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) to radiolabel the cyclic pentapeptide c(RGDfK) with 68Ga or 64Cu. NODAGA-c(RGDfK) was labeled at room temperature with both radionuclides within 10 min. Incubation at 95°C for up to 30 min was used for the other conjugates. The affinity profile of the metallopeptides was evaluated by a cell-based receptor-binding assay. Small-animal PET studies and biodistribution studies were performed in nude mice bearing subcutaneous U87MG glioblastoma xenografts. Results: The conjugates were labeled with a radiochemical purity greater than 97% and specific activities of 15–20 GBq/μmol. The affinity profile was similar for all metallopeptides and comparable to the reference standard c(RGDfV). In the biodistribution studies, all compounds demonstrated a relatively similar tumor and normal organ uptake at 1 h after injection that was comparable to published data on 18F-labeled RGD peptides. At 18 h after injection, however, 64Cu-NODAGA-c(RGDfK) and 64Cu-CB-TE2A-c(RGDfK) showed up to a 20-fold increase in tumor-to-organ ratios. PET studies demonstrated high-contrast images of the U87MG tumors at 18 h, confirming the biodistribution data. Conclusion: The ease of radiolabeling makes 68Ga-NODAGA-c(RGDfK) an attractive alternative to 18F-labeled RGD peptides. The high tumor-to-background ratios of 64Cu-NODAGA-c(RGDfK) and 64Cu-CB-TE2A-c(RGDfK) at 18 h warrant testing of 64Cu-labeled RGD peptides in patients.

Development of a Real-time RT-PCR Assay for Detecting EGFRvIII in Glioblastoma Samples

Purpose: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may be warranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge. Experimental Design: We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization. Results: The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00). Conclusion: This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.

18F-FDOPA PET and PET/CT Accurately Localize Pheochromocytomas

Successful treatment of pheochromocytoma requires accurate diagnosis and localization of tumors. Herein, we investigated the accuracy of PET using 3,4-dihydroxy-6-18F-fluoro-phenylalanine (18F-FDOPA), an amino acid transporter substrate, as an independent marker for detection of benign and malignant pheochromocytomas. Methods: The study comprised 25 consecutive patients (9 men, 16 women) whose median age was 51 y (range, 25–68 y), with known or suspected pheochromocytoma. Eleven patients underwent standardized 18F-FDOPA PET and 14 patients underwent 18F-FDOPA PET/CT studies, with a median of 511 MBq of 18F-FDOPA (range, 206–625 MBq). Two readers, unaware of the reports of other imaging studies and clinical data, analyzed all scans visually and quantitatively (maximum standardized uptake value [SUVmax] and maximum transverse diameter). Histology and long-term clinical follow-up served as the gold standard. Correlation between SUVmax of tumors and biochemical markers was evaluated. SUVmax of the benign and malignant tumors was compared. Results: Seventeen patients underwent surgery. Histology confirmed pheochromocytoma or paraganglioma in 11 cases (8 adrenal, including 2 malignant tumors, and 3 extraadrenal, including 1 malignant tumor). The diagnosis of pheochromocytoma was established by follow-up in 2 additional patients (1 adrenal and 1 unknown location) and ruled out in 6 patients. Visual analysis detected and localized pheochromocytoma in 11 of 13 patients without false-positive results (sensitivity, 84.6%; specificity, 100%; accuracy, 92%). These lesions had an SUVmax of 2.3–34.9 (median, 8.3). Evaluation of the false-negative cases revealed a 13 × 5 mm lesion with an SUVmax of 1.96 in 1 case; no lesion was localized in the second case using multiple additional modalities. Spearman nonparametric analysis did not show statistically significant correlation between SUVmax of the tumors and biochemical markers. The Mann–Whitney nonparametric test did not demonstrate a statistically significant difference between the SUVmax of 18F-FDOPA in malignant and benign tumors. Conclusion: 18F-FDOPA PET and PET/CT are highly sensitive and specific tools that can provide additional independent information for diagnosis and localization of benign and malignant pheochromocytomas.

Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100

Acute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvβ3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin-expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.

Noninvasive Imaging of αVβ3 Function as a Predictor of the Antimigratory and Antiproliferative Effects of Dasatinib

Src family kinases (SFKs) are commonly deregulated in cancer cells. Among other functions, SFKs are critical for cellular migration and invasion. SFK inhibitors are being studied as targeted cancer drugs, but there are no biomarkers for noninvasive assessment of SFK inhibition. The aim of this study was to evaluate whether imaging of alpha(V)beta(3) integrin activity with positron emission tomography (PET) and [(64)Cu]DOTA-cyclo-(Arg-Gly-Asp-dPhe-Lys) {[(64)Cu]DOTA-c(RGDfK)} can be used for monitoring response to the SFK inhibitor dasatinib. Severe combined immunodeficient mice bearing U87MG xenografts were gavaged daily over 72 hours with 72 or 95 mg/kg of dasatinib or vehicle. Tumor uptake of [(64)Cu]DOTA-c(RGDfK) was measured by small-animal PET. In parallel, fluorodeoxyglucose (FDG) scans were performed to assess tumor metabolism in response to dasatinib treatment. Dasatinib significantly (P<0.0001) reduced [(64)Cu]DOTA-c(RGDfK) uptake by up to 59% in U87MG xenografts [2.10+/-0.14% injected dose/gram (ID/g) in the 95 mg/kg group and 3.12+/-0.18% ID/g in the 72 mg/kg group, versus 5.08+/-0.80% ID/g in controls]. In contrast, tumor FDG uptake showed no significant reduction with dasatinib therapy (8.13+/-0.45% ID/g in treated versus 10.39+/-1.04% ID/g in controls; P=0.170). Histologically, tumors were viable at the time of the follow-up PET scan but showed inhibition of focal adhesion kinase. Continued dasatinib treatment resulted in a significant inhibition of tumor growth (tumor size on day 10 of therapy: 21.13+/-2.60 mm(2) in treated animals versus 122.50+/-17.68 mm(2) in controls; P=0.001). [(64)Cu]DOTA-c(RGDfK) may provide a sensitive means of monitoring tumor response to SFK inhibition in alpha(V)beta(3)-expressing cancers early in the course of therapy.

Derivation of a Compartmental Model for Quantifying 64Cu-DOTA-RGD Kinetics in Tumor-Bearing Mice

Radiolabeled arginine-glycine-aspartate (RGD) peptides are increasingly used in preclinical and clinical studies to assess the expression and function of the αvβ3 integrin, a cellular adhesion molecule involved in angiogenesis and tumor metastasis formation. To better understand the PET signal obtained with radiolabeled RGD peptides, we have constructed a compartmental model that can describe the time–activity curves in tumors after an intravenous injection. Methods: We analyzed 60-min dynamic PET scans obtained with 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA)-RGD in 20 tumor-bearing severe combined immunodeficient (SCID) mice after a bolus dose (18,500 kBq [500 μCi]), using variations of the standard 2-compartment (4k) tissue model augmented with a compartment for irreversible tracer internalization. αvβ3 binding sites were blocked in 5 studies with a coinjection of cold peptide. In addition, 20 h after injection, static PET was performed on 9 of 20 mice. We fitted 2k (k3 = k4 = 0), 3k (k4 = 0), 4k, and 4kc (k4 = constant) models to the PET data and used several criteria to determine the best model structure for describing 64Cu-DOTA-RGD kinetics in mice. Akaike information criteria (AIC), calculated from model fits and the ability of each model to predict tumor concentration 20 h after tracer injection, were considered. Results: The 4kc model has the best profile in terms of AIC values and predictive ability, and a constant k4 is further supported by Logan–Patlak analysis and results from iterative Bayesian parameter estimation. The internalization compartment allows quantification of the putative tracer internalization rate for each study, which is estimated here to be approximately an order of magnitude less than k3 and thus does not confound the apparent specific binding of the tracer to the tumor integrin during the first 60 min of the scan. Analysis of specific (S) and nonspecific or nondisplaceable (ND) binding using fitted parameter values showed that the 4kc model provided expected results when comparing αvβ3 blocked and nonblocked studies. That is, specific volume of distribution, [VS = (K1k3)/(k2k4)], is much higher than is nondisplaceable volume of distribution, [VND = (K1/k2)], in nonblocking studies (2.2 ± 0.6 vs. 0.85 ± 0.14); VS and VND are about the same in the blocking studies (0.46 ± 1.6 vs. 0.56 ± 0.09). Also, the ratio of static tumor and plasma measurements at 60 and 10 min [CT(60)/CP(10)] is highly correlated (RS = 0.92) to tumor VS. Conclusion: We have developed and tested a compartmental model for use with the 64Cu-DOTA-RGD PET tracer and demonstrated its potential as a tool for analysis and design of preclinical and clinical imaging studies.

Targeted Radiotherapy of Prostate Cancer with a Gastrin-Releasing Peptide Receptor Antagonist Is Effective as Monotherapy and in Combination with Rapamycin

The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. Methods: To determine the effect of treatment with rapamycin and radiotherapy with a novel 177Lu-labeled GRPr antagonist (177Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and 177Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on 177Lu-RM2 tumor uptake, in vivo small-animal PET studies with 68Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of 177Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with 177Lu-RM2 alone or after pretreatment with rapamycin. Results: Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with 177Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of 177Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. Conclusion: Radiotherapy using a 177Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

Somatostatin Receptor–Targeted Radiopeptide Therapy with 90Y-DOTATOC and 177Lu-DOTATOC in Progressive Meningioma: Long-Term Results of a Phase II Clinical Trial

Meningiomas express members of the somatostatin receptor family. The present study assessed the long-term benefits and harm of somatostatin-based radiopeptide therapy in meningioma patients. Methods: Patients with progressive unresectable meningioma were treated with 90Y-DOTATOC and 177Lu-DOTATOC until tumor progression or permanent toxicity occurred. Multivariable Cox regression analyses were used to study predictors of survival. Results: Overall, 74 treatment cycles were performed on 34 patients. Stable disease was achieved in 23 patients. Severe hematotoxicity occurred in 3 patients, and severe renal toxicity in 1 patient. Mean survival was 8.6 y from the time of recruitment. Stable disease after treatment (hazard ratio, 0.017 vs. progressive disease; 95% confidence interval, 0.001–0.35; n = 34; P = 0.01) and high tumor uptake (hazard ratio, 0.046 vs. intermediate or low tumor uptake; 95% confidence interval, 0.004–0.63; n = 34; P = 0.019) were associated with longer survival. Conclusion: 90Y-DOTATOC and 177Lu-DOTATOC are promising tools for treating progressive unresectable meningioma, especially in cases of high tracer uptake in the tumor.

The prognostic and predictive value of sstr2-immunohistochemistry and sstr2-targeted imaging in neuroendocrine tumors.

PurposeOur aim was to assess the prognostic and predictive value of somatostatin receptor 2 (sstr2) in neuroendocrine tumors (NETs).MethodsWe established a tissue microarray and imaging database from NET patients that received sstr2-targeted radiopeptide therapy with yttrium-90-DOTATOC, lutetium-177-DOTATOC or alternative treatment. We used univariate and multivariate analyses to identify prognostic and predictive markers for overall survival, including sstr2-imaging and sstr2-immunohistochemistry.ResultsWe included a total of 279 patients. In these patients, sstr2-immunohistochemistry was an independent prognostic marker for overall survival (HR: 0.82, 95 % CI: 0.67 – 0.99, nu2009=u2009279, pu2009=u20090.037). In DOTATOC patients, sstr2-expression on immunohistochemistry correlated with tumor uptake on sstr2-imaging (nu2009=u2009170, pu2009<u20090.001); however, sstr2-imaging showed a higher prognostic accuracy (positive predictive value: +27xa0%, 95 % CI: 3 – 56xa0%, pu2009=u20090.025). Sstr2-expression did not predict a benefit of DOTATOC over alternative treatment (pu2009=u20090.93).ConclusionsOur results suggest sstr2 as an independent prognostic marker in NETs. Sstr2-immunohistochemistry correlates with sstr2-imaging; however, sstr2-imaging is more accurate for determining the individual prognosis.