Murine Model Of HER2 Research Articles

Abstract 568: Activation of the RXR nuclear receptor regulates CD4 T cell activity in a murine model of HER2+ breast cancer

Abstract The role of CD4 T cells for anti-tumor responses is less recognized than CD8 cells. Evidence suggests that CD4 T cells can act directly as antitumor cells or direct the maturation and activation of dendritic cells (DCs). The retinoid X receptor (RXR) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors. RXR and its heterodimers have profound effects on the function of myeloid cells, but effects on T cells are less known. Treatment with the RXR agonist MSU42011 (100 mg/Kg diet) reduced tumor burden in a HER2+ model of breast cancer (p=0.0068) and in a KRAS-driven model of lung cancer (p=0.0015). However, when human lung cancer cells were implanted into immunodeficient mice, MSU42011 failed to reduce tumor burden, suggesting that the anti-tumor properties of MSU42011 are dependent on a functional immune system. After 10 days of treatment, the percentage of CD4, CD62L+ T cells in HER2+ mammary tumors treated with MSU42011 was significantly higher compared to the control group (2.41 vs 3.78, p=0.05), as analysed by flow cytometry. Additionally, tumor antigen presenting cells (CD11c+, CD11b+, IA-IE+) and DCs (CD11c+, IA-IE+) had increased expression of PDL1 in the MSU42011 group when compared with controls (MFI=4016 vs 5225, p=0.069; 5484 vs 7140, p=0.045, respectively). RT-PCR analysis of whole tumors showed a significant increase in mRNA expression of interferon (IFN; p=0.0026) and IL12a (p=0.0011) in treated mice. Similar increases in IFN and IL12a were found in the murine model of lung cancer. Next, T cells were depleted to determine if required for the anti-tumor effects of MSU42011. CD8 cells depletion did not reverse the anti-tumor efficacy of MSU42011. CD4 depletion in mice treated with MSU42011 rescued tumor growth (p=0.016 MSU42011 vs MSU42011+anti-CD4), as tumors of mice treated with MSU42011 and anti-CD4 antibodies for 10 days grew at the same rate as tumors treated with control plus or minus anti-CD4 antibodies. Tumors treated with MSU42011 and anti-CD4 antibodies also showed reduced levels of IL12a compared with MSU42011 alone (2.28 vs 0.87, p>0.05), as determined by RT-PCR.To dissect the effects of RXR activation on CD4 and DC cells, primary DCs, alone or co-cultured with T cells and conditioned media from cancer cells, were treated with MSU42011 (100 nM). This treatment increased PDL1 and IL12 mRNA expression in DCs. T cells, co-cultured with DC and conditioned media and treated with MSU42011, expressed higher IL2 mRNA levels. MSU42011 also increased expression of IFN in CD3 T cells. In conclusion, MSU42011 increased recruitment and activation of CD4 T cells in vitro and in HER2+ mammary tumors. These data, in combination with our previous work showing that RXR agonists reduce expression of tumor-promoting FOXP3 T cells in vitro and in vivo, confirm a strong correlation between RXR pharmacologic activation and CD4 T cell activation within the tumor microenvironment. Citation Format: Ana Sofia Leal, Karen T. Liby. Activation of the RXR nuclear receptor regulates CD4 T cell activity in a murine model of HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 568.

Optimizing combination therapy in a murine model of HER2+ breast cancer

Human epidermal growth factor receptor 2 positive (HER2+) breast cancer is frequently treated with drugs that target the HER2 receptor, such as trastuzumab, in combination with chemotherapy, such as doxorubicin. However, an open problem in treatment design is to determine the therapeutic regimen that optimally combines these two treatments to yield optimal tumor control. Working with data quantifying temporal changes in tumor volume due to different trastuzumab and doxorubicin treatment protocols in a murine model of human HER2+ breast cancer, we propose a complete framework for model development, calibration, selection, and treatment optimization to find the optimal treatment protocol. Through different assumptions for the drug–tumor interactions, we propose ten different models to characterize the dynamic relationship between tumor volume and drug availability, as well as the drug–drug interaction. Using a Bayesian framework, each of these models are calibrated to the dataset and the model with the highest Bayesian information criterion weight is selected to represent the biological system. The selected model captures the inhibition of trastuzumab due to pre-treatment with doxorubicin, as well as the increase in doxorubicin efficacy due to pre-treatment with trastuzumab. We then apply optimal control theory (OCT) to this model to identify two optimal treatment protocols. In the first optimized protocol, we fix the maximum dosage for doxorubicin and trastuzumab to be the same as the maximum dose delivered experimentally, while trying to minimize tumor burden. Within this constraint, optimal control theory indicates the optimal regimen is to first deliver two doses of trastuzumab on days 35 and 36, followed by two doses of doxorubicin on days 37 and 38. This protocol predicts an additional 45% reduction in tumor burden compared to that achieved with the experimentally delivered regimen. In the second optimized protocol we fix the tumor control to be the same as that obtained experimentally, and attempt to reduce the doxorubicin dose. Within this constraint, the optimal regimen is the same as the first optimized protocol but uses only 43% of the doxorubicin dose used experimentally. This protocol predicts tumor control equivalent to that achieved experimentally. These results strongly suggest the utility of mathematical modeling and optimal control theory for identifying therapeutic regimens maximizing efficacy and minimizing toxicity.

Abstract 5050: Optimizing delivery of combination targeted and chemotherapy in a murine model of HER2+ breast cancer

Abstract The combination of targeted therapy to the human epidermal growth factor receptor 2 (HER2) and chemotherapy in HER2+ breast cancer is known to increase time to progression, duration of response, and survival rates. Previously, we established a murine model of human HER2+ breast cancer, and investigated whether the sequence in which two drugs are delivered affected the treatment outcome [1]. However, this combination therapy can cause cardiac toxicity. While the cardiac damage induced by trastuzumab does not appear to be dose dependent and it is reversible, doxorubicin cardiotoxicity is cumulative, dose dependent, and irreversible. Thus, rigorously identifying a treatment protocol that simultaneously maintains tumor control and reduces the total dose of doxorubicin, would potentially decrease the side effects experienced by patients receiving these treatments, while maintaining high treatment efficacy. We propose a complete framework for model development, calibration, selection, and treatment optimization to find the optimal treatment protocol for HER2+ breast cancer using a trastuzumab-doxorubicin combination. To do so, we constructed a family of ten mathematical models designed to characterize the dynamics of tumor growth and treatment response. In particular, the models included tumor-drug and drug-drug interactions. Using a Bayesian framework, each of these models were calibrated to the data obtained in [1], and the most parsimonious model as determined by the Bayesian information criterion was selected to represent the system. We then applied optimal control theory (while considering uncertainty in the model parameters) to systematically identify the optimal treatment protocol using the selected model. We had two major findings. First, we kept the total dose of both drugs fixed to that which was employed in the experiments in [1], and identified a treatment schema where the tumors showed complete regression without increasing the overall amount of therapy. Then, we found the treatment schema with the least amount of drug delivered (decreased by 58%) that resulted in the same tumor kinetics as the experimental results. Thus, we employed mathematical modeling and an in vivo model of breast cancer to rigorously and systematically identify treatment protocols that can improve tumor control with the same total dose, or achieve the same tumor control with less than half the doxorubicin dose. Predictions can now be tested experimentally. This has a significant impact for the design of combination therapy for HER2+ breast cancer. [1] Sorace, A.G., Quarles, C.C., Whisenant, J.G., Hanker, A.B., McIntyre, J.O., Sanchez, V.M. and Yankeelov, T.E., 2016. Trastuzumab improves tumor perfusion and vascular delivery of cytotoxic therapy in a murine model of HER2+ breast cancer: preliminary results. Breast cancer research and treatment, 155(2), pp.273-284. Citation Format: Ernesto A.B.F. Lima, Reid Wyde, Anna G. Sorace, Thomas E. Yankeelov. Optimizing delivery of combination targeted and chemotherapy in a murine model of HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5050.

Abstract 3138: Identification of therapy-sensitive tumor phenotypes using quantitative MRI habitats in a preclinical model of HER2+ breast cancer

Abstract Introduction: Heterogeneity of the tumor microenvironment influences therapeutic delivery and efficacy, presenting a significant challenge in cancer treatment. Quantitative magnetic resonance imaging (MRI) can spatially resolve intratumoral heterogeneity into physiologically-distinct subregions, or habitats. We use quantitative MRI habitats to elucidate intertumoral heterogeneity through identification of tumor imaging phenotypes, and longitudinally evaluate treatment response in a murine model of HER2+ breast cancer. Methods: BT474 cells were subcutaneously implanted in athymic nude mice (n = 62). Once tumors reached ~235 mm3, mice were randomly assigned to trastuzumab (10 mg/kg) or saline control groups, and treated on days 0 and 3. Diffusion weighted (DW) and dynamic contrast-enhanced (DCE) MRI data were collected on days 0 (pre-treatment), 1 and 4. Apparent diffusion coefficients (ADC, a measure of cell density) were calculated from DW-MRI. DCE-MRI data was modeled to extract the extravascular, extracellular volume fraction, ve, and the volume transfer coefficients, Ktrans and kep, which correspond to the rate of contrast agent wash-in and wash-out, respectively. Habitats were identified through hierarchical clustering of tumor voxel data (ADC, ve, Ktrans, and kep). Tumor composition was quantified as percent tumor volume comprised by each habitat. Finally, tumors were described by their baseline tumor composition and clustered to identify unique tumor imaging phenotypes. Treatment response was measured using percent change in tumor volume over time. Results: Clustering of the MRI data yielded three habitats: low-vascularity low-cellularity (LV-LC), low-vascularity high-cellularity (LV-HC), high-vascularity high-cellularity (HV-HC). Two distinct tumor imaging phenotypes were identified from clustering, designated as Type 1 and Type 2. Type 1 tumors showed higher relative HV-HC tumor volume and lower relative LV-HC and LV-LC tumor volume, compared to Type 2 tumors (p < 0.05). Type 1 tumors treated with trastuzumab showed a decrease in tumor volume at day 4 (p < 0.05) compared to control. Type 2 tumors treated with trastuzumab showed no significant changes in tumor volume compared to control. Treated Type 1 tumors showed a significant increase in LV-LC percent tumor volume at day 4, compared to day 0 (30% vs. 21%, p < 0.01). Treated Type 2 tumors showed a significant decrease in LV-HC percent tumor volume at day 4, compared to day 0 (25% vs. 46%, p < 0.05). Discussion & Conclusions: Quantitative MRI habitats can be used to identify tumor phenotypes with differing response to treatment. The Type 1 phenotype may confer increased therapeutic sensitivity, demonstrated by improved response to trastuzumab. Clinical application of this approach could improve understanding of tumor pathology and therapeutic sensitivity for patients with HER2+ breast cancer. Citation Format: Anum S. Kazerouni, David A. Hormuth, Tessa Davis, Meghan J. Bloom, John Virostko, Thomas E. Yankeelov, Anna G. Sorace. Identification of therapy-sensitive tumor phenotypes using quantitative MRI habitats in a preclinical model of HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3138.

Anti-HER2 induced myeloid cell alterations correspond with increasing vascular maturation in a murine model of HER2+ breast cancer

BackgroundTherapy targeted to the human epidermal growth factor receptor type 2 (HER2) is used in combination with cytotoxic therapy in treatment of HER2+ breast cancer. Trastuzumab, a monoclonal antibody that targets HER2, has been shown pre-clinically to induce vascular changes that can increase delivery of chemotherapy. To quantify the role of immune modulation in treatment-induced vascular changes, this study identifies temporal changes in myeloid cell infiltration with corresponding vascular alterations in a preclinical model of HER2+ breast cancer following trastuzumab treatment.MethodsHER2+ tumor-bearing mice (N = 46) were treated with trastuzumab or saline. After extraction, half of each tumor was analyzed by immunophenotyping using flow cytometry. The other half was quantified by immunohistochemistry to characterize macrophage infiltration (F4/80), vascularity (CD31 and α-SMA), proliferation (Ki67) and cellularity (H&E). Additional mice (N = 10) were used to quantify differences in tumor cytokines between control and treated groups.ResultsImmunophenotyping showed an increase in macrophage infiltration 24 h after trastuzumab treatment (P ≤ 0.05). With continued trastuzumab treatment, the M1 macrophage population increased (P = 0.02). Increases in vessel maturation index (i.e., the ratio of α-SMA to CD31) positively correlated with increases in tumor infiltrating M1 macrophages (R = 0.33, P = 0.04). Decreases in VEGF-A and increases in inflammatory cytokines (TNF-α, IL-1β, CCL21, CCL7, and CXCL10) were observed with continued trastuzumab treatment (P ≤ 0.05).ConclusionsPreliminary results from this study in a murine model of HER2+ breast cancer show correlations between immune modulation and vascular changes, and reveals the potential for anti-HER2 therapy to reprogram immunosuppressive components of the tumor microenvironment. The quantification of immune modulation in HER2+ breast cancer, as well as the mechanistic insight of vascular alterations after anti-HER2 treatment, represent novel contributions and warrant further assessment for potential clinical translation.

Abstract 1946: Quantifying trastuzumab-induced alterations of intratumoral heterogeneity using MRI-derived tumor subregions in the preclinical setting

Abstract Introduction: Heterogeneity in the tumor microenvironment affects therapeutic delivery, providing a major challenge for cancer treatment. We investigate the ability of multi-parametric, voxel-based characterizations of tumor heterogeneity from quantitative magnetic resonance imaging (MRI) to spatially resolve physiological tumor subregions of response to anti-HER2 targeted therapy in a murine model of HER2+ breast cancer. Methods: BT474 cells were implanted in nude athymic mice (n=20) and tumors grown to ~225 mm3. Mice were randomly assigned to saline control or trastuzumab groups, with treatments given on days 0 and 3. Dynamic contrast enhanced (DCE) MRI and diffusion weighted (DW) MRI data were collected on days 0 (pre-treatment), 1 and 4. Tumors, excised on day 4, were processed for histology with anti-CD31 staining. Apparent diffusion coefficients (ADC, a measure of cell density) were extracted from DW-MRI data. DCE-MRI data was modeled to extract the extravascular, extracellular volume fraction, ve, and the volume transfer coefficients, Ktrans and kep, which correspond to the rate of wash-in and wash-out of contrast agent, respectively. Hierarchical clustering of tumor voxel data (ADC, ve, Ktrans, and kep) was used to identify physiological tumor subregions. The contribution of each subregion to tumor volume was quantified as percent tumor volume for each mouse and time point. Image analysis of histology data was used to generate vessel area and nuclei maps for whole-slice histology data. CD31 stained slices were subsequently divided into physiological subregions in terms of vessel and nuclei density, and the contribution of each subregion to the whole-slice area was quantified as percent tumor area. Results: Hierarchical clustering of the MRI data yielded four clusters: low vascularity - low cellularity (LV-LC), low vascularity - high cellularity (LV-HC), high vascularity - low cellularity (HV-LC), high vascularity - high cellularity (HV-HC). At day 0, no significant differences in cluster percent tumor volume were observed between control and treated tumors (p>0.05). At day 4, a significant decrease in LV-HC percent tumor volume was observed in treated tumors compared to control (mean 13.7% vs. 35.0%, p=0.04). Histology subregion analysis corroborated in vivo imaging findings, with treated tumors having significantly lower LV-HC percent tumor area compared to control (mean 26.8% vs 53.6%, p<0.01). Conclusion: High-dimensional analysis of quantitative MRI parameter maps can be utilized to identify physiological subregions of tumor response, and can be biologically validated using histology data. The results suggest trastuzumab therapy decreases the hypoxic (LV-HC) tumor volume. Quantifying tumor microenvironment alterations in response to therapy can potentially be used to predict response for patients with HER2+ breast cancer. Citation Format: Anum Syed, Jennifer Whisenant, Anna Sorace, Thomas Yankeelov. Quantifying trastuzumab-induced alterations of intratumoral heterogeneity using MRI-derived tumor subregions in the preclinical setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1946.

Characterizing Trastuzumab-Induced Alterations in Intratumoral Heterogeneity with Quantitative Imaging and Immunohistochemistry in HER2+ Breast Cancer

The purpose of this study is to investigate imaging and histology-based measurements of intratumoral heterogeneity to evaluate early treatment response to targeted therapy in a murine model of HER2+ breast cancer. BT474 tumor-bearing mice (N = 30) were treated with trastuzumab or saline and imaged longitudinally with either dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) or 18F-fluoromisonidazole (FMISO) positron emission tomography (PET). At the imaging study end point (day 4 for MRI or 7 for PET), each tumor was excised for immunohistochemistry analysis. Voxel-based histogram analysis was performed on imaging-derived parametric maps (i.e., Ktrans and ve from DCE-MRI, SUV from 18F-FMISO-PET) of the tumor region of interest to measure heterogeneity. Image processing and histogram analysis of whole tumor slice immunohistochemistry data were performed to validate the in vivo imaging findings. Trastuzumab-treated tumors had increased heterogeneity in quantitative imaging measures of cellularity (ve), with a mean Kolmogorov-Smirnov (K-S) distance of 0.32 (P = .05) between baseline and end point distributions. Trastuzumab-treated tumors had increased vascular heterogeneity (Ktrans) and decreased hypoxic heterogeneity (SUV), with a mean K-S distance of 0.42 (P < .01) and 0.46 (P = .047), respectively, between baseline and study end points. These observations were validated by whole-slice immunohistochemistry analysis with mean interquartile range of CD31 distributions of 1.72 for treated and 0.95 for control groups (P = .02). Quantitative longitudinal changes in tumor cellular and vascular heterogeneity in response to therapy may provide evidence for early prediction of response and guide therapy for patients with HER2+ breast cancer.

Abstract 924: A mathematical-experimental approach for predicting host responses in a preclinical model for trastuzumab-treated HER2+ breast cancer

Abstract Introduction: Trastuzumab, a targeted therapy for human epidermal growth factor receptor 2 (HER2) positive breast cancer, induces cell cycle arrest and inhibits HER2 expression. Trastuzumab has been shown to improve vascular delivery of subsequent cytotoxic therapies, but the mechanism by which it regulates tumor-associated angiogenesis is not well characterized. Therefore, we developed an integrated, mathematical-experimental approach to systematically investigate the interactions of tumor-growth characteristics—vasculature, immune response, hypoxia, and necrosis—and to evaluate their effects on tumor response to treatment in a murine model of HER2+ breast cancer. Experimental: Mice (N=102) were injected subcutaneously with HER2-overexpressing BT474 breast cancer cells and treated with trastuzumab (10 mg/kg) or saline. Tumor volumes for two separate cohorts of treated and control mice were recorded longitudinally, and either mice were quantitatively imaged or tumors extracted for histology over seven days. Necrosis, vasculature, and hypoxia were characterized by: immunohistochemistry for percent necrosis, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for the percentage of well-vascularized tissue, and 18F-fluoromisonidazole positron emission tomography (FMISO-PET) for percent hypoxia. Preliminary immune (myeloid) cell infiltration was quantified using immunofluorescence of F4/80 and CD11c. Mathematical Model: A system of five coupled, ordinary differential equations describing the temporal variation in tumor growth, vasculature, hypoxia, necrosis, and immune response was calibrated using data for tumor volume and percentages of well-vascularized tissue, hypoxia, and necrosis. After validation using a second tumor volume data set, uncertainty analysis was used to verify plausible overlap between the model's simulations and the experimental data when considering calibration error, and sensitivity analysis identified critical parameters for experimental estimation and potential model reductions. Results and Discussion: The calibrated model was used to predict immune response behavior over time and yielded different results between the two groups—stagnant versus increasing immune component values for control versus treated mice, respectively. Preliminary immunofluorescence data support the immunological predictions of the model; in particular, F4/80 and CD11c co-staining in the total tissue is greater in trastuzumab treated than control tumors (p &amp;lt; 0.03). Furthermore, differences in the calibrated parameter sets indicate several experimentally testable hypotheses, including increased cross-talk between immune cells and vasculature as a mechanism for vascular stabilization due to trastuzumab therapy. We acknowledge the support of CPRIT RR160005 and NCI R01CA186193. Citation Format: Angela M. Jarrett, Meghan Bloom, Wesley Godfrey, Anum Syed, David A. Ekrut, Lauren I. Ehrlich, Thomas E. Yankeelov, Anna G. Sorace. A mathematical-experimental approach for predicting host responses in a preclinical model for trastuzumab-treated HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 924.

Abstract P3-05-16: A mathematical model for predicting immune response in a trastuzumab treated HER2+ breast cancer animal model: Preliminary efforts

Abstract Introduction: Trastuzumab is a targeted antibody to the human epidermal growth factor receptor 2 (HER2) that induces cell cycle arrest and is used in the treatment of HER2+ positive breast cancer. We have recently presented in vivo evidence in a murine model that trastuzumab also improves vascular delivery of subsequent cytotoxic therapies. The mechanism by which trastuzumab and the immune system interact to regulate tumor-associated angiogenesis is not well characterized. Therefore, we offer a preliminary report on a mathematical framework to systematically investigate the potential interactions among the immune response, tumor cells, vasculature, and other environmental factors based on experimental data for the BT474 murine model of HER2+ breast cancer. Experimental: BT474 breast cancer cells were implanted subcutaneously into athymic nude mice. After tumors reached 250 mm3, mice were treated with trastuzumab or saline, tumor volumes were recorded, and tumors were extracted at various times over seven days. Immunohistochemistry for treated and control tumors were evaluated for 0, 1, 3, 4, and 7 days post trastuzumab treatment. Histology data includes: percent hypoxia (pimonidazole), percent necrosis, and vascular maturation index (VMI, ratio of alpha-smooth muscle actin to total vessel counts as stained with CD31). Ongoing studies are quantifying immune cell infiltration through immunofluorescent imaging of F4/80 and CD11c expression. Modeling: We developed a system of five coupled, ordinary differential equations that accounts for the temporal variation in tumor growth, vasculature, hypoxia, necrosis, and immune response. The general immune response component corresponds to the mouse's pro-inflammatory responses, as T cell driven responses are absent in this murine model. Uncertainty analysis was performed to verify plausible overlap between the model's predictions and the experimental data—where local and global samplings of all parameters were used to generate potential model results to be compared to the data for both control and treated mouse sets. Sensitivity analysis was performed using Sobol' Indices to determine the driving parameters of the system to identify target parameters for experimental estimation. The model parameters were calibrated using mean and standard deviations for the available data (tumor volume, VMI, percentage of hypoxia, and percentage of necrosis) for each experimental time point to predict the differences of the immune component between the treated and control tumors. Results and Discussion: The model is well behaved and can be calibrated with the available data. The model predicts distinct differences for the immune response between the control and treated groups—showing increasing versus decreasing immune component values over time for treated versus control results, respectively. Preliminary results from the immunofluorescent imaging data support the immunological predictions of the model; in particular, the amount of immune infiltration (i.e., more immune cells) in necrotic areas is greater in the treated than the untreated tumors (p&amp;lt;0.025). We acknowledge the support of CPRIT RR160005 and NCI R01CA186193. Citation Format: Jarrett AM, Yankeelov TE, Ehrlich LI, Godfrey W, Sorace AG. A mathematical model for predicting immune response in a trastuzumab treated HER2+ breast cancer animal model: Preliminary efforts [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-16.

Abstract P4-02-02: Increased tumor perfusion following treatment with trastuzumab as measured by contrast-enhanced ultrasound

Abstract Introduction: The primary purpose of this study is to determine if the order of dosing in standard-of-care (SOC) combination therapy for HER2+ breast cancer has an effect on the perfusion characteristics within the tumor. Improving the intratumoral delivery of cytotoxic systemic therapy is a significant challenge in advancing cancer treatment. Knowledge of the vascular changes following SOC treatments could enable optimizing their order and timing, potentially leading to significantly improved response. Currently, one SOC regimen for HER2+ breast cancer treatment is doxorubicin administered for 3-4 cycles prior to trastuzumab. Our goal is to quantitatively map the changes in perfusion in response to different combinations of trastuzumab plus doxorubicin treatment through imaging in a murine model of HER2+ breast cancer. Experimental Design: BT474 breast cancer cells (1×107) were subcutaneously implanted into mice (n = 12) and randomly assigned into three treatment groups: two doses of trastuzumab (10 mg/kg) followed by doxorubicin (1.5 mg/kg), doxorubicin prior to trastuzumab (same total drug dosage as group 1), and saline. After tumors reached ~225 mm3, animals were imaged with contrast-enhanced ultrasound (CEUS) (VisualSonics Vevo 770, Definity microbubbles) before treatment (day 0), and on days 1, 3, 4, and 7. Treatment occurred on days 0, 3 and 4. Percent change (from baseline, day 0 scans) of the CEUS signal intensity quantified from the functional vasculature (surrogate for vessel perfusion) following contrast injection were measured for each animal for each day. Tumors were extracted on day 7, and sectioned, paraffin-embedded, and stained with CD31, alpha-SMA and H&amp;E. Results: Tumors treated with trastuzumab initially exhibited a significant increase in CEUS signal intensity (from the functional vasculature) on day 1 compared to tumors initially treated with doxorubicin (p &amp;lt; 0.01). Additionally, compared to the control tumors, tumors treated with trastuzumab prior to doxorubicin revealed a significant increase in perfusion (change in signal intensity of functional vasculature) of contrast agent on days 3 (p = 0.01), 4 (p = 0.001) and day 7 (p &amp;lt; 0.01). There were no significant differences in the doxorubicin treated first group and the controls on any of the days (p &amp;gt; 0.25). Qualitative differences were noted between control and treated groups for alpha-SMA, no apparent differences were noted in microvessel density. Conclusion: Trastuzumab significantly improves a tumor's vascular perfusion in this HER2+ breast cancer model. Doxorubicin dosing prior to treatment with trastuzumab may potentially be hindering the intratumoral delivery of the subsequently delivered, targeted therapy. Improving the tumor's functional vasculature by altering the order of dosing of these combination therapies by giving trastuzumab prior to cytotoxic therapy has potential to enhance both the delivery and the effectiveness of these combination therapies. These data indicate a potential pathway to optimize therapeutic efficacy for individual HER2+ breast cancer patients. Citation Format: Sorace AG, Barnes SL, Quarles CC, McIntyre JO, Yankeelov TE. Increased tumor perfusion following treatment with trastuzumab as measured by contrast-enhanced ultrasound [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-02-02.

Abstract P4-12-12: Quantitative in vivo imaging of intratumoral heterogeneity to assess tumor response to trastuzumab treatment in a preclinical model of HER2+ breast cancer

Abstract Introduction: Intratumoral heterogeneity is associated with variable treatment response and poorer patient prognosis. Initial changes in intratumoral cellular and vascular heterogeneity after treatment could serve as an early metric for treatment response and be used to guide and optimize therapy for improved patient outcome. Quantitative, noninvasive imaging can characterize, in 3D, tumor response to treatment by evaluating longitudinal alterations in heterogeneity. Our goal is to quantitatively image the changes in intratumoral cellular and vascular heterogeneity in response to trastuzumab in a murine model of HER2+ breast cancer. Experimental Design: Data from two independent cohorts of mice were used in this study. Mice were implanted subcutaneously with BT474 breast cancer cells (1×107) and randomly assigned to trastuzumab treated (10 mg/kg) or saline control groups. After tumors reached ~225 mm3, intraperitoneal injections of trastuzumab or saline were given on days 0 and 3. In the first cohort of mice (n=14), quantitative dynamic contrast enhancement magnetic resonance imaging (DCE-MRI) assessed tumor response on days 0 (prior to treatment), 1 and 4. In this study, we focused on the calculated extravascular extracellular space, measured using DCE-MRI parameter, ve. In the second cohort of mice (n=10), 18F-flouromisonidazole positron emission tomography (FMISO-PET) imaging was utilized to assess tumor hypoxia on days 0 (prior to treatment), 1, 3, 4, and 7 through changes in standardized uptake value (SUV). Longitudinal changes in heterogeneity were assessed using voxel based histogram analysis of ve or SUV. Statistical analysis (R Studio) compared histogram analyses (full-width half-maximum, range, kurtosis, and standard deviation) of control and treated groups. Tumor size measurements obtained at the conclusion of the experiment were used to validate imaging findings of tumor response to treatment. Results: DCE-MRI revealed increased heterogeneity in extravascular extracellular space after trastuzumab treatment, as quantified by a significant increase in the FWHM of ve voxel distributions at day 4 post-treatment (p=0.03). Additionally, the FMISO-PET data showed a significant decrease in hypoxic regional heterogeneity in treated tumors, as quantified by percent change (normalized to baseline) in standard deviation and range of SUV tumor voxel distributions on days 3 (p=0.02), 4 (p=0.01), and 7 (p&amp;lt;0.001) compared to saline controls. Conclusion: Trastuzumab improved oxygen delivery throughout the tumor as quantified by decreased hypoxia heterogeneity (narrowing of distributions) of the FMISO-PET SUV map. Likewise, the increased heterogeneity (widening of distributions) of the DCE-MRI ve parametric map, reflects a more heterogeneous cellularity interpreted to arise from enhanced cell death following trastuzumab treatment. Ongoing analysis of heterogeneity of corresponding histology slides will be used to validate the imaging measures of heterogeneity. DCE-MRI and FMISO-PET reveal quantitative longitudinal changes in tumor cellular and vascular heterogeneity, and may be able to predict response and guide therapy for patients with HER2+ breast cancer. Citation Format: Syed A, Quarles CC, McIntyre JO, Barnes SL, Yankeelov TE, Sorace AG. Quantitative in vivo imaging of intratumoral heterogeneity to assess tumor response to trastuzumab treatment in a preclinical model of HER2+ breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-12.

In Vivo Imaging of Tumor Metabolism and Acidosis by Combining PET and MRI-CEST pH Imaging.

The vast majority of cancers exhibit increased glucose uptake and glycolysis regardless of oxygen availability. This metabolic shift leads to an enhanced production of lactic acid that decreases extracellular pH (pHe), a hallmark of the tumor microenvironment. In this way, dysregulated tumor pHe and upregulated glucose metabolism are linked tightly and their relative assessment may be useful to gain understanding of the underlying biology. Here we investigated noninvasively the in vivo correlation between tumor 18F-FDG uptake and extracellular pH values in a murine model of HER2+ breast cancer. Tumor extracellular pH and perfusion were assessed by acquiring MRI-CEST (chemical exchange saturation transfer) images on a 3T scanner after intravenous administration of a pH-responsive contrast agent (iopamidol). Static PET images were recorded immediately after MRI acquisitions to quantify the extent of 18F-FDG uptake. We demonstrated the occurrence of tumor pHe changes that report on acidification of the interstitial fluid caused by an accelerated glycolysis. Combined PET and MRI-CEST images reported complementary spatial information of the altered glucose metabolism. Notably, a significant inverse correlation was found between extracellular tumor pH and 18F-FDG uptake, as a high 18F-FDG uptake corresponds to lower extracellular pH values. These results show how merging the information from 18F-FDG-uptake and extracellular pH measurements can improve characterization of the tumor microenvironment. Cancer Res; 76(22); 6463-70. ©2016 AACR.

Quantitative [18F]FMISO PET Imaging Shows Reduction of Hypoxia Following Trastuzumab in a Murine Model of HER2+ Breast Cancer.

Evaluation of [18F]fluoromisonidazole ([18F]FMISO)-positron emission tomography (PET) imaging as a metric for evaluating early response to trastuzumab therapy with histological validation in a murine model of HER2+ breast cancer. Mice with BT474, HER2+ tumors, were imaged with [18F]FMISO-PET during trastuzumab therapy. Pimonidazole staining was used to confirm hypoxia from imaging. [18F]FMISO-PET indicated significant decreases in hypoxia beginning on day 3 (P < 0.01) prior to changes in tumor size. These results were confirmed with pimonidazole staining on day 7 (P < 0.01); additionally, there was a significant positive linear correlation between histology and PET imaging (r 2 = 0.85). [18F]FMISO-PET is a clinically relevant modality which provides the opportunity to (1) predict response to HER2+ therapy before changes in tumor size and (2) identify decreases in hypoxia which has the potential to guide subsequent therapy.

Abstract 4237: Decreased hypoxia in a HER2+ breast cancer model following trastuzumab therapy

Abstract Introduction: Hypoxic tumors demonstrate treatment resistance with an increased risk of metastasis. Therefore, alleviating hypoxia has potential to improve efficiency of combination therapies. The primary goal of this study is to quantify alterations in hypoxia in response to trastuzumab in a murine model of HER2+ breast cancer through imaging and histology. Experimental Design: Mice were implanted subcutaneously with BT474 breast cancer cells (107) and randomly assigned into treated (10 mg/kg trastuzumab) or control (saline) groups. After tumors reached ∼250 mm3, animals (n = 32) were utilized to identify longitudinal changes in functional vascular through an intravenous injection of Hoechst 33342 nuclear stain (immediately prior to sacrifice) and hypoxia through pimonidazole intravenous injection (one hour prior to sacrifice). Tumors were extracted for immunofluorescence between days 0 through 7. Tumor sections were flash frozen and stained with anti-pimonidazole and propidium iodide (nuclear counterstain). Additionally, a set of tumors (n = 36) were sacrificed for immunohistochemistry (days 0 through 7), formalin fixed and stained for CA-IX. All stained sections were scanned in high resolution (20×) and quantitatively analyzed with Leica SCN400 software. Another cohort of animals (n = 10) were imaged with 18F-FMISO (fluoromisonidazole) PET between days 0 through 7 and quantified via mean tracer concentration (%ID/g). Results: Immunohistochemistry revealed significantly increased hypoxia in the control group compared to treated on days 3 (p = 0.03) and 7 (p = 0.002), as measured through CA-IX staining. Additionally, on day 4, functional vascular delivery was increased while hypoxia (pimonidazole) decreased in treated tumors compared to control. 18F-FMISO PET imaging corroborated histology findings with significantly decreased hypoxia in treated tumors compared to control tumors on day 7 (-47%; p&amp;lt;0.0001). Conclusion: Trastuzumab has been shown to decrease hypoxia, as measured through gold-standard immunofluorescence. Additionally, this trastuzumab-induced improvement in tumor hypoxia can be measured via clinically relevant, noninvasive imaging (18F-FMISO PET). Temporarily improving the tumor's functional vasculature and decreasing hypoxia during trastuzumab treatment has potential to enhance the effectiveness of combination therapies. Identifying windows of improved vascular and cellular normalization with noninvasive medical imaging provide opportunity to decrease resistance to standard-of-care treatments and optimize therapeutic timing and regimens. Citation Format: Anna G. Sorace, C. Chad Quarles, Violeta Sanchez, Thomas E. Yankeelov. Decreased hypoxia in a HER2+ breast cancer model following trastuzumab therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4237.

Abstract P6-01-03: Trastuzumab-induced hypoxia changes in a HER2+ murine model of breast cancer

Abstract Introduction: The primary goal of this study is to quantitatively map the changes in perfusion and hypoxia in response to trastuzumab treatment through imaging and immunohistochemical studies in a murine model of HER2+ breast cancer. Correlating quantitative imaging data with pathological validation of vessel architecture will identify the temporal changes in vascular function, which will enable optimizing the order and timing of multi-modal (i.e., trastuzumab, chemo- and radiation) therapy, leading to significantly improved anticancer response. Experimental Design: Mice were implanted subcutaneously with BT474 breast cancer cells (1×107) and randomly assorted into groups: treated (10 mg/kg trastuzumab) or control (saline). After tumors reached ∼225 mm3, animals (n = 20) were imaged with dynamic contrast enhanced-MRI (7.0 T MRI) before treatment (day 0), and 24 hours after each treatment (day 1 and day 4). Pharmacokinetic parameters, Ktrans and ve, were extracted. Subgroups of animals were sacrificed for histology between days 0 through 7 (n = 36). Tumor sections were paraffin-embedded and stained with CA-IX, CD31, α-SMA, Ki67 and H&amp;E. Slides were scanned in high resolution (20×) and quantitatively analyzed with Leica SCN400 software. Another cohort of animals (n = 32) was utilized to identify longitudinal changes in functional vascular (Hoechst 33342) and hypoxia (pimonidazole) through immunofluorescence. Agents were injected prior to sacrifice between days 0 through 7 and tumors were immediately frozen for processing. Results: Treated tumors exhibited a significant increase in Ktrans (p = 0.03) on day 4 compared to controls, indicative of heightened vessel perfusion and/or permeability. Additionally on day 4, treated tumors exhibited a significant increase in ve (p = 0.01), the extravascular extracellular volume fraction, indicating increased cell death. Significant decreases in Ki67 proliferation staining (p = 0.02) and tumor volume at day 7 (p = 0.04) in the treated group confirmed tumor response to trastuzumab. Immunohistochemical analyses revealed treated tumors have a significant decrease in CD31 microvessel density staining (p = 0.01), with a simultaneous significant increase in α-SMA pericyte coverage staining (p = 0.05) on day 4; thus, there was an overall increase in the "vessel maturation index" (ratio of α-SMA to CD31 staining) compared to controls (p = 0.01). CA-IX staining demonstrated increased hypoxia in the control group compared to treated, showing significant increases on day 3 (p = 0.03) and day 7 (p = 0.002). Qualitative differences were noted between control and treated groups for both functional vasculature and pimonidazole hypoxia fluorescent staining. Conclusion: Increased intratumoral vascular delivery (Ktrans) with a simultaneous increase in vessel maturation (immunohistochemistry) are exhibited on day 4 post trastuzumab treatment. Additionally, decreased hypoxia is revealed in treated tumors compared to controls. Hypoxic tumor cells show resistance to both radiation and chemotherapy, therefore temporarily improving the tumor's functional vasculature and decreasing hypoxia during trastuzumab treatment has potential to enhance the effectiveness of these combination therapies. Citation Format: Sorace AG, Quarles CC, Yankeelov TE. Trastuzumab-induced hypoxia changes in a HER2+ murine model of breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-01-03.

Trastuzumab improves tumor perfusion and vascular delivery of cytotoxic therapy in a murine model of HER2+ breast cancer: preliminary results.

To employ in vivo imaging and histological techniques to identify and quantify vascular changes early in the course of treatment with trastuzumab in a murine model of HER2+ breast cancer. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to quantitatively characterize vessel perfusion/permeability (via the parameter K (trans) ) and the extravascular extracellular volume fraction (v e ) in the BT474 mouse model of HER2+ breast cancer (N = 20) at baseline, day one, and day four following trastuzumab treatment (10 mg/kg). Additional cohorts of mice were used to quantify proliferation (Ki67), microvessel density (CD31), pericyte coverage (α-SMA) by immunohistochemistry (N = 44), and to quantify human VEGF-A expression (N = 29) throughout the course of therapy. Longitudinal assessment of combination doxorubicin ± trastuzumab (N = 42) tested the hypothesis that prior treatment with trastuzumab will increase the efficacy of subsequent doxorubicin therapy. Compared to control tumors, trastuzumab-treated tumors exhibited a significant increase in K (trans) (P = 0.035) on day four, indicating increased perfusion and/or vessel permeability and a simultaneous significant increase in v e (P = 0.01), indicating increased cell death. Immunohistochemical and ELISA analyses revealed that by day four the trastuzumab-treated tumors had a significant increase in vessel maturation index (i.e., the ratio of α-SMA to CD31 staining) compared to controls (P < 0.001) and a significant decrease in VEGF-A (P = 0.03). Additionally, trastuzumab dosing prior to doxorubicin improved the overall effectiveness of the therapies (P < 0.001). This study identifies and validates improved perfusion characteristics following trastuzumab therapy, resulting in an improvement in trastuzumab-doxorubicin combination therapy in a murine model of HER2+ breast cancer. This data suggests properties of vessel maturation. In particular, the use of DCE-MRI, a clinically available imaging method, following treatment with trastuzumab may provide an opportunity to optimize the scheduling and improve delivery of subsequent cytotoxic therapy.

Abstract 1492: Trastuzumab-induced normalization in a HER2+ murine model of breast cancer

Abstract Introduction: The primary goal of this study is to quantitatively map the vascular and cellular response to trastuzumab treatment in a murine model of HER2+ breast cancer. We report longitudinal dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and immunohistochemical studies hypothesized to identify timing windows of trastuzumab-induced vascular normalization. Experimental Design: Mice were subcutaneously implanted with BT474 breast cancer cells and randomly assigned to treated (10 mg/kg trastuzumab) and control (saline) groups. After tumors reached ∼225 mm3, animals (n = 20) were imaged (7.0 T MRI) before treatment (day 0), and 24 hours after each treatment (day 1 and day 4). Subgroups were sacrificed for histology between days 0 through 10 (n = 45). DCE-MRI analysis yielded parametric maps of blood vessel perfusion and permeability (Ktrans) and the extravascular extracellular volume fraction (ve). Tumor sections were paraffin-embedded and stained with CD31, α-SMA, Ki67 and H&amp;E. Slides were scanned in high resolution (20×) and quantitatively analyzed with Leica SCN400 software. Results: Compared to controls, treated tumors exhibited a significant increase in Ktrans (p = 0.03) on day 4, indicative of enhanced vessel perfusion and/or permeability. Additionally at day 4, treated tumors exhibited a significant increase in extravascular extracellular volume fraction measured via ve (p = 0.01), indicating increased cell death. These significant changes occurred prior to a significant change in tumor size at day 4 (p = 0.11). Decreased Ki67 proliferation staining (p = 0.02) at day 3 and a significant decrease in tumor volume at day 7 (p = 0.04) in the treated group confirmed tumor response to trastuzumab. The increase in Ktrans suggests properties of vessel normalization with potential to improve delivery of systemic therapies. Immunohistochemical analyses showed treated tumors have a significant decrease in microvessel density (CD31 staining, p &amp;lt; 0.01), with a simultaneous significant increase in α-SMA staining (pericyte coverage, p = 0.05) on day 4; thus, there was an overall increase in the “vessel maturation index” (ratio of α-SMA to CD31 staining) compared to controls (p = 0.01). Conclusion: These concordant (though preliminary) imaging and histological data suggest that trastuzumab treatment promotes vascular normalization, providing motivation for future studies designed to improve the efficacy of combination therapies in HER2+ breast cancer. For example, DCE-MRI has the potential to identify windows of vascular normalization thereby indicating optimal timing for treatment dosing with a cytotoxic therapy. As trastuzumab is currently given clinically in combination with various chemotherapies for HER2+ cancer, trastuzumab-induced vascular normalization could be utilized to improve drug delivery efficiency and enhance overall patient response without increasing dose or systemic toxicity. Citation Format: Anna G. Sorace, Jennifer G. Whisenant, J. Oliver McIntyre, Thomas E. Yankeelov. Trastuzumab-induced normalization in a HER2+ murine model of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1492. doi:10.1158/1538-7445.AM2015-1492

Assessing reproducibility of diffusion-weighted magnetic resonance imaging studies in a murine model of HER2 + breast cancer

Background and purposeThe use of diffusion-weighted magnetic resonance imaging (DW-MRI) as a surrogate biomarker of response in preclinical studies is increasing. However, before a biomarker can be reliably employed to assess treatment response, the reproducibility of the technique must be established. There is a paucity of literature that quantifies the reproducibility of DW-MRI in preclinical studies; thus, the purpose of this study was to investigate DW-MRI reproducibility in a murine model of HER2+ breast cancer. Materials and methodsTest–Retest DW-MRI scans separated by approximately six hours were acquired from eleven athymic female mice with HER2+ xenografts using a pulsed gradient spin echo diffusion-weighted sequence with three b values [150, 500, and 800s/mm2]. Reproducibility was assessed for the mean apparent diffusion coefficient (ADC) from tumor and muscle tissue regions. ResultsThe threshold to reflect a change in tumor physiology in a cohort of mice is defined by the 95% confidence interval (CI), which was±0.0972×10-3mm2/s (±11.8%) for mean tumor ADC. The repeatability coefficient defines this threshold for an individual mouse, which was±0.273×10-3mm2/s. The 95% CI and repeatability coefficient for mean ADC of muscle tissue were±0.0949×10-3mm2/s (±8.30%) and±0.266×10-3mm2/s, respectively. ConclusionsMean ADC of tumors is reproducible and appropriate for detecting treatment-induced changes on both an individual and mouse cohort basis.

Abstract 3985: Sorafenib modulates macrophages to promote T helper type 1 immunity.

Abstract Multiple regulatory processes within the tumor microenvironment work in concert to limit local anti-tumor immunity, and it is likely that integrative cancer therapies that target multiple cellular components in the tumor will be required to optimize antitumor immunity. Sorafenib is a promiscuous small molecule tyrosine kinase inhibitor developed as an anti-angiogenesis agent. We have previously shown that Sorafenib treatment can shift bone-marrow derived macrophages activated with lipopolysaccharide (LPS) and prostaglandin E2 (PGE2) from the pro-tumorigenic IL-10 secreting phenotype to the anti-tumor IL-12 secreting phenotype. Here, we extend these studies to explore the effect of Sorafenib on tumor-associated macrophages (TAMs) in a murine model of HER2+ breast cancer. Sorafenib treatment effectively delays the outgrowth of HER-2-overexpressing tumors in FVB/N mice relative to vehicle-treated controls. Immunohistochemical analysis revealed increased infiltration of F480+ macrophages in Sorafenib-treated tumors. F480+ TAMs isolated from Sorafenib-treated tumors had greater levels of IL-12 gene expression, and secreted higher levels of monocyte chemotactic protein-1 (MCP-1); there was no difference in IL-10 gene expression. Furthermore, F480+ TAMs isolated from Sorafenib-treated tumors demonstrated a superior ability to stimulate CD4+ T cell proliferation, and intracellular cytokine staining of tumor infiltrating lymphocytes isolated from Sorafenib-treated tumors showed an increase in CD4+ IFNγ-secreting T cells. Taken together, these data suggest that Sorafenib may alter the activation state of TAMs to increase pro-immunogenic signals and promote T helper type 1 immunity in the tumor microenvironment. Citation Format: Melek M. Sunay, James M. Leatherman, Gang Chen, Leisha A. Emens. Sorafenib modulates macrophages to promote T helper type 1 immunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3985. doi:10.1158/1538-7445.AM2013-3985

Reproducibility of Static and Dynamic 18F-FDG, 18F-FLT, and 18F-FMISO MicroPET Studies in a Murine Model of HER2+ Breast Cancer

The objective of this study is to determine the reproducibility of static 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG), 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT), and [(18)F]-fluoromisonidazole ((18)F-FMISO) microPET measurements, as well as kinetic parameters returned from analyses of dynamic (18)F-FLT and (18)F-FMISO data. HER2+ xenografts were established in nude mice. Dynamic data were acquired for 60min, followed by a repeat injection and second scan 6h later. Reproducibility was assessed for the percent-injected dose per gram (%ID/g) for each radiotracer, and with kinetic parameters (K (1) -k (4) , K ( i )) for (18)F-FLT and (18)F-FMISO. The value needed to reflect a change in tumor physiology is given by the 95% confidence interval (CI), which is ±14, ±5, and ±6% for (18)F-FDG (n = 12), (18)F-FLT (n = 11), and (18)F-FMISO (n = 11) %ID/g, respectively. V ( d ) (=K (1) /k (2)), k (3), and K (FLT) are the most reproducible (18)F-FLT (n = 9) kinetic parameters, with 95% CIs of ±18, ±10, and ±18%, respectively. V ( d ) and K (FMISO) are the most reproducible (18)F-FMISO kinetic parameters (n = 7) with 95% CIs of ±16 and ±14%, respectively. Percent-injected dose per gram measurements are reproducible and appropriate for detecting treatment-induced changes. Kinetic parameters have larger threshold values, but are potentially sufficiently reproducible to detect treatment response.