Suboptimal Sensitivity and Specificity of PET and Other Gross Imaging Techniques in Assessing Lymph Node Metastasis

Quantification of Positron Emission Tomography Data Using Simultaneous Estimation of the Input Function: Validation with Venous Blood and Replication of Clinical Studies Abstract Purpose To determine if one venous blood sample can substitute full arterial sampling in quantitative modeling for multiple positron emission tomography (PET) radiotracers using simultaneous estimation of the input function (SIME). Procedures Participants underwent PET imaging with [11C]ABP688, [11C]CUMI-101, and [11C]DASB. Full arterial sampling and additional venous blood draws were performed for quantification with the arterial input function (AIF) and SIME using one arterial or venous (vSIME) sample. Results Venous and arterial metabolite-corrected plasma activities were within 6 % of each other at varying time points. vSIME- and AIF-derived outcome measures were in good agreement, with optimal sampling times of 12 min ([11C]ABP688), 90 min ([11C]CUMI-101), and 100 min ([11C]DASB). Simulation-based power analyses revealed that SIME required fewer subjects than the AIF method to achieve statistical power, with significant reductions for [11C]CUMI-101 and [11C]DASB with vSIME. Replication of previous findings and test-retest analyses bolstered the simulation analyses. Conclusions We demonstrate the feasibility of AIF recovery using SIME with one venous sample for [11C]ABP688, [11C]CUMI-101, and [11C]DASB. This method simplifies PET acquisition while allowing for fully quantitative modeling, although some variability and bias are present with respect to AIF-based quantification, which may depend on the accuracy of the single venous blood measurement.

Optical Imaging of Triple-Negative Breast Cancer Cells in Xenograft Athymic Mice Using an ICAM-1-Targeting Small-Molecule Probe Abstract Purpose The development of early, accurate diagnostic strategies for triple-negative breast cancer (TNBC) remains a significant challenge. Intercellular adhesion molecule-1 (ICAM-1) overexpressed in human TNBC cells is a potential molecular target and biomarker for diagnosis. In this study, small-molecule probe (denoted as γ3-Cy5.5) constructed with a short 17-mer linear peptide (γ3) and near-infrared fluorescence (NIRF) dye cyanine 5.5 (Cy5.5) was used to detect the expression of ICAM-1 in vitro and in vivo, and to diagnose TNBC via NIRF imaging. Procedures Western blotting and flow cytometric analysis were used for the detection of ICAM-1 expression in MDA-MB-231 and MCF-7 cells. The cytotoxicity of the small-molecule probe γ3-Cy5.5 was detected using the CCK8 assay. The in vitro targeting of the small-molecule probe γ3-Cy5.5 was verified via flow cytometry and a laser scanning confocal microscope. Finally, the targeting of small-molecule probe in vivo and ex vivo was observed by NIRF imaging. Results Western blotting and flow cytometry demonstrate that ICAM-1 was highly expressed in the MDA-MB-231 TNBC cell line. Laser confocal microscopy and flow cytometry results show that TNBC cells have an increased cellular uptake of γ3-Cy5.5 compared to the control probe γ3S-Cy5.5. With in vivo NIRF, a significantly higher Cy5.5 signal appeared in the tumors of mice administered γ3-Cy5.5 than those treated with γ3S-Cy5.5. The target-to-background ratio observed on the NIRF images was significantly higher in the γ3-Cy5.5 group (10.2, 13.6) compared with the γ3S-Cy5.5 group (4.4, 4.0) at 1 and 2 h, respectively. Conclusions This is the first report of the use of ICAM-1-specific small-molecule probe for in vivo NIRF optical imaging of TNBC. This method provides a noninvasive and specific strategy for the early diagnosis of TNBC.

Metabolic Changes in Different Stages of Liver Fibrosis: In vivo Hyperpolarized 13 C MR Spectroscopy and Metabolic Imaging Abstract Purpose The objective was to assess metabolic changes in different stages of liver fibrosis using hyperpolarized C-13 magnetic resonance spectroscopy (MRS) and metabolic imaging. Procedures Mild and severe liver fibrosis were induced in C3H/HeN mice (n = 14) by injecting thioacetamide (TAA). Other C3H/HeN mice (n = 7) were injected with phosphate buffer saline (PBS) (7.4 pH) as normal controls. Hyperpolarized C-13 MRS was performed on the livers of the mice, which was accompanied by intravoxel incoherent motion (IVIM) diffusion-weighted imaging with 12 b values. The differential metabolite ratios, apparent diffusion coefficient values, and IVIM parameters among the three groups were analyzed by a one-way analysis of variance test. Results The ratios of [1-13C]lactate/pyruvate, [1-13C]lactate/total carbon (tC), [1-13C]alanine/pyruvate, and [1-13C] alanine/tC were significantly higher in both the mild and severe fibrosis groups than in the normal control group (p < 0.05). While the [1-13C]lactate/pyruvate and [1-13C]lactate/tC ratios were not significantly different between mild and severe fibrosis groups, the ratios of [1-13C]alanine/pyruvate and [1-13C]alanine/tC were significantly higher in the severe fibrosis group than in the mild fibrosis group (p < 0.05). In addition, D* showed a significantly lower value in the severe fibrosis group than in the normal or mild fibrosis groups and negatively correlated with the levels of [1-13C] lactate and [1-13C]alanine. Conclusions Our findings suggest that it might be possible to differentiate mild from severe liver fibrosis using the cellular metabolic changes with hyperpolarized C-13 MRS and metabolic imaging.

PET/CT Imaging of NSCLC with a α v β 6 Integrin-Targeting Peptide Abstract Purpose Targeted therapies are regarded as promising approaches to increase 5-year survival rate of non-small cell lung cancer (NSCLC) patients. Here, we investigated the clinical value of the αvβ6 integrin-specific peptide SFITGv6 as a diagnostic reagent targeting NSCLC. Methods Affinity and binding properties of [125I]SFITGv6 or [177Lu]SFITGv6 for αvβ6 integrin-expressing NSCLC cell lines were evaluated in cell culture experiments including competition, kinetic, internalization, and efflux. To confirm αvβ6 integrin specificity in vivo small-animal positron emission tomography (PET) imaging using [68Ga]SFITGv6 as radiotracer and biodistribution of [177Lu]SFITGv6 in NCI-H2009 and NCI-H322 tumor-bearing mice was performed. Finally, to distinguish between benign and malignant lesions [68Ga]SFITGv6 was applied as radiotracer for PET/x-ray computed tomography (CT) imaging of NSCLC patients with unclear diagnosis upon routinely performed 2-deoxy-2-[18F]flouro-D-glucose ([18F]FDG)-PET/CT. The biodistribution of the SFITGv6-ligand in different organs and tumor lesions of NSCLC patients was quantified 1 h and 3 h after injection measuring standard uptake values (SUV)max. Results In vitro experiments revealed a significant time-dependent SFITGv6 binding of up to 33 % to αvβ6 integrin-expressing the cell lines NCI-H2009, NCI-H322, NCI-H292, NCI-H358, and high affinity (IC50-mean 3.1 nM) to NCI-H2009 and NCI-H322. Moreover, a fast internalization of approximately 66 % by NCI-H2009 and NCI-H322 cells was observed. Small-animal PET imaging and biodistribution experiments of NCI-H2009 and NCI-H322 xenografts demonstrated an increased tumor-specific accumulation of SFITGv6 40 to 60 min after injection. Finally, PET/CT scans of NSCLC patients after [18F] FDG injection followed by [68Ga]SFITGv6 application revealed correlating images. Comparing the uptake of [68Ga]SFITGv6 and [18F] FDG both PET/CT-examinations presented with significantly increased SUVmax values in histologically proven NSCLC lesions, but a generally higher accumulation of [18F] FDG was noticed. Conclusions Even if SFITGv6 demonstrates excellent affinity and specificity for αvβ6 integrin-expressing NSCLC cell lines and several NSCLC xenografts [18F]FDG-PET/CT provides an advantage over [68Ga]SFITGv6-PET/CT for the diagnosis of NSCLC patients.

Imaging of the Mouse Lymphatic Sinus during Early Stage Lymph Node Metastasis Using Intranodal Lymphangiography with X-ray Micro-computed Tomography Abstract Purpose Lymph node (LN) metastasis is detected prior to distant metastasis in many types of cancer. Detecting early stage LN metastasis can improve treatment outcomes. However, there are few clinical imaging modalities capable of diagnosing metastatic LNs of clinical N0 status (i.e., before their volume increases) with high precision. Here, we report a new method for diagnosing metastatic LNs of clinical N0 status in a mouse model of LN metastasis. Procedures The method involved using intranodal lymphangiography with x-ray micro-computed tomography (micro-CT). Contrast agent was injected into an upstream LN to deliver it to a downstream LN, which was then removed and analyzed by micro-CT. Results We found that using an intranodal injection rate of 10–60 μl/min filled the lymphatic sinus of the downstream LN with contrast agent, although the accumulation of contrast agent in the upstream LN increased with a faster injection rate. Furthermore, breast cancer cells growing in the lymphatic sinus of the downstream LN (which was of clinical N0 status) impeded the flow of contrast agent from the upstream LN, resulting in areas deficient of contrast agent in the metastatic downstream LN. The formation of defect areas in the downstream LN manifested as a difference in position between the centroid of the entire LN area and the centroid of the region that filled with contrast agent. Conclusion The present study indicates that intranodal lymphangiography with micro-CT has the potential to be used as a new method for diagnosing metastatic LNs of clinical N0 status.

PIK3CA Mutational Status Is Associated with High Glycolytic Activity in ER+/HER2− Early Invasive Breast Cancer: a Molecular Imaging Study Using [ 18 F]FDG PET/CT Abstract Purpose In PIK3CA mutant breast cancer, downstream hyperactivation of the PI3K/AKT/mTOR pathway may be associated with increased glycolysis of cancer cells. The purpose of this study was to investigate the functional association of PIK3CA mutational status and tumor glycolysis in invasive ER+/HER2− early breast cancer. Procedures This institutional review board-approved retrospective study included a dataset of 67 ER+/HER2− early breast cancer patients. All patients underwent 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/X-ray computed tomography ([18F]FDG PET/CT) and clinico-pathologic assessments as part of a prospective study. For this retrospective analysis, pyrosequencing was used to detect PIK3CA mutations of exons 4, 7, 9, and 20. Tumor glucose metabolism was assessed semi-quantitatively with [18F]FDG PET/CT using maximum standardized uptake values (SUVmax). SUVmax values were corrected for the partial volume effect, and metabolic tumor volume was calculated using the volume of interest automated lesion growing function 2D tumor size, i.e., maximum tumor diameter was assessed on concurrent pre-treatment contrast-enhanced magnetic resonance imaging. Results PIK3CA mutations were present in 45 % of all tumors. Mutations were associated with a small tumor diameter (p < 0.01) and with low nuclear grade (p = 0.04). Glycolytic activity was positively associated with nuclear grade (p = 0.01), proliferation (p = 0.002), regional lymph node metastasis (p = 0.015), and metabolic tumor volume (p = 0.001) but not with tumor size/T-stage. In invasive ductal carcinomas, median SUVmax was increased in PIK3CA-mutated compared to wild-type tumors; however, this increase did not reach statistical significance (p = 0.05). Multivariate analysis of invasive ductal carcinomas revealed [18F]FDG uptake to be independently associated with PIK3CA status (p = 0.002) and nuclear tumor grade (p = 0.046). Size, volume, and regional nodal status had no influence on glycolytic activity. PIK3CA mutational status did not influence glycolytic metabolism in lobular carcinomas. Glycolytic activity and PIK3CA mutational status had no significant influence on recurrence-free survival or disease-specific survival. Conclusions In ER+/HER2− invasive ductal carcinomas of the breast, glucose uptake is independently associated with PIK3CA mutations. Initial data suggest that [18F]FDG uptake reflects complex genomic alterations and may have the potential to be used as candidate biomarker for monitoring therapeutic response and resistance mechanisms in emerging therapies that target the PI3K/AKT/mTOR pathway.

PET Imaging of HER2-Positive Tumors with Cu-64-Labeled Affibody Molecules Abstract Purpose Previous studies has demonstrated the utility of human epidermal growth factor receptor type 2 (HER2) as an attractive target for cancer molecular imaging and therapy. An affibody protein with strong binding affinity for HER2, ZHER2:342, has been reported. Various methods of chelator conjugation for radiolabeling HER2 affibody molecules have been described in the literature including N-terminal conjugation, C-terminal conjugation, and other methods. Cu-64 has recently been extensively evaluated due to its half-life, decay properties, and availability. Our goal was to optimize the radiolabeling method of this affibody molecule with Cu-64, and translate a positron emission tomography (PET) probe with the best in vivo performance to clinical PET imaging of HER2-positive cancers. Procedures In our study, three anti-HER2 affibody proteins-based PET probes were prepared, and their in vivo performance was evaluated in mice bearing HER2-positive subcutaneous SKOV3 tumors. The affibody analogues, Ac-Cys-ZHER2:342, Ac-ZHER2:342(Cys39), and Ac-ZHER2:342-Cys, were synthesized using the solid phase peptide synthesis method. The purified small proteins were site-specifically conjugated with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris- aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA). The resulting DOTA-affibody conjugates were then radiolabeled with Cu-64. Cell uptake assay of the resulting PET probes, [64Cu]DOTA-Cys-ZHER2:342, [64Cu]DOTA-ZHER2:342(Cys39), and [64Cu]DOTA-ZHER2:342-Cys, was performed in HER2-positive human ovarian SKOV3 carcinoma cells at 4 and 37 °C. The binding affinities of the radiolabeled peptides were tested by cell saturation assay using SKOV3 cells. PET imaging, biodistribution, and metabolic stability studies were performed in mice bearing SKOV3 tumors. Results Cell uptake assays showed high and specific uptake by incubation of Cu-64-labeled affibodies with SKOV3 cells. The affinities (KD) of the PET radio probes as tested by cell saturation analysis were in the low nanomolar range with the ranking of [64Cu]DOTA-Cys-ZHER2:342 (25.2 ± 9.2 nM) ≈ [64Cu]DOTA-ZHER2:342-Cys (32.6 ± 14.7 nM) > [64Cu]DOTA-ZHER2:342(Cys39) (77.6 ± 22.2 nM). In vitro stability and in vivo metabolite analysis study revealed that all three probes were stable enough for in vivo imaging applications, while [64Cu]DOTA-Cys-ZHER2:342 showed the highest stability. In vivo small-animal PET further demonstrated fast tumor targeting, good tumor accumulation, and good tumor to normal tissue contrast of all three probes. For [64Cu]DOTA-Cys-ZHER2:342, [64Cu]DOTA-ZHER2:342(Cys39), and [64Cu]DOTA-ZHER2:342-Cys, tumor uptake at 24 h are 4.0 ± 1.0 % ID/g, 4.0 ± 0.8 %ID/g, and 4.3 ± 0.7 %ID/g, respectively (mean ± SD, n = 4). Co-injection of the probes with non-labeled anti-HER2 affibody proteins confirmed in vivo specificities of the compounds by tumor uptake reduction. Conclusions The three Cu-64-labeled ZHER2:342 analogues all display excellent HER2 targeting ability and tumor PET imaging quality. Although varied in the position of the radiometal labeling of these three Cu-64-labeled ZHER2:342 analogues, there is no significant difference in tumor and normal tissue uptakes among the three probes. [64Cu]DOTA-Cys-ZHER2:342 stands out as the most superior PET probe because of its highest affinities and in vivo stability.

Prognostic Impact of Intratumoral Heterogeneity Based on Fractal Geometry Analysis in Operated NSCLC Patients Abstract Purpose To determine the heterogeneity of glucose uptake applying fractal analysis on positron emission tomography/computed tomography (PET/CT) with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) images in patients with non-small cell lung carcinoma (NSCLC) before surgery, and to assess whether this heterogeneity was associated with disease-free survival (DFS). Procedures [18F]FDG PET/CT scans of 113 patients’ prior surgery were retrospectively revised. PET DICOM images were analyzed for fractal geometry using a ad hoc software to automatically determine the following indexes: (a) mean intensity value (MIV), (b) standard deviation (SD), (c) relative dispersion (RD), (d) three-dimensional (3D) histogram of the fractal dimension (3D HIST FR DIM), and (e) fractal dimension in 3D (3D-FD). All the fractal indexes were subsequently compared with metabolic parameters and disease-free survival (DFS). Results We found a significant correlation between 3D-FD and SUVmax, SUVmean, MTV, and TLG. Additionally, positive correlations between MIV, SD, and all metabolic parameters were also detected. Patients with high 3D-FD tumor (≥ 1.62) showed significantly higher values of SUVmax, SUVmean, MTV, and TLG than those with lower 3D-FD. In univariate analysis, median 3D-FD and median TLG were significantly associated with DFS (p = 0.04 and p = 0.03, respectively). These findings were confirmed on log-rank test. On multivariate analysis, among age, stage disease, histotype, 3D-FD, and metabolic parameters, only 3D-FD was identified as independent prognostic factor for DFS (p = 0.032; HR 0.418, 95 % CI 0.189–0.926). 3D-FD was different between adenocarcinoma and squamous cell carcinoma (1.60 versus 1.88, p = 0.014), and 3D-FD value was found higher in advanced stage disease. Conclusions Metabolic heterogeneity determined applying fractal principles on PET images can be considered as a novel imaging biomarker for survival in patients with NSCLC.

Quantification of [ 18 F]UCB-H Binding in the Rat Brain: From Kinetic Modelling to Standardised Uptake Value Abstract Purpose [18F]UCB-H is a specific positron emission tomography (PET) biomarker for the Synaptic Vesicle protein 2A (SV2A), the binding site of the antiepileptic drug levetiracetam. With a view to optimising acquisition time and simplifying data analysis with this radiotracer, we compared two parameters: the distribution volume (Vt) obtained from Logan graphical analysis using a Population-Based Input Function, and the Standardised Uptake Value (SUV). Procedures Twelve Sprague Dawley male rats, pre-treated with three different doses of levetiracetam were employed to develop the methodology. Three additional kainic acid (KA) treated rats (temporal lobe epilepsy model) were also used to test the procedure. Image analyses focused on: (i) length of the dynamic acquisition (90 versus 60 min); (ii) correlations between Vt and SUV over 20-min consecutive time-frames; (iii) and (iv) evaluation of differences between groups using the Vt and the SUV; and (v) preliminary evaluation of the methodology in the KA epilepsy model. Results A large correlation between the Vt issued from 60 to 90-min acquisitions was observed. Further analyses highlighted a large correlation (r > 0.8) between the Vt and the SUV. Equivalent differences between groups were detected for both parameters, especially in the 20–40 and 40–60-min time-frames. The same results were also obtained with the epilepsy model. Conclusions Our results enable the acquisition setting to be changed from a 90-min dynamic to a 20-min static PET acquisition. According to a better image quality, the 20–40-min time-frame appears optimal. Due to its equivalence to the Vt, the SUV parameter can be considered in order to quantify [18F]UCB-H uptake in the rat brain. This work, therefore, establishes a starting point for the simplification of SV2A in vivo quantification with [18F]UCB-H, and represents a step forward to the clinical application of this PET radiotracer.