Molecular Imaging and Biology

Correction to: Imaging of Tumor Spheroids, Dual-Isotope SPECT, and Autoradiographic Analysis to Assess the Tumor Uptake and Distribution of Different Nanobodies This article was corrected/updated to include the complete graphic legend of Fig. 3.

Evaluation of Glycolytic Response to Multiple Classes of Anti-glioblastoma Drugs by Noninvasive Measurement of Pyruvate Kinase M2 Using [ 18 F]DASA-23 Abstract Purpose Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, the key process of tumor metabolism. PKM2 is found in high levels in glioblastoma (GBM) cells with marginal expression within healthy brain tissue, rendering it a key biomarker of GBM metabolic re-programming. Our group has reported the development of a novel radiotracer, 1-((2-fluoro- 6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA- 23), to non-invasively detect PKM2 levels with positron emission tomography (PET). Procedure U87 human GBM cells were treated with the IC50 concentration of various agents used in the treatment of GBM, including alkylating agents (temozolomide, carmustine, lomustine, procarbazine), inhibitor of topoisomerase I (irinotecan), vascular endothelial and epidermal growth factor receptor inhibitors (cediranib and erlotinib, respectively) anti-metabolite (5-fluorouracil), microtubule inhibitor (vincristine), and metabolic agents (dichloroacetate and IDH1 inhibitor ivosidenib). Following drug exposure for three or 6 days (n = 6 replicates per condition), the radiotracer uptake of [18F]DASA-23 and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) was assessed. Changes in PKM2 protein levels were determined via Western blot and correlated to radiotracer uptake. Results Significant interactions were found between the treatment agent (n = 12 conditions total comprised 11 drugs and vehicle) and the duration of treatment (3- or 6-day exposure to each drug) on the cellular uptake of [18F]DASA-23 (p = 0.0001). The greatest change in the cellular uptake of [18F]DASA-23 was found after exposure to alkylating agents (p < 0. 0001) followed by irinotecan (p = 0. 0012), erlotinib (p = 0. 02), and 5-fluorouracil (p = 0. 005). Correlation of PKM2 protein levels and [18F]DASA-23 cellular uptake revealed a moderate correlation (r = 0.44, p = 0.15). Conclusions These proof of principle studies emphasize the superiority of [18F]DASA-23 to [18F]FDG in detecting the glycolytic response of GBM to multiple classes of anti-neoplastic drugs in cell culture. A clinical trial evaluating the diagnostic utility of [18F]DASA-23 PET in GBM patients (NCT03539731) is ongoing.

Comparison of [ 18 F]Fluoroethyltyrosine PET and Sodium MRI in Cerebral Gliomas: a Pilot Study Abstract Purpose Positron emission tomography (PET) using O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) improves the diagnostics of cerebral gliomas compared with conventional magnetic resonance imaging (MRI). Sodium MRI is an evolving method to assess tumor metabolism. In this pilot study, we explored the relationship of [18F]FET-PET and sodium MRI in patients with cerebral gliomas in relation to the mutational status of the enzyme isocitrate dehydrogenase (IDH). Procedures Ten patients with untreated cerebral gliomas and one patient with a recurrent glioblastoma (GBM) were investigated by dynamic [18F]FET-PET and sodium MRI using an enhanced simultaneous single-quantum- and triple-quantum-filtered imaging of 23Na (SISTINA) sequence to estimate total (NaT), weighted non-restricted (NaNR, mainly extracellular), and restricted (NaR, mainly intracellular) sodium in tumors and normal brain tissue. [18F]FET uptake and sodium parameters in tumors with a different IDH mutational status were compared. After biopsy or resection, histology and the IDH mutational status were determined neuropathologically. Results NaT (p = 0.05), tumor-to-brain ratios (TBR) of NaT (p = 0.02), NaNR (p = 0.003), and the ratio of NaT/NaR (p < 0.001) were significantly higher in IDH-mutated than in IDH-wild-type gliomas (n = 5 patients each) while NaR was significantly lower in IDH-mutated gliomas (p = 0.01). [18F]FET parameters (TBR, time-to-peak) were not predictive of IDH status in this small cohort of patients. There was no obvious relationship between sodium distribution and [18F]FET uptake. The patient with a recurrent GBM exhibited an additional radiation injury with strong abnormalities in sodium MRI. Conclusions Sodium MRI appears to be more strongly related to the IDH mutational status than are [18F]FET-PET parameters. A further evaluation of the combination of the two methods in a larger group of high- and low-grade gliomas seems promising.

Quantitative and Qualitative Improvement of Low-Count [ 68 Ga]Citrate and [ 90 Y]Microspheres PET Image Reconstructions Using Block Sequential Regularized Expectation Maximization Algorithm Abstract Purpose There are several important positron emission tomography (PET) imaging scenarios that require imaging with very low photon statistics, for which both quantitative accuracy and visual quality should not be neglected. For example, PET imaging with the low photon statistics is closely related to active efforts to significantly reduce radiation exposure from radiopharmaceuticals. We investigated two examples of low-count PET imaging: (a) imaging [90Y]microsphere radioembolization that suffers the very small positron emission fraction of Y-90's decay processes, and (b) cancer imaging with [68Ga]citrate with uptake time of 3–4 half-lives, necessary for visualizing tumors. In particular, we investigated a type of penalized likelihood reconstruction algorithm, block sequential regularized expectation maximization (BSREM), for improving both image quality and quantitative accuracy of these low-count PET imaging cases. Procedures The NEMA/IEC Body phantom filled with aqueous solution of Y-90 or Ga-68 was scanned to mimic the low-count scenarios of corresponding patient data acquisitions on a time-of-flight (TOF) PET/magnetic resonance imaging system. Contrast recovery, background variation, and signal-to-noise ratio were evaluated in different sets of count densities using both conventional TOF ordered subset expectation (TOF-OSEM) and TOF-BSREM algorithms. The regularization parameter, beta, in BSREM that controls the tradeoff between image noise and resolution was evaluated to find a value for improved confidence in image interpretation. Visual quality assessment of the images obtained from patients administered with [68Ga]citrate (n = 6) was performed. We also made preliminary visual image quality assessment for one patient with [90Y]microspheres. In Y-90 imaging, the effect of 511-keV energy window selection for minimizing the number of random events was also evaluated. Results Quantitatively, phantom images reconstructed with TOF-BSREM showed improved contrast recovery, background variation, and signal-to-noise ratio values over images reconstructed with TOF-OSEM. Both phantom and patient studies of delayed imaging of [68Ga]citrate show that TOF-BSREM with beta = 500 gives the best tradeoff between image noise and image resolution based on visual assessment by the readers. The NEMA-IQ phantom study with [90Y]microspheres shows that the narrow energy window (460–562 keV) recovers activity concentrations in small spheres better than the regular energy window (425–650 keV) with the beta value of 2000 using the TOF-BSREM algorithm. For the images obtained from patients with [68Ga]citrate using TOF-BSREM with beta = 500, the visual analogue scale (VAS) was improved by 17 % and the Likert score was increased by 1 point on average, both in comparison to corresponding scores for images reconstructed using TOF-OSEM. Conclusion Our investigation shows that the TOF-BSREM algorithm improves the image quality and quantitative accuracy in low-count PET imaging scenarios. However, the beta value in this algorithm needed to be adjusted for each radiopharmaceutical and counting statistics at the time of scans.

Retrospective Brain Motion Correction in Glutamate Chemical Exchange Saturation Transfer (GluCEST) MRI Abstract Purpose To evaluate the feasibility of motion correction in glutamate chemical exchange saturation transfer (GluCEST) imaging, using a rat model of epileptic seizure. Procedures Epileptic seizure was induced in six male Wistar rats by intraperitoneal injection of kainic acid (KA). CEST data were obtained using a 7.0 T Bruker MRI scanner before and 3 h after KA injection. Retrospective motion correction was performed in CEST images using a gradient-based motion correction (GradMC) algorithm. GluCEST signals in the hippocampal regions were quantitatively evaluated with and without motion correction. Results Calculated GluCEST signals differed significantly between the pre-KA injection group, regardless of motion-correction implementation, and the post-KA injection group with motion correction (3.662 ± 1.393 % / 3.726 ± 1.982 % for pre-KA injection group with/without motion correction vs. 6.996 ± 1.684 % for post-KA injection group with motion correction; all P < 0.05). Conclusions Our results clearly show that GradMC can be used in CEST imaging for efficient correction of seizure-like motion. The GradMC can be further implemented in various CEST imaging techniques to increase the accuracy of analysis.

Optical Redox Imaging of Fixed Unstained Muscle Slides Reveals Useful Biological Information Abstract Purpose Optical redox imaging (ORI) technique images cellular autofluorescence of nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp containing FAD, i.e., flavin adenine dinucleotide). ORI has found wide applications in the study of cellular energetics and metabolism and may potentially assist in disease diagnosis and prognosis. Fixed tissues have been reported to exhibit autofluorescence with similar spectral characteristics to those of NADH and Fp. However, few studies report on quantitative ORI of formalin-fixed paraffin-embedded (FFPE) unstained tissue slides for disease biomarkers. We investigate whether ORI of FFPE unstained skeletal muscle slides may provide relevant quantitative biological information. Procedures Living mouse muscle fibers and frozen and FFPE mouse muscle slides were subjected to ORI. Living mouse muscle fibers were imaged ex vivo before and after paraformaldehyde fixation. FFPE muscle slides of three mouse groups (young, mid-age, and muscle-specific overexpression of nicotinamide phosphoribosyltransferase (Nampt) transgenic mid-age) were imaged and compared to detect age-related redox differences. Results We observed that living muscle fiber and frozen and FFPE slides all had strong autofluorescence signals in the NADH and Fp channels. Paraformaldehyde fixation resulted in a significant increase in the redox ratio Fp/(NADH + Fp) of muscle fibers. Quantitative image analysis on FFPE unstained slides showed that mid-age gastrocnemius muscles had stronger NADH and Fp signals than young muscles. Gastrocnemius muscles from mid-age Nampt mice had lower NADH compared to age-matched controls, but had higher Fp than young controls. Soleus muscles had the same trend of change and appeared to be more oxidative than gastrocnemius muscles. Differential NADH and Fp signals were found between gastrocnemius and soleus muscles within both mid-aged control and Nampt groups. Conclusion Aging effect on redox status quantified by ORI of FFPE unstained muscle slides was reported for the first time. Quantitative information from ORI of FFPE unstained slides may be useful for biomedical applications.

Mapping pH at Cancer Cell Surfaces Abstract Purpose To develop a tool to measure the pH at the surfaces of individual cells. Procedures The SNARF pH-sensitive dye was conjugated to a pHLIP® peptide (pH-Low Insertion Peptide) that binds cellular membranes in tumor spheroids. A beam splitter allows simultaneous recording of two images (580 and 640 nm) by a CCD camera. The ratio of the two images is converted into a pH map resolving single spheroid cells. An average pH for each cell is calculated and a pH histogram is derived. Results Surface pH depends on cellular glycolytic activity, which was varied by adding glucose or deoxy-glucose. Glucose was found to decrease the surface pH relative to the pH of the bulk solution. The surface pH of metastatic cancer cells was lower than that of non-metastatic cells indicating a higher glycolytic activity. Conclusions Our method allows cell surface pH measurement and its correlation with cellular glycolytic activity.

Magnetic Resonance Imaging of Hard Tissues and Hard Tissue Engineered Bio-substitutes Abstract Magnetic resonance imaging (MRI) is a non-invasive diagnostic imaging tool based on the detection of protons into the tissues. This imaging technique is remarkable because of high spatial resolution, strong soft tissue contrast and specificity, and good depth penetration. However, MR imaging of hard tissues, such as bone and teeth, remains challenging due to low proton content in such tissues as well as to very short transverse relaxation times (T2). To overcome these issues, new MRI techniques, such as sweep imaging with Fourier transformation (SWIFT), ultrashort echo time (UTE) imaging, and zero echo time (ZTE) imaging, have been developed for hard tissues imaging with promising results reported. Within this article, MRI techniques developed for the detection of hard tissues, such as bone and dental tissues, have been reviewed. The main goal was thus to give a comprehensive overview on the corresponding (pre-) clinical applications and on the potential future directions with such techniques applied. In addition, a section dedicated to MR imaging of novel biomaterials developed for hard tissue applications was given as well.

Prognostic Value of O-(2-[ 18 F]Fluoroethyl)-L-Tyrosine PET/CT in Newly Diagnosed WHO 2016 Grade II and III Glioma Abstract Purpose The use of [18F]fluoroethyl)-l-tyrosine ([18F]FET) positron emission tomography/computed tomography (PET/CT) has proven valuable in brain tumor management. This study aimed to investigate the prognostic value of radiotracer uptake in newly diagnosed grade II or III gliomas according to the current 2016 World Health Organization (WHO) classification. Procedures A total of 35 treatment-naive patients (mean age, 48 ± 17 years) with histologically proven WHO grade II or III gliomas as defined by the current 2016 WHO classification were included. Static PET/CT imaging was performed 20 min after intravenous [18F]FET injection. Images were assessed visually and semi-quantitatively using regions of interest for both tumor (SUVmax, SUVmean) and background (BKGmean) to calculate tumor-to-background (TBR) ratios. The association among histological results, molecular markers (including isocitrate dehydrogenase enzyme and methylguanine-DNA methyltransferase status), clinical features (age), and PET findings was tested and compared with outcome (progression-free [PFS] and overall survival [OS]). Results Fourteen patients presented with grade II (diffuse astrocytoma n = 10, oligodendroglioma n = 4) and 21 patients with grade III glioma (anaplastic astrocytoma n = 15, anaplastic oligodendroglioma n = 6). Twenty-seven out of the 35 patients were PET-positive (grade II n = 8/14, grade III n = 19/21), with grade III tumors exhibiting significantly higher amino acid uptake (TBRmean and TBRmax; p = 0.03 and p = 0.02, respectively). PET-negative lesions demonstrated significantly prolonged PFS (p = 0.003) as compared to PET-positive gliomas. PET-positive disease had a complementary value in prognostication in addition to patient age, glioma grade, and molecular markers. Conclusions Amino acid uptake as assessed by [18F]FET-PET/CT imaging is useful as non-invasive read-out for tumor biology and prognosis in newly diagnosed, treatment-naive gliomas according to the 2016 WHO classification.

Impact of rs12917 MGMT Polymorphism on [ 18 F]FDG-PET Response in Pediatric Hodgkin Lymphoma (PHL) Abstract Purpose The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is an important component of the DNA repair machinery. MGMT removes O6-methylguanine from the DNA by transferring the methyl group to a cysteine residue in its active site. Recently, we detected the single nucleotide polymorphism (SNP) rs12917 (C/T) in the MGMT sequence adjacent to the active site in Hodgkin lymphoma (HL) cell line KM-H2. We now investigated whether this SNP is also present in other HL cell lines and patient samples. Furthermore, we asked whether this SNP might have an impact on metabolic response in 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ([18F]FDG-PET), and on overall treatment outcome based on follow-up intervals of at least 34 months. Procedures We determined the frequency of this MGMT polymorphism in 5 HL cell lines and in 29 pediatric HL (PHL) patients. The patient cohort included 17 female and 12 male patients aged between 4 and 18 years. After characterization of the sequence, we tested a possible association between rs12917 and age, gender, Ann Arbor stage, treatment group, metabolic response following two courses of OEPA (vincristine, etoposide, prednisone, and doxorubicin) chemotherapy, radiotherapy indication, and relapse status. Results We detected the minor T allele in four of five HL cell lines. 11/29 patients carried the minor T allele whereas 18/29 patients showed homozygosity for the major C allele. Interestingly, we observed significantly better metabolic response in PHL patients carrying the rs12917 C allele resulting in a lower frequency of radiotherapy indication. Conclusion MGMT polymorphism rs12917 seems to affect chemotherapy response in PHL. The prognostic value of this polymorphism should be investigated in a larger patient cohort.