Chapter 2. Experimental Approach
2.1 Modular Synthetic Approach to TMIA Synthesis
Our group is interested in a peptide-based modular approach to synthesizing imagingagents. Imaging or contrast modules for each imaging method are synthesized, and the relevant imaging and targeting modules are coupled together. 57 An amino acid backbone such as lysine is favorable due to its primary amine side chain. This functional group is easily coupled to a chelating group (such as DOTA) for chelation of a metal that is useful for imaging. Peptides are preferable over original small molecules that could possibly be designed. They have known advantages in medicinal chemistry due to their relatively small, compact structures and their bioavailability and biostability. 58 Because the reagents and coupling mechanisms for peptides are fairly well known, we can focus efforts on developing new TMIAs, rather than developing new reactions.
PET/MRI is an emerging imaging technology that allows for the acquisition of multiple MRI parameters simultaneously with PET data. In this review, we’ll address the technical
requirements of PET/MRI including protocols and tracers, the potential of integrated localized breast PET/MRI exams, and possible applications of whole body PET/MRI in breast cancer patients. Currently, PET/MRI can be performed on sequential and integrated PET/MRI scanners but, as not all practices can access these dedicated machines, several studies look at PET and MRI exams that are performed separately on separate scanners within a short time frame. This practice likely provides similar clinical data, although exact co-localization for iso-voxel analysis, currently performed only in research, is not possible. In PET/MRI, the MRI sequences are flexible and can be customized according to the aim of the exam. The most commonly used radiotracer is 18 F-FDG, however, tracers that image hypoxia and drug targets such as estrogen receptors and HER2 are in development and may increase the utility of PET/MR. For dedicated breast PET/MRI, a potential advantage over standard breast MRI alone may be the
Although researchers have made great strides toward understanding the biological processes under- lying cancer pathology, this has not led to major improvements in the management of the disease.
Development of new treatments to combat cancer remains imperative. Nanosized therapies show promise to improve tumor treatment response by localizing therapy while reducing treatment-related toxicity. Understanding how nanotherapies are taken up and cause their effects at the intact tumor level in vivo will complement ex vivo histological and in vitro biochemical studies and facilitate the translation of nanotherapy treatments to the clinic. Currently, few in vivo methods exist to study nanotherapy uptake and response at the intact tumor scale. Magnetic resonance imaging (MRI) and positron emission tomography (PET) are imaging methods that provide different but comple- mentary information about the tumor microenvironment and nanotherapy uptake/response. Direct spatiotemporal correlation of PET and MRI data via their simultaneous acquisition has the poten- tial to be powerfully synergistic, especially for the study of physiological processes that are time sensitive or where good spatial coregistration of the multimodal data is important. As the field of hybrid PET/MRI is still in its infancy, with only a handful of active systems worldwide, it is vital that continued PET/MRI technology development be pursued to realize its full potential.
Conversely, the quantitative DWI parameters such as b- values and ADCs of malignant vs. benign lesions didn’t show any statistical difference between malignant and non- malignant lesions. This finding is partially in contradiction to the current literature, which states that DWI is able to differentiate benign from malignant lesions in primary staging of head and neck cancers using ADC with different threshold values [ 6 , 11 ], and is also able to differentiate changes related to treatment from loco-regional recurrence [ 32 , 38 ]. One potential explanation for these conflicting results is that our b-values are partly lower than previously reported in the literature (800 vs. 1,000–2,000). It is known that the ADC value decreases when the b-value increases beyond 1,000 s/ mm2 [ 30 ]. The decrease in the observed ADC with an in- creasing b-value is explained by the decay of biexponential signal intensity [ 30 ]. Thus, this requires further investigation as to whether higher b-values should be used in head and neck cancer protocols. The problem with higher b-values is that MR imaging time increases and integration into a clinically acceptable PET/MRI protocol is thus even more difficult.
This study was approved by the institutional review board, and written informed consent was obtained from all pa- tients. This study was performed under an Investigational New Drug approval from the Food and Drug Administra- tion as part of a trial prospectively evaluating the accuracy of 68 Ga-PSMA-11 for the detection of prostate cancer (NCT02611882). Inclusion in the trial required a PSA doubling time of less than 12 months, and these patients have been reported as part of a change in management analysis . Fifty-five consecutive patients who under- went 68 Ga-PSMA-11 PET/MRI for BCR from March 2016 to September 2016 were evaluated. All of the patients were previously reported in a study evaluating how 68 Ga- PSMA-11 PET changed management in patients with BCR . This prior study surveyed the referring clini- cians to determine the effect that the imaging results would have on patient management but did not compare
positron emission tomography (PET)/CT was introduced into routine clinical practice and currently is considered a main diagnostic imaging tool for follow-up of HNC as well. It combines the functional information of PET with the morphological features of ceCT for the imaging of tumours with increased glucose metabolism [ 4 – 6 ]. How- ever, PET/CT has some disadvantages, such as the use of ionizing radiation, partly reduced image quality from metal dental artefacts and it partly needs iodinated contrast for higher diagnostic overall specificity. Moreover, it has lim- ited ability to identify cystic/necrotic lymph nodes and to thoroughly assess infiltration of neighbouring structures, mainly perineural spread [ 7 ]. Furthermore, PET/CT has a limited role in the evaluation of locoregional recurrence in the first weeks after radiation therapy. There is general consensus to set the optimum time for PET/CT to 8– 12 weeks after the end of treatment, thereby reducing both false-positive and false-negative findings, while the latter is related to the presence of undetectable microscopic residual disease [ 8 ]. Awareness and recognition of the imaging appearances of post-treatment changes is critical for the radiologist to decide whether these require conser- vative management or more active treatment [ 9 ].
supradiaphagmatic lymph nodes (5.2%), bones (35.9%), local recurrence (35.1%) and other organs (e.g., lung, liver) with 5.2% (4). Different than was previously thought, nodal recurrence is more common than local recurrence and not only involves usual lymph node stations (obturator, external iliac, internal iliac and common iliac) but also involves atypical pelvic atypical. Mesorectal lymph node involvement is one of the most common atypical nodal stations in the pelvis, with 15.8% detection rate according to Hijazi et al. (39). As demonstrated in some studies (4,39), PSMA PET/CT can detect lymph nodes that measure less than 5 mm in the short- axis diameter, explaining its better sensitivity for nodal detec- tion than CT or MRI. Regarding systemic spread of PCa to bone, few papers have demonstrated the clear superiority of PSMA PET for detecting bone lesions compared to bone scinti- graphy (BS), and that BS did not have significant additional diagnostic value in BCR scenario (27,40,41). However, focal bone PSMA uptake alone should not be immediately con- sidered metastasis; moreover, if moderate/mild uptake, which has to be correlated with CT morphology, is noted, it can decrease the possibility of false positives (40), as a few cases of benign lesions with PSMA uptake have been reported (42,43). Literature has confirmed the abovementioned findings, which have deep clinical implications. As recommended in current guidelines, a negative conventional imaging evalua- tion suggests that a patient is suitable for salvage radio- therapy of the prostate bed; however, PSMA imaging could significantly change treatment decisions by detecting small pelvic lymph nodes or bone metastasis. Recent study by Hope et al. has shown that PSMA imaging had a significant impact for evaluating BCR in 53% of patients and led to major changes in management and avoided unnecessary imaging studies (BS, CT and MRI) and invasive procedures (5). In this study, most of the major changes involved conversion Figure 2 - Evaluation of BCR in a 68-year-old patient treated with prostatectomy with a Gleason 7 adenocarcinoma (8 years) and prostate bed radiation (3 years), with a current PSA level of 0.29 ng/ml. 68 Ga-PSMA-fused PET/MRI MIP images (A) demonstrated 2
However, the specificity decreases at the same time (from 65.9 % to 31.7 %). The current literature is controversial regarding ADC thresholds in oncological imaging. Razek
and co-workers have shown that ADC values for residual or recurrent head and neck tumors were significantly lower than that for post-treatment changes . Other publications have demonstrated that ADC was significantly higher in metastatic lymph nodes than in benign lymphadenopathy . However, the ADC thresholds usually differ when analyzing HNC be- fore treatment and after therapy . Since our population includes patients referred for primary staging and for follow- up/recurrence imaging, identifying one ideal threshold value that could fit both at the same time is practically impossible.
Figure 3: Lymph nodes. (A) Top row shows transverse contrast-enhanced CT scan, fusion image, and fibroblast-activation protein inhibitor PET scan with axillary level 1 lymph node metastasis and putative 6-mm internal mammary lymph node metastasis in patient 19. Middle row shows transverse contrast-enhanced supine fat-saturated T1- weighted MRI scan, fusion image, and PET scan in patient 17. Morphologically enlarged PET-negative level 1 axillary node was identified as sentinel lymph node and resected.
Pathologic findings did not reveal metastasis. Instead, metastatic spread was found in PET-positive, enlarged interpectoral lymph node at US-guided needle biopsy. Bottom row shows transverse contrast-enhanced supine fat-saturated T1-weighted MRI scan, fusion image, and PET scan in patient 15. Uptake in 6-mm level 2 lymph node was found in addition to known metastases at axillary level 1. All PET scans are scaled to standardized uptake value (SUV) on scale of 0–10. (B) Box plots show maximum SUV (SUV max ) of benign lymph nodes in contralateral axillary level 1 and lymph node metastases most adjacent to breast in 13 patients according to grade, receptor status, and histologic type.
DWI, in which the imaging signal depends on the degree of compression of extracellular space due to cell enlargement and increased cell density in tumor tissue, has become especially pop- ular due to its high lesion-to-background contrast and its ability to quantify treatment-induced changes in diffusivity through cal- culation of apparent diffusion coefﬁcients (ADCs). Increased dif- fusivity on DWI as re ﬂected by an ADC increase has been shown to correspond to treatment-induced cell death (necrosis) in mela- noma as well as colon cancer xenografts. 10,11 Although limited by some technical artifacts—especially in the mediastinum, where the combination of cardiac and respiratory motion repre- sents a challenge; and in the lower neck, where RF ﬁeld inhomo- geneity is frequently observed —DWI has largely replaced gadolinium-based contrast-enhanced (CE-) T 1 -weighted sequences for whole-body imaging of hematological malignancies in clinical practice. This is because CE-MRI does not appear to have clear advantages over unenhanced MR sequences in terms of lesion detection or staging 12,13 —eg, in the study by Arendt et al, 13 agreement between unenhanced and contrast-enhanced
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Abstract: A novel nanocarrier system was designed and developed with key components uniquely structured at the nanoscale for early cancer diagnosis and treatment. In order to per- form magnetic resonance imaging, hydrophilic superparamagnetic maghemite nanoparticles (NPs) were synthesized and coated with a lipophilic organic ligand. Next, they were entrapped into polymeric NPs made of biodegradable poly(lactic-co-glycolic acid) linked to polyethylene glycol. In addition, resulting NPs have been conjugated on their surface with a 2,2′-(7-(4-((2- aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ligand for subsequent 68 Ga incorporation. A cell-based cytotoxicity assay has been employed to verify the in vitro cell viability of human pancreatic cancer cells exposed to this nanosystem. Finally, in vivo positron emission tomography-computerized tomography biodistribution studies in healthy animals were performed.
Earlier, it was believed that the information from MRI image can be used for defining the region of interest in PET data (Evans et al. 1988). However, Atlases are used to map certain regions that can be registered to the subject ’ s organ. Also, the atlases of human body are employed for determining the bio-distribution of tumours and metastases. The multimodal atlas can be acquired from a 3D slicer module for simple evaluation of the available image. This module has been validated with a publicly available soft tissue sarcoma information from the CancerImaging Archive (Rackerseder et al. 2017). Beyond this visualisation technique, retrospective analysis of structural and metabolic neuro-imaging can be performed using the free access software such as MRIcron, BrainSuite, BioImageSuite, ImageJ, FSL, Amide and MeVIS Lab. Usually, BrainSuite is used for stripping off the skull and creating the binary cerebral volume mask, hence, it will be processed with FSL FAST software. Also, the co-registration techniques can be achieved using BrainImageSuite. In recent time, Yuankai Zhu et al. evaluated the glucose metabolism in epileptic paediatric patients with the visual assessment and Statistical Parametric Mapping (SPM) (Fig. 11) (Zhu et al. 2017). While SPM is designed to perform segmentation of brain tissues consisting of Grey Matter (GM), White Matter (WM) and Cerebrospinal Fluid, the FSL and Brainsuite can segment sub-cortical structures also. Also, the performance of FSL was influenced by image noise and intensity non-homogenity (Kazemi and Noorizadeh 2014). Also, PMOD (version 3.5, PMOD Technologies, Zurich, Switzerland) was used in parametric images of Dopamine receptors (D 1 R) distribution volume ratio (DVR) and binding potential (BP ND ) of the PET data from
Pancreatic cancer was also considered in PET/MRI nanoprobes development by Locatelli et al (2012) , and more recently Kim et al  created a colon tumor-targeting nanoagent in 2013.
Also, PET-MRI nanoprobes have been used in macrophage imaging for diag- nosing atherosclerosis. This disease is characterized by the formation of plaques within the arteries walls, resulting from the accumulation of fatty substances, such as cholesterol and triglyceride, and calcium. The normal blood circula- tion is affected, creating a thrombus and a chronic inflammatory response in the affected area, activating macrophages and white blood cells towards the atherosclerotic plaque. Therefore, in 2010 Jarret et al  and also by the year of 2013 Majmudar and coworkers  created PET/MRI nanocarriers that target monocytes and/or macrophages present in atherosclerotic plaques.
In both SPECT and PET, the exogenous contrast agent is referred to as a radiotracer.
It is comprised of a radioactive atom that emits gamma-rays upon its radioactive decay;
and a molecule that has biological relevance. The biological relevance can include affinity to certain types of cells, or properties that facilitate or prevent diffusion across a membrane. In general, these radiotracer properties allow detection of cell populations, measurement of filtration or blood flow, and, by extension, the differentiation of diseased tissues from healthy tissues. In many cases, abnormal tracer uptake can be indicative of disease processes such as cancer (35) or tissue damage, as would be the case following a heart attack (36). SPECT and PET are often referred to as functional imaging modalities, since they show tissue health, rather than anatomical structure. SPECT and PET differ in that the latter relies upon the detection of pairs of gamma-rays, in contrast to Single Photon Emission Computed Tomography.
3.j. Method development versus yield determination
In this project, the goal was primarily to devise a route to peptides that a student in our lab could utilize to effectively synthesize TMIAs. The specific stretch goal was to develop a method to produce product. It is important to recognize that there are two factors which makes yield determination difficult: a) at each step about 3-5 mg of resin was removed in order to verify each intermediate in the entire synthesis. The purity of this intermediate was verified by an extracted wavelength chromatogram at 265nm, which is the lambda max of the Fmoc group. In each case it was verified by total ion current in LC- MS that there were minimal, or no impurities related to any peptides, protected or deprotected amino acids or peptide combinations of those. b) at various points in the synthesis the remaining resin was divided into smaller portions in order to test out new procedures. As each portion becomes smaller, aliquots removed for analysis become more of a factor. For this reason, yield determination was not a priority in this research. Rather, relative purity of products after cleavage in each step and the final products were important.
glucose loading, and the hyperinsulinemic-euglycemic clamp technique is most often used. It is recommended to combine FDG PET with a perfusion study (e.g., N-13 ammo- nia PET or Tc-99 m sestamibi SPECT) in order to identify hibernating myocardium (mis- match between decreased perfusion and increased glucose consumption). Based on a meta-analysis, FDG PET has a 92% sensitivity among viability imaging approaches and an acceptable specificity of 63% (Schinkel et al. 2007). LGE MRI is often used as an alterna- tive for viability imaging, even though the term viability is entirely misleading since scarred tissue is actually imaged. The theory behind this approach is that dysfunctional myocardium is more likely to recover the higher the subendocardial proportion of non-scarred (= viable) myocardium. The technique makes use of the fact that Gd-chelate-based contrast agents have a reduced wash-out from areas with increased extravascular space (such as the fibrotic tissue in a myocardial scar) in comparison to a fast wash-out from “ healthy ” non-scarred myocardium (Klein et al. 2004; Klein et al. 2007). Even though the approaches are quite different (molecularimaging of an increased glucose consumption in viable cells vs. extracellular contrast agent accumulation in fi- brotic tissue), a good agreement regarding myocardial viability assessment has been dem- onstrated (Klein et al. 2002). Benefits of LGE MRI over FDG PET include the lack of ionizing radiation, the highly reproducible accumulation of Gd-based contrast agents in scar tissue, independently from the metabolic state of the patient (which may be an im- portant factor in certain patients such as diabetics), and the high in-plane resolution of MRI of about 1 – 3 mm, which may permit the distinction between thinned myocardium and scarring (e.g., in the case of a reduced FDG uptake) or the depiction of tiny areas of myocardial scar, which are also known to carry prognostic significance even in patients without overt myocardial infarction (Kim et al. 2000; Kwong et al. 2006). Studies on viabil- ity imaging using hybrid FDG PET/MRI are still scarce. In the three available studies, agreements between FDG PET and MRI were reported as moderate to substantial (Fig. 5) (Priamo et al. 2017; Rischpler et al. 2015; Nensa et al. 2013). In one study with a very small patient cohort, adding FDG PET to LGE MRI resulted in a reclassification of about 19% of segments (Priamo et al. 2017). However, no follow-up to validate the actual impact in terms of wall motion recovery was available.
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MEDISO Medical Imaging Systems
Mediso Medical Imaging Systems with headquarters in Budapest is a dynamic supplier of nuclear medicine and modern hybrid imaging techniques to healthcare and research institutes around the world. The company was founded in 1990 by experts of the leading research and manufacturing company of the region carrying on their proficiency in developing and producing nuclear equipment since 1960. At present Mediso continue its legacy in researching innovative medical imaging technologies and developing cutting edge preclinical and clinical imaging systems.
bone marrow, which leads to prolonged blood half-life and leaking across hyperpermeable tumor microvessels. The nanoparticles slowly accumulate in the interstitium of malignant tumors, where they are phagocytosed by TAM. This phagocytosis is a slow process, requiring delayed imaging for macrophage depiction at 24 hours after iron oxide injection [102-105]. At 24 hours postcontrast, experimental data revealed a specific cellular uptake and MR enhancement of ferumoxytol in TAM isolated from adenocarcinomas [125, 126]. No or minimal ferumoxytol uptake was noted in cancer cells . The differential high ferumoxytol uptake by TAM and low or absent uptake by cancer cells is the basis for successful TAM imaging. Several studies in animal models and patients have shown that ferumoxytol nanoparticles are compartmentalized in TAMs at 24 hours post injection (p.i.). On these 24 hour delayed scans, the negative (dark) tumor enhancement on T2-weighted MR imaging studies correlated with TAM distribution on histopathology . Cellular uptake of iron oxide nanoparticles led to a decreasing T1-signal effect, but persistent T2-signal effect on MR images . This “decoupling” of T1- and T2-signal effects on MR images was indicative of intracellular compartmentalization. Recently, this concept has been translated to first-in-human clinical trials and showed that ferumoxytol-MRI can quantify TAM quantities in patients with glioblastoma , osteosarcoma and lymphoma . Within each tumor group, T2* signal enhancement on MR images correlated significantly with the density of CD68+ and CD163+ TAM (P < 0.05) [126, 128].
demonstrated. Results of the MAP reconstruction method were consistent with the results of other researchers.
The MAP reconstruction method presented in Chapter 5 is essentially a modern update of the classical Bayesian estimation criterion for anatomically-guided PET. The key difference is the recognition that in a data-rich environment such as medical imaging, the anatomical guidance need not come from a single scan nor be purely anatomical in nature. Unbridled from processing constraints by parallel computing, the question of prior incorporation into the reconstruction process is no longer limited to which hyperparameter to tune for best results but which data provide useful features. This presupposes a holistic, data-centric approach to medical imaging in which a Bayesian estimation criterion may prove outdated. Data-centric approaches to problem solving increasingly rely on machine learning algorithms to organically derive solutions to classification, regression, and detection problems. These algorithms are modeled after the connection patterns of neurons and rely heavily on truthed datasets to train neural networks to produce desired output. The E-phantom platform could prove useful as a training tool for machine learning algorithms since it offers researchers the type of varied and truthed training data required of deep learning methods.
specifically to AD type neurodegenerative disease than other types, especially in the early stages of dementia. While most other tauopathies are characterized by the presence of both neuronal and glial tau pathologies . Besides the PETimaging findings at molecular-level, alterations of tau-mediat- ed brain functions had been investigated with several MRI met- rics . These MRI multiparametric findings using rTg4510 mouse model of taupathy had observed changes in brain atro- phy (remarkable), cerebral blood flow (elevation during inter- vention), diffusion tensor imaging (DTI, downstream or later stage from formation of tau lesions) and amide proton transfer (decreased amide proton transfer-APT metabolism). We have recently investigated multiple MRI and convention- al PETimaging biomarkers (amyloid and FDG) for differen- tiating early AD from normal controls and MCI; including structural and functional connectivity based on MRI (fcMRI), small-worldness analysis with MRI cortical thickness metric to better differentiate several stages of early dementia, and be- tween MRI metrics such as white matter lesion load (a marker for cerebrovascular risk), functional activity and PET amyloid load for better understanding and defining the underlying neuropathological changes of brain circuits and association with phenotypic data [17-21]. Due to the relatively new devel- opment of the tau tracer, In vivo associations between PET tau and MRI metrics have not been reported and confirmed in hu- man subjects yet. The between-modality interactions includ- ing human brain structural and functional networks undergo robust changes in normal aging and preclinical stages of AD, suggesting that neuroimaging metrics variability and integra- tion from various networks in different scales might be useful to monitor brain changes.