This work was supported by a grant from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, grant number R01 HD081123-01A1. Anuj Pareek is a visiting Ph.D. student from the Department of Radiology, Aarhus University Hospital, Denmark. The authors acknowledge technologists Dawn Holley and Harsh Gandhi from the PET/MRI Metabolic Service Centre for their assistance with the acquisition of PET/MRI scans. We thank members of the Daldrup-Link lab, the PET/MRI service center, the Molecular Imaging Program at Stanford, the Radiological Science Lab, the Pediatric Radiology Section, the Stanford Cancer Institute and the pediatric oncology team at Lucile Packard Children's Hospital for helpful discussions and support of this project.

All methods presented here have been established under a research study, which was approved by the institutional review board of Stanford University. The &#34;off label&#34; use of ferumoxytol was performed under an investigator-initiated investigational new drug application (IND 111,154).
1. 24 h Prior to PET/MRI Scan: Patient Safety Screening
<ol>
	<li>Screen the patient for potential magnetic field hazards using an MRI safety questionnaire. If the administration of intravenous MRI contrast agent is planned, check contraindications, such as history of allergies and kidney function, among others.</li>
	<li>Inform the patient of purpose, procedure, risks and benefits prior to the scan.</li>
	<li>Weigh the patient for calculation of radiotracer and contrast agent dose.</li>
	<li>Ask the patient to fast for at least 4 hours before the scan.</li>
</ol>
2. 1-2 h Before the PET/MRI Scan: Patient Preparation
<ol>
	<li>Ask the patient to remove all metallic objects and change into a gown. If there are any implanted metallic objects, make sure that they are safe for MRI scanning. Use a metal detector as needed. Do not perform the PET/MRI if there is a concern for patient safety.</li>
	<li>Place an intravenous line in a peripheral superficial vein or access a port-catheter.</li>
	<li>Dilute ferumoxytol (5 mg Fe/kg bodyweight) 1:4 in saline, and infuse slowly over at least 15 min according to FDA protocols10. 
	Note: Ferumoxytol can be used &#34;off label&#34; as a contrast agent through an investigator-initiated IND, because it provides long lasting vascular and tissue enhancement for the duration of the entire scan. Alternatively, a cyclic gadolinium chelate can be administered directly before start of the PET/MRI scan. For patients with liver lesions, gadoxetate disodium may be chosen.</li>
	<li>Check the patient&#39;s blood-glucose levels with a glucometer.If the patient&#39;s blood-glucose levels are above 200 mg/dL, consider re-scheduling the PET/MRI scan.</li>
	<li>45 min prior to the PET/MRI scan, administer 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) at a dose of 2 MBq/kg bodyweight in 1 mL of saline through the intravenous line.</li>
</ol>
3. PET/MR Image Acquisition
Note: For this protocol, we used a 3.0 Tesla GE Signa integrated PET/MRI system bundled with MP24_R03 scanner software.
<ol>
	<li>Save the protocol for the whole-body scan (Table 1) and local tumor scan (Table 2) on the scanner, so that these can be easily retrieved without losing much time to search sequences or adjust protocols.</li>
	<li>For patients with lymphoma, prescribe a whole-body scan only, either extending from head to toe or extending from head to mid-thigh level, according to the clinical oncologist&#39;s request. For all other patients, prescribe a whole-body scan from head to toe followed by a separate local tumor scan.
	<ol>
		<li>If a tumor is already appropriately centered in one of the whole-body PET slabs, prescribe and run the dedicated MRI sequences during the whole-body scan in order to save time; e.g., an abdominal tumor can be scanned with the whole-body coil.</li>
	</ol>
	</li>
	<li>To enable easy retrieval and review of PET/MRI data for the Radiologist, save the &#34;diagnostic&#34; images under one accession number. Later (see step 4.3.5) save the source images under a second accession number in the PACS system.</li>
	<li>Use the same localizers throughout the scan and make sure the patient doesn&#39;t move for the duration of the scan. This enables co-registration of the acquired PET and MRI data later.</li>
	<li>Prescribe the necessary MRI sequences within each PET slab. The integrated PET/MRI system then allows for the simultaneous acquisition of PET data and MRI data.</li>
	<li>Ask the patient to void directly before entering the scanner room.</li>
	<li>Whole-Body PET/MRI Scan
	<ol>
		<li>Position the patient on the PET/MRI table in a supine position. If the patient cannot lay supine, scan the head supine and the whole body in prone position. For very tall patients, acquire the lower legs in a second step. For patients with bone or soft tissue sarcoma, make sure to cover the whole affected bone from joint to joint (e.g., whole femur from hip to knee joint).</li>
		<li>Close a breathing belt around the patient. Then position a head coil and 2-3 surface coils on the neck, chest, abdomen, pelvis and extremities of the patient.</li>
		<li>Acquire whole-body localizers in three planes; axial, coronal and sagittal (Table 1) using the generic localizer protocol. Select the number of stations according to patient height. Scanning direction can be either from head to feet or feet towards head.</li>
		<li>Prescribe the entire protocol before starting the whole-body scan. Streamline this process and use a pre-saved protocol so that children do not lose patience.</li>
		<li>Prescribe axial PET slabs first, using the maximum axial field of view (FOV, typically 25 cm) and 3 min data acquisition. Depending on patient size, 4-5 slabs will cover head to mid-thigh and 9-10 slabs will cover the whole body.</li>
		<li>In the scanner software, enable two separate reconstructions for the PET data: one non-attenuation corrected (NAC) PET and one MRI attenuation corrected (MAC) PET. Use a matrix size of 192 x 192. Use 28 subsets with 2 iterations. The PET/MRI software allows for attenuation correction accounting for patient, patient cradle and dedicated MRI coils using sequences described under 3.7.7 below.</li>
		<li>Fill each PET slab with a low-resolution Liver Acquisition with Volume Acquisition (LAVA) sequencefor Attenuation Correction (MRAC) and a higher resolution LAVA sequence for anatomical co-registration of 18F-FDG-PET data. Diffusion-Weighted Images (DWI) can be added as deemed appropriate (Table 1).</li>
		<li>In the Task menu select the PET Task, click Add Task, click Add Postprocessing, choose MRAC Autobind (User) and then click Setup which will bring up an additional menu. Select all consecutive Water MRAC for the PET slabs and click Accept. This will enable the software to automatically merge the MRAC.</li>
		<li>Apply similar steps to merge the DWI. In this case, choose the first PET slab and click on the DWI sequence. Then click Add Task and follow the same additional steps as in 3.7.8.</li>
		<li>Add a dedicated T2-sequence for evaluation of the lungs and/or mediastinum (Table 1).</li>
	</ol>
	</li>
	<li>Local PET/MRI scan
	<ol>
		<li>Place an appropriate coil over the primary tumor. For extremity tumors, a flex coil may be used. For bone tumors, make sure to cover the adjacent joint (e.g., for an osteosarcoma of the distal femur metaphysis, include the distal femoral epiphysis and knee joint). For primary tumors of the neck, chest, abdomen and pelvis, placement of coils for local tumor and whole body staging can be done at once (see step 3.7.2). The separate materials/equipment list provides an overview of PET-compatible MRI coils available on the scanner.</li>
	</ol>
	</li>
</ol>
4. Image Data Processing
Note: To obtain the merged whole-body PET/MRI scan, the following four steps must be done: 1) reconstruct the PET image data, 2) merge the individual MRI data acquisitions to a single image volume, 3) co-register the PET to the MRI data, and 4) generate a PET whole-body maximum intensity projection (MIP). The scanner software greatly simplifies these steps.
<ol>
	<li>Merging of individual MRI data
	<ol>
		<li>Select all individual MRI data sets of a specific sequence, e.g.,LAVA 1 through 9, and press the Autobind button. The software then constructs a merged MRI data set. Save the merged MRI data set. The software automatically names the data set and adds it to the series list.</li>
	</ol>
	</li>
	<li>Co-registration of PET and MRI data
	<ol>
		<li>To co-register the merged MAC PET-data and merged LAVA-data, select each data set which needs to be co-registered and press the ImageQC button. The software then shows both the individual non-co-registered data sets and the co-registered data set in two planes in six separate windows.</li>
		<li>Select the MRI data and window-level appropriately, since this cannot be adjusted once the co-registered scan is finalized and saved.</li>
		<li>Similarly select the co-registered PET/MRI data set and window-level the PET data. It is also possible to adjust opacity, zoom and position of the co-registered data before saving.</li>
		<li>Choose the appropriate slice orientation for the co-registered data set, i.e., axial or coronal.</li>
		<li>Lastly, save the adjusted co-registered PET/MRI data set:
		<ol>
			<li>Choose Batch in the Film/Save menu. Select Loop and scroll all the way to the most cranial slice and click Set Start, then scroll down to the most caudal slice and click Set End.</li>
			<li>In the description field, choose an appropriate filename. Use a consistent and easily recognizable file name for all studies, so that the radiologist and clinician can easily find a specific data set (e.g., PET-LAVA Whole-Body).</li>
			<li>Click Preview and let the scan scroll from head to toe while checking that the final result is acceptable in terms of fusion accuracy and window levels. Close the preview and then select OK to start rendering the PET/MRI scan. The rendering process can be followed by observing the image index track bar.</li>
		</ol>
		</li>
		<li>Follow the same steps to co-register the MAC PET-data and the dedicated T2 MRI sequence for evaluation of the lungs. Since the PET-data FOV extends beyond the MRI, set the start and end of the scan to include only the co-registered part. 
		Note: The local PET/MRI can be co-registered following the same steps.</li>
	</ol>
	</li>
	<li>Generate PET whole-body MIP overview
	<ol>
		<li>To generate a PET whole-body maximum intensity projection (MIP), select the MAC PET and any MRI data set and press the ImageQC button.</li>
		<li>Double-click on the window with PET data only, and hover the cursor in the upper left corner to select View Type, and then select VR. This will create a 3D reconstruction of the PET data. Again, hover the mouse in the upper left corner to select rendering mode and then select HD MIP to finally create the PET whole-body MIP overview.</li>
		<li>To save the MIP overview choose Batch in the Film/Save menu and select Rotate. Adjust the number of images to 60. In the description field choose an appropriate file name. Click Preview and check that the final result is acceptable. Close the preview and then select OK to start rendering the MIP overview. Make sure the rendering is complete before you exit.</li>
		<li>Send the diagnostic image data sets to the institutions Picture Archiving and Communication System (PACS) by selecting the appropriate series and click on the desired Network in the Destinations menu.</li>
		<li>To save the source images under a different accession number, click Edit Patient and change the accession number. Then repeat the send procedure.</li>
	</ol>
	</li>
</ol>
5. Image Analysis and SUV Measurement
<ol>
	<li>Load the co-registered PET/MRI data in an FDA-approved medical imaging viewer to read and analyze the scan. Commercial PET/MRI viewers allow for measurement of the maximum Standardized Uptake Values (SUV) in 3D. 
	Note: For this specific protocol, we used MIM version 6.6.</li>
	<li>Choose Calculate SUV statistics from the tool bar. Position the region of interest (ROI) around the tissue exhibiting pathological 18F-FDG uptake on the co-registered PET/MRI. The ROI diameter can be enlarged by holding and dragging with the right mouse button. The software will determine both the mean and maximum SUV value. For patients with lymphoma, measure the liver and mediastinal blood pool as a standard of reference. Follow institution-specific guidelines for reporting of SUV measurements.</li>
</ol>

<ol>
	<li>Pichler, B. J., Judenhofer, M. S., Wehrl, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET/MRI+hybrid+imaging:+Devices+and+initial+results.">PET/MRI hybrid imaging: Devices and initial results.</a> Eur. Radiol. 18 (6), 1077-1086 (2008).</li><li>Torigian, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+/+MR+Imaging+:+Technical+Aspects+and+Potential+Clinical+Applications.">PET / MR Imaging : Technical Aspects and Potential Clinical Applications.</a> Radiology. 267 (1), 26-44 (2013).</li><li>Daldrup-Link, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+PET/MR+Can+Add+Value+For+Children+With+Cancer.">How PET/MR Can Add Value For Children With Cancer.</a> Curr. Radiol. Rep. 5 (3), (2017).</li><li>Muehe, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+Provide+Gadolinium-Free+PET/MR+Cancer+Staging+of+Children+and+Young+Adults+in+Less+than+1+h:+the+Stanford+Approach.">How to Provide Gadolinium-Free PET/MR Cancer Staging of Children and Young Adults in Less than 1 h: the Stanford Approach.</a> Mol Imaging Biol. , (2017).</li><li>Spick, C., Herrmann, K., Czernin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-FDG+PET/CT+and+PET/MRI+perform+equally+well+in+cancer+patients:+Evidence+from+studies+in+more+than+2300+patients.">18F-FDG PET/CT and PET/MRI perform equally well in cancer patients: Evidence from studies in more than 2300 patients.</a> J. Nucl. Med. 57 (310), 1-38 (2016).</li><li>Sher, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+sequential+PET/MRI+in+comparison+with+PET/CT+of+pediatric+lymphoma:+A+prospective+study.">Assessment of sequential PET/MRI in comparison with PET/CT of pediatric lymphoma: A prospective study.</a> Am. J. Roentgenol. 206 (3), 623-631 (2016).</li><li>Jadvar, H., Colletti, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+Advantage+of+PET/MRI.">Competitive Advantage of PET/MRI.</a> Eur. J. Radiol. 83 (1), 84-94 (2014).</li><li>Wan, J. C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsies+come+of+age:+towards+implementation+of+circulating+tumour+DNA.">Liquid biopsies come of age: towards implementation of circulating tumour DNA.</a> Nat. Rev. Cancer. 17 (4), 223-238 (2017).</li><li>Grant, A. M., Deller, T. W., Khalighi, M. M., Maramraju, S. H., Delso, G., Levin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NEMA+NU+2-2012+performance+studies+for+the+SiPM-based+ToF-PET+component+of+the+GE+SIGNA+PET/MR+system.">NEMA NU 2-2012 performance studies for the SiPM-based ToF-PET component of the GE SIGNA PET/MR system.</a> Med. Phys. 43 (5), 2334-2343 (2016).</li><li>. FDA Drug Safety Communication: FDA strengthens warnings and changes prescribing instructions to decrease the risk of serious allergic reactions with anemia drug Feraheme (ferumoxytol) Available from: <a target="_blank" href="https://www.fda.gov/Drugs/DrugSafety/ucm440138.htm">https://www.fda.gov/Drugs/DrugSafety/ucm440138.htm</a> (2015)</li><li>Mattsson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+Dose+to+Patients+from+Radiopharmaceuticals:+a+Compendium+of+Current+Information+Related+to+Frequently+Used+Substances.">Radiation Dose to Patients from Radiopharmaceuticals: a Compendium of Current Information Related to Frequently Used Substances.</a> Ann. ICRP. 44 (2 Suppl), 7-321 (2015).</li></ol>

The authors have nothing to disclose.

We have shown a step-by-step protocol for PET/MRI studies of pediatric cancer patients. The most critical part of the protocol is time-efficient planning and prescribing the PET-slabs and MRI sequences with the correct parameters and in the correct consecutive order before initiating the whole-body scan. This enables continuous acquisition of the entire body. Efficient scanning is especially important in the pediatric setting, where unsedated patients can easily lose patience and either s...

Integrated Positron Emission Tomography (PET)/Magnetic Resonance Imaging (MRI) enables cancer staging and treatment monitoring with high sensitivity, high soft tissue contrast, and added metabolic information1,2,3,4. In adult patients, PET/MRI performed equally well as PET/CT for staging of established cancers5,6,7. In the future, liquid biopsies will likely lead to earlier detection of cancer development (e.g., through transcriptomes and circulating DNA) and require more sensitive imaging tests than are currently available to find small tumors in the body8. This might put PET/MRI into a superior position to evaluate the whole body and detect cancers in anatomical areas that have been classically evaluated with MRI alone, such as the brain, neck, abdomen/pelvis,&#160;and musculoskeletal system.
For pediatric patients, PET/MRI has several advantages over PET/CT: First, PET/MRI provides a markedly reduced radiation exposure of the patient by up to 74%4. This can be achieved by using ionizing radiation-free MRI instead of CT technologies for anatomical co-registration of PET data. In addition, the increased sensitivity of modern PET detector systems9 and longer PET data acquisition during an MRI scan enables significant reduction of administered radiotracer doses by 30-50% compared to current PET/CT protocols4. Second, the possibility of combining staging scans of the primary tumor and the whole body saves time and avoids duplicate sedations for some patients, such as patients with bone and soft tissue sarcomas. However, a &#34;one stop&#34; staging scan is only clinically feasible if all PET/MRI data (local tumor and whole body) can be acquired in an efficient manner and if the abundance of resultant image data is presented in an easily digestible format to the radiologist. The protocol presented here will provide step-by-step instructions for the acquisition of PET/MRI data that can be used for clinical staging of children with cancer, with particular attention to specific needs of the pediatric population.

<table border="0" cellpadding="0" cellspacing="0" width="835">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Integrated PET/MRI scanner</td>
			<td style="width:189px;"></td>
			<td style="width:143px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">SIgna PET/MR</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;"></td>
			<td>3.0 T integrated PET/MRI scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Software</td>
			<td style="width:189px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">MP24_R03</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;"></td>
			<td>PET/MRI scanner software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">MIM software version 6.6.13</td>
			<td style="width:189px;">MIM Software Inc.</td>
			<td style="width:143px;"></td>
			<td>PET/MRI analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Contrast Agents</td>
			<td style="width:189px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ferumoxytol</td>
			<td style="width:189px;">AMAG Pharmaceuticals</td>
			<td style="width:143px;"></td>
			<td>Iron Oxide nanoparticles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">18F-FDG</td>
			<td style="width:189px;">-</td>
			<td style="width:143px;"></td>
			<td>2-Deoxy-2-(18F)fluoro-D-glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">MRI coils available on the scanner</td>
			<td style="width:189px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nova 32 Channel Head coil</td>
			<td style="width:189px;">Nova&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neocoil 1H 16 channel GEM Flex Large Array</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">M7000SK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neocoil 1H 16 channel &#160;GEM Flex Medium Array</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">M7000SL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neocoil 1H 16 channel GEM Flex Small Array</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">M7000SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Everest Central Molecular Imaging Array (CMA)</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">M8000RB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Everest Head Neck Unit</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">M8000CB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Everest Lower and Upper Anterior Array</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">M8000CC &#38; M8000CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Invivo 1H 8 channel High Res Brain Array</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">M8000RA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.0T HD Breast Array Coil</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">M7000GG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.0T Split-Top Head TR Single Ch Coil</td>
			<td style="width:189px;">GE</td>
			<td style="width:143px;">G6000BH</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-body pet/mri of pediatric patients: the details that matter

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both high soft tissue contrast and added metabolic information. Integrated PET/MRI has shown to be valuable in the clinical setting and has many promising future applications. The protocol presented here will provide step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI data in children with cancer. It also provides instructions on how to combine a whole-body staging scan with a local tumor scan for evaluation of the primary tumor. The focus of this protocol is to be both comprehensive and time-efficient, which are two ubiquitous needs for clinical applications. This protocol was originally developed for children above 6 years, or old enough to comply with breath-hold instructions, but can also be applied to patients under general anesthesia. Similarly, this protocol can be modified to fit institutional preferences in terms of choice of MRI pulse sequences for both the whole-body scan and local tumor assessment.

We demonstrate an integrated whole-body (head to toe) data set of MRI, PET and fused PET/MR images, respectively, respectively, of a 10 year-old boy with status post resection of an undifferentiated sarcoma of the 12th rib, who presented with pulmonary and cardiac metastases (Figure 1). The data set shown here was acquired within 40 min. The effective dose of the patient during this exam was 3.3 mSv11.
For patients who rece...

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Whole-body PET/MRI of Pediatric Patients: The Details That Matter

This article provides comprehensive step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI scans for cancer staging of pediatric patients. The protocol was developed for children above 6 years, or old enough to comply with breath-hold instructions, but can be used for general anesthesia patients as well.

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both ...

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Cancer Research

14.3K Views.  Stanford University. This article provides comprehensive step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI scans for cancer staging of pediatric patients. The protocol was developed for children above 6 years, or old enough to comply with breath-hold instructions, but can be used for general anesthesia patients as well.

Video: Whole-body PET/MRI of Pediatric Patients: The Details That Matter

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is typically performed via transfemoral access (TFA). However, transradial access (TRA) is preferred in coronary artery interventions due to decreased complications and mortality. Whether the advantages of TRA can be applied to TACE required investigation.
Patients receiving TRA TACE at a single center were retrospectively enrolled for study. Procedural details, technical success, radial artery occlusion (RAO) rate, and access site-related bleeding complications were evaluated. From October 2017 to October 2018, 112 patients underwent 160 TRA TACE procedures. The overall technical success rate was 95.0% (152/160). The rate of crossover from TRA to TFA was 1.9%. No access site-related bleeding complications were found in any cases. Asymptomatic RA occlusion occurred in three patients (2.7%). Compared with TFA, TRA can increase safety and patient satisfaction while decreasing access site-related bleeding complications. Moreover, TRA interventions can benefit patients with advanced age, obesity, or a high risk of bleeding complications.

Transarterial chemoembolization (TACE) is the standard therapy for patients in the intermediate stage of hepatocellular carcinoma and is typically performed through femoral artery access. Compared with transfemoral access, transradial access (TRA) can decrease the rate of bleeding complications and improve patient tolerance. A method is presented here to perform transarterial chemoembolization via radial artery access.

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is ...

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Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...