The authors acknowledge all the staff involved in 68Ga-DOTATATE PET/CT imaging at the department of Nuclear Medicine at the Netherlands Cancer Institute.

1. General radiation and radiopharmaceutical safety
<ol>
	<li>Ensure that radioactive materials are only worked with and handled by trained personnel. The dose to hospital staff, patients, and everyone else present should always be kept as low as reasonably achievable (ALARA).</li>
	<li>Regarding the preparation of radiopharmaceuticals, adhere to national laws, regulations, and guidelines, such as Good Manufacturing Practice (GMP). 
	CAUTION: The following protocol is for the 68Ga-DOTATATE PET/CT imaging of adults only and is not suitable for children or pregnant women.</li>
</ol>
2. Preparations required prior to the labeling of 68Ga-DOTATATE
<ol>
	<li>Elute the 68Ge/68Ga generator with hydrochloric acid (HCl) according to the manufacturer&#8217;s specifications, between 4 and 24 h prior to the start of labeling 68Ga-DOTATATE.</li>
</ol>
3. Labeling of 68Ga-DOTATATE
NOTE: The preparation for and labeling of 68Ga-DOTATATE takes 90 min and should be started 2 h prior to patient administration, to allow for quality control. The labeling module should be placed in a lead shielding that can be closed during the labeling process to ensure radiation protection of personnel. If a registered kit is used, then the summary of the product characteristics (SMPC) must be followed or cross-validated locally with the presented protocol.
<ol>
	<li>Place the 68Ga labeling kit on the labeling module according to the manufacturer&#8217;s specifications. Place the three manifolds on the corresponding module units. Attach the solutions provided in the 68Ga labeling kit to the manifolds.</li>
	<li>Prepare the final vial in a sterile environment, such as a downflow unit or laminar flow cabinet.</li>
	<li>Place a nonvented 0.22 &#181;m filter underneath a vented 0.22 &#181;m filter and attach the nonvented filter to a sterile needle (20 G). Place the needle with the two filters attached in a 30 mL sterile vial. Place a vented 0.2 &#181;m bent filter with a needle (22 G) in the same sterile vial as per step 3.2 to allow venting.</li>
	<li>Attach the sterile vial with the nonvented filter to the output of the labeling module and place the vial in the lead shielding sufficient for positron emitters.</li>
	<li>Attach the output of the 68Ga/68Ge generator to the input of the labeling module.</li>
	<li>Dissolve 50 &#181;g of HA-DOTATATE (DOTA-3-iodo-Tyr3-octreotate) or 20 &#181;g of DOTATATE (DOTA-0-Tyr3-octreotate) peptide in 1.5 mL of 1.5 M HEPES buffer solution provided in the kit and place it in the reaction vial.</li>
	<li>Close the lead shielding around the labeling module and start the production of 68Ga-DOTATATE via the tablet computer attached to the labeling module.</li>
	<li>Wait until the synthesis of 68Ga-DOTATATE is finished (~36 min).</li>
	<li>After labeling, remove the needles with filters from the glass vial and close the lead shielding around the vial.</li>
	<li>Test the integrity of the nonvented 0.22 &#181;m filter as follows.
	<ol>
		<li>Fill a syringe (10 mL) with air and place the syringe on top of the filter. Place the needle attached to the filter in a tube filled with water.</li>
		<li>Force the air through the filter and needle and determine when bubbles begin to form. The air should be compressed to &#60;20% of the original volume.</li>
	</ol>
	</li>
	<li>Measure the activity of 68Ga-DOTATATE produced by placing the vial in a dose calibrator and note the activity reference time (ART).</li>
	<li>In a sterile environment such as a laminar flow cabinet, remove 0.5 mL of 68Ga-DOTATATE from the vial for quality control and prepare syringes for patient administration. 
	NOTE: Place the solution with 68Ga-DOTATATE in lead shielding. In this protocol, the diluted solution does not have to be shielded due to the low levels of activity and short exposure time. However, a radiation risk assessment should be performed prior to performing quality control of the 68Ga-DOTATATE.</li>
</ol>
4. Quality control of 68Ga-DOTATATE prior to patient administration
NOTE: The quality control of 68Ga-DOTATATE takes 30 min and should be started 30 min prior to patient administration. The described dilutions for stock solutions lead to &#60;5% dead time in the measurement equipment. This can vary between different equipment and should be tested prior to performing quality control of the 68Ga-DOTATATE. The European Pharmacopoeia describes the quality control of gallium edotreotide injections based on the following release criteria: appearance = clear and colorless; pH = 4.0&#8211;8.0; sterility; endotoxins &#60;175 IU per administered volume; ethanol &#60;10% v/v; radionuclide purity &#62;99.9% of total activity; radiochemical purity &#62;91% of total activity; absence of other impurities; HEPES &#60;200 &#181;g per administered volume14. All tests have been evaluated during the validation of the preparation method. For routine quality control, a selected subset of tests (based on trend monitoring) is performed and described below. The solid-phase extraction in this protocol has been cross-validated with and obtains the same results as the high-performance liquid chromatography method described in the European Pharmacopoeia. This was performed based on GMP.
<ol>
	<li>Prepare the following solutions in advance.
	<ol>
		<li>Prepare a 1 M ammonium acetate solution by dissolving 3.9 g of ammonium acetate in 50 mL of water. The solution can be stored at room temperature for up to 2 weeks.</li>
		<li>Prepare 5 mM ethylenediaminetetraacetic acid (EDTA) by dissolving 0.1 g of EDTA in 50 mL of water. The solution can be stored at room temperature for up to 1 year.</li>
		<li>Prepare 50:50 methanol:1 M ammonium acetate. The solution can be stored at room temperature for up to 24 h.</li>
	</ol>
	</li>
	<li>Visually inspect the final 68Ga-DOTATATE product to ensure that it is a colorless liquid without any particles.</li>
	<li>Measure the pH of the 68Ga-DOTATATE solution with a pH indicator strip. The pH should lie between 6.5 and 7.5.</li>
	<li>Measure 68Ga colloids through instant thin-layer chromatography (ITLC).
	<ol>
		<li>Add 500 &#181;L of water and 20 &#181;L of 68Ga-DOTATATE to prepare a stock solution and homogenize (68Ga colloids [GC]) by carefully shaking the vial.</li>
		<li>Cut a strip of ITLC- silica gel (SG) glass fiber paper of at least 7 cm long and 1 cm wide. 
		NOTE: Only use clean ITLC-SG paper without damages. If the paper is damaged, the components traveling with the solvent can be hindered and the results will be inaccurate.</li>
		<li>Add 5 &#181;L of GC 1.5 cm from the bottom of the ITLC-SG paper and place it in a tube containing 2 mL of 50:50 methanol:1 M ammonium acetate. Ensure that the 68Ga-DOTATATE does not come into contact with the liquid. Close the tubes to prevent evaporation.</li>
		<li>Wait several minutes until the solvent has traveled at least 5 cm above where the 68Ga-DOTATATE was applied. Cut the paper in half and place the bottom and upper halves in separate tubes (BH1 and UH1).</li>
		<li>Place the UH1 and BH1 tubes in a well counter and determine the number of counts in each vial in the 400&#8211;600 keV energy window for 30 s, to determine the colloid percentage (see the calculations in steps 4.4.7&#8211;4.4.9).</li>
		<li>Repeat steps 4.4.2&#8211;4.4.5 to acquire BH2 and UH2..</li>
		<li>Perform a background measurement of an empty well counter and determine the number of counts in the 400&#8211;600 keV energy window for 30 s.</li>
		<li>Correct the counts in the UH1, UH2, BH1 and BH2 for decay and background (determined in step 4.4.7) to obtain UH1cor, UH2cor, BH1cor, BH2cor. &#916;t is the time difference between the measurement of the sample and the ART (determined in step 3.11) in minutes. 
		<img alt="Equation 1" src="/files/ftp_upload/59358/59358eq1.jpg" /></li>
		<li>Calculate the number of 68Ga colloids with the following formula; note that this should be less than 3%. UH1cor, UH2cor, BH1cor, BH2cor are the corrected values obtained in step 4.4.8. 
		<img alt="Equation 2" src="/files/ftp_upload/59358/59358eq2.jpg" /></li>
	</ol>
	</li>
	<li>Determine the 68Ga ions through column separation.
	<ol>
		<li>Make a stock solution by diluting 20 &#181;L of 68Ga-DOTATATE in 1 mL of 5 mM EDTA.</li>
		<li>Prepare a C-18 cartridge by slowly flushing it with 1 mL of 100% ethanol, followed by 1 mL of sterile water.</li>
		<li>Prepare a sample (S) by diluting 10 &#181;L of the stock solution in 1 mL of sterile water, placing it in a well counter, and determining the number of counts in the 400&#8211;600 keV energy window for 30 s.</li>
		<li>Flush the sample slowly (5&#8211;10 mL/min) through the C-18 cartridge with a syringe and collect the remaining solution (C). Rinse the sample tube with 1 mL of water and flush this through the column in the collection tube.</li>
		<li>Place the collecting tube (C), the empty sample tube (E), and the syringe (Sy) in a well counter and determine the number of counts in each of them in the 400&#8211;600 keV energy window for 30 s. Use this to estimate the ion percentage (see the calculations in steps 4.5.7 and 4.5.8).</li>
		<li>Repeat steps 4.5.2&#8210;4.5.5.</li>
		<li>Correct the counts for decay and background (determined in step 4.4.7) in C, S, E, and Sy to determine C&#8217;, S&#8217;, E&#8217; and Sy&#8217;, to determine the number of counts at the ART with the following formula, in which &#916;t is the time difference between the measured sample and the ART in minutes. 
		<img alt="Equation 3" src="/files/ftp_upload/59358/59358eq3.jpg" /></li>
		<li>Calculate the 68Ga ion percentage with the following formula; note that this should be less than 2%. 
		<img alt="Equation 4" src="/files/ftp_upload/59358/59358eq4.jpg" /></li>
	</ol>
	</li>
	<li>Determine the radiopharmaceutical purity by calculating the total amount of 68Ga-DOTATATE with the following formula, which should be at least 91%. 
	<img alt="Equation 5" src="/files/ftp_upload/59358/59358eq5.jpg" /></li>
</ol>
5. Periodical quality control of 68Ga-DOTATATE after patient administration
NOTE: This should be performed &#62;48 h after the preparation of 68Ga-DOTATATE to allow for the decay of 68Ga.
<ol>
	<li>Prepare the following solutions in advance.
	<ol>
		<li>Prepare HEPES reference solution by dissolving 20 mg of HEPES in 50 mL of sterile water. The solution can be stored at room temperature for up to 6 months. 
		NOTE: This is based on the maximum recommended HEPES dose of 200 &#181;g per administered volume.</li>
		<li>Prepare 25:75 v/v water:acetonitrile.</li>
	</ol>
	</li>
	<li>Determine HEPES concentration in the final product (weekly).
	<ol>
		<li>Transfer 3 &#181;L of 68Ga-DOTATATE solution in steps of 1 &#181;L to an ITLC-SG F254 paper of at least 8 cm length. Use a blow dryer to dry the paper in between the 1 &#181;L applications.</li>
		<li>Repeat step 5.2.1 with the HEPES reference solution.</li>
		<li>Place the strips in a solvent of 25:75 water:acetonitrile. Ensure that the applied solutions do not come into contact with the liquid.</li>
		<li>Wait several minutes until the solvent has traveled to at least 2/3 of the paper length.</li>
		<li>Develop the paper for at least 4 min in a closed chamber with iodine crystals.</li>
		<li>Visually assess the outcome; a yellow spot should appear. The spot on the paper with 68Ga-DOTATATE should be less intense than that of the reference solution.</li>
	</ol>
	</li>
	<li>Determine 68Ge breakthrough in the final product (monthly).
	<ol>
		<li>Prepare a sample with 200 &#181;L of 68Ga-DOTATATE solution (G). Place it in a well counter and determine the number of counts in each in the 400&#8211;600 keV energy window for 30 s.</li>
		<li>Repeat step 5.3.1.</li>
		<li>Correct the counts in G to determine G&#8217; for decay, to determine the number of counts at the ART with the following formula, in which &#916;t is the time difference between the measured sample and the ART in minutes. 
		<img alt="Equation 6" src="/files/ftp_upload/59358/59358eq6.jpg" /></li>
		<li>Calculate the 68Ge breakthrough (MBq/MBq), which should be less than 0.001%, with the following formula. 
		<img alt="Equation 7" src="/files/ftp_upload/59358/59358eq7.jpg" /></li>
	</ol>
	</li>
	<li>Determine the sterility of the final product (monthly).
	<ol>
		<li>Add the remaining 68Ga-DOTATATE solution to a tryptic soy broth (TSB) medium. Incubate for 14 d at 30&#8211;35 &#176;C.</li>
		<li>Check that the TSB medium is a clear liquid after the incubation period.</li>
	</ol>
	</li>
</ol>
6. Patient preparation and administration of 68Ga-DOTATATE
NOTE: The injected activity in this protocol provides good quality images with the PET/CT system available and the imaging protocol as described in section 7. With other imaging systems and protocols, the injected activity should be optimized.
<ol>
	<li>In advance, send the appointment and patient folder with information about 68Ga-DOTATATE PET/CT by mail to each patient. Confirm every appointment by telephone 1 day in advance.</li>
	<li>Food and drinks are not restricted prior to 68Ga-DOTATATE PET/CT imaging. Advise patients to drink an extra 1 L of water in the 2 h prior to imaging. It is also recommended patients do not bring children or pregnant women with them to the nuclear medicine department.</li>
	<li>On the day of the 68Ga-DOTATATE PET/CT, have the patients check in at the department of nuclear medicine 60 min prior to the imaging. Take a short medical history.</li>
	<li>Inquire about the date of the last somatostatin analog administration. This is not a contraindication for 68Ga-DOTATATE PET/CT but should be noted.</li>
	<li>Place an intravenous cannula in the arm and flush it with saline to verify the placement of the cannula.</li>
	<li>Inject 100 MBq of 68Ga-DOTATATE 45 min prior to the PET/CT imaging.</li>
</ol>
7. PET/CT imaging
<ol>
	<li>Place patient with the arms above their head on the PET/CT scanner. Instruct the patient to remain still throughout the exam.</li>
	<li>Acquire a survey image and select the scan area from the pituitary gland to the mid-thigh, unless otherwise specified due to clinical indications.</li>
	<li>Perform a low-dose CT scan with 40 mAs, 140 keV, and 5 mm slices for attenuation correction and anatomical correlation.</li>
	<li>Perform a PET scan with 150 s per bed position, starting at the head of the patient.</li>
	<li>Reconstruct the CT images with filtered back projection and 5 mm slices.</li>
	<li>Reconstruct the PET images with BLOB-OS-TF, with three iterations and 33 subsets with a voxel size of 4 mm x 4 mm x 4 mm.</li>
	<li>Send all images to the local picture archiving and communication system (PACS).</li>
</ol>

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P., 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=Localistion+of+endocrine-related+tumours+with+radioiodinated+analogue+of+somatostatin.">Localistion of endocrine-related tumours with radioiodinated analogue of somatostatin.</a> The Lancet. 333 (8632), 242-244 (1989).</li><li>Krenning, E. P., 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=Somatostatin+receptor+scintigraphy+with+[111In-DTPA-D-Phe1]-+and+[123I-Tyr3]-octreotide:+the+Rotterdam+experience+with+more+than+1000+patients.">Somatostatin receptor scintigraphy with [111In-DTPA-D-Phe1]- and [123I-Tyr3]-octreotide: the Rotterdam experience with more than 1000 patients.</a> European Journal of Nuclear Medicine. 20 (8), 716-731 (1993).</li><li>Balon, H. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=68Ga-DOTATATE+Compared+with+111In-DTPA-Octreotide+and+Conventional+Imaging+for+Pulmonary+and+Gastroenteropancreatic+Neuroendocrine+Tumors:+A+Systematic+Review+and+Meta-Analysis.">68Ga-DOTATATE Compared with 111In-DTPA-Octreotide and Conventional Imaging for Pulmonary and Gastroenteropancreatic Neuroendocrine Tumors: A Systematic Review and Meta-Analysis.</a> Journal of Nuclear Medicine. 57 (6), 872-878 (2016).</li><li>Yang, J., 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=Diagnostic+role+of+Gallium-68+DOTATOC+and+Gallium-68+DOTATATE+PET+in+patients+with+neuroendocrine+tumors:+a+meta-analysis.">Diagnostic role of Gallium-68 DOTATOC and Gallium-68 DOTATATE PET in patients with neuroendocrine tumors: a meta-analysis.</a> Acta Radiologica. 55 (4), 389-398 (2014).</li><li>Barrio, 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=The+Impact+of+Somatostatin+Receptor-Directed+PET/CT+on+the+Management+of+Patients+with+Neuroendocrine+Tumor:+A+Systematic+Review+and+Meta-Analysis.">The Impact of Somatostatin Receptor-Directed PET/CT on the Management of Patients with Neuroendocrine Tumor: A Systematic Review and Meta-Analysis.</a> Journal of Nuclear Medicine. 58 (5), 756-761 (2017).</li><li>Strosberg, J. 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The authors have nothing to disclose.

This protocol describes the production and subsequent PET/CT imaging of 68Ga-DOTATATE. In order for the efficient use of each produced batch of 68Ga-DOTATATE, an optimal workflow with strict timing is required. Since the half-life of 68Ga is 68 min, a relatively small time delay of 15 min leads to a 15% loss of radioactivity. This requires active communication between the production facility, the personnel administrating the dose to the patient, and the PET/CT technician. Also, patients s...

NETs are a heterogeneous group of tumors that arises from neuroendocrine cells. They can occur at almost any location in the body but are most common in the gastrointestinal tract, pancreas, and lung1. Although NETs are a rare disease, their incidence in the United States has risen from 1.09 per 100,000 people in 1973 to 6.98 per 100,000 people in 20122. For an accurate diagnosis and staging of a NET, 68Ga-DOTATATE PET/CT is the standard of care. This protocol describes the production and quality control of 68Ga-DOTATATE, as well as patient preparation and the acquisition of PET/CT images.
Well-differentiated NETs are characterized by an overexpression of somatostatin receptors1. Somatostatin analogs that bind to this receptor can be labeled with a radioactive isotope to allow for nuclear medicine imaging. At first, iodine-123 was used with gamma camera imaging, which was soon replaced by indium-111 (111In)3,4. 111In-octreotide scintigraphy was the golden standard for nuclear medicine NET imaging for over a decade5. Meanwhile, technical advances were made in PET, which has a higher sensitivity and resolution than gamma camera imaging. For NETs, somatostatin analogs coupled to the positron emitter 68Ga, such as 68Ga-DOTATATE, were developed6.
68Ga-somatostatin receptor (68Ga-SRS) PET/CT is the current modality of choice in nuclear medicine imaging of well-differentiated NETS. The superiority of 68Ga-SRS PET/CT over 111In-octreotide has been demonstrated in several studies7,8. The reported sensitivity and specificity lie around 90%-95% and 85%-100%, respectively9,10. A meta-analysis has shown that 68Ga-SRS PET/CT leads to a change in management in 44% of cases, even if preceded by 111In-octreotide scintigraphy11. In guidelines, 68Ga-SRS PET/CT is now recommended over 111In-octreotide scintigraphy for NET imaging, and it is also approved by the Food and Drug Administration and European Medicines Agency12. A guideline for tumor imaging with 68Ga-conjugated peptides is also available13.
This protocol details the radiolabeling of 68Ga-DOTATATE (conform the quality control requirements of the European Pharmacopoeia14), patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

<table border="0" cellpadding="0" cellspacing="0" style="width:713px;" width="713">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetonitrile</td>
			<td style="width:156px;">Biosolve</td>
			<td style="width:119px;">012007</td>
			<td style="width:223px;">&#62; 99.9 %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium acetate</td>
			<td style="width:156px;">Merck</td>
			<td style="width:119px;">101116</td>
			<td>&#8805; 98 %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aqua / Water for injections</td>
			<td style="width:156px;">Braun</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Automated labeling system</td>
			<td style="width:156px;">Scintomics</td>
			<td style="width:119px;"></td>
			<td>GRP 3V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C-18 cartridge</td>
			<td style="width:156px;">Waters</td>
			<td style="width:119px;">WAT023501</td>
			<td>Sep-Pak C18 Plus Light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dose calibrator</td>
			<td style="width:156px;">Veenstra Instruments</td>
			<td style="width:119px;"></td>
			<td>VIK-202-5051</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:156px;">Merck</td>
			<td style="width:119px;">324503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:156px;">Sigma Aldrich</td>
			<td style="width:119px;">32221-M</td>
			<td>&#8805; 99.8 %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ga-68 labeling kit</td>
			<td style="width:156px;">ABX</td>
			<td style="width:119px;">SC-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ge-68/Ga-68 generator</td>
			<td style="width:156px;">Eckert &#38; Ziegler</td>
			<td style="width:119px;"></td>
			<td>1850 MBq</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HA-DOTATATE</td>
			<td style="width:156px;">Scintomics</td>
			<td style="width:119px;">GRPC/R-000095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl 0.1M for elution</td>
			<td style="width:156px;">ABX</td>
			<td style="width:119px;">HCl-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:156px;">Sigma Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td>&#8805; 99.5 %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodine</td>
			<td style="width:156px;">Sigma Aldrich</td>
			<td style="width:119px;">207772</td>
			<td>&#8805; 99.8 %, solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ITLC-SG F254 plates</td>
			<td style="width:156px;">Merck</td>
			<td style="width:119px;">105735</td>
			<td>TLC Silica gel 60 F254</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ITLC-SG paper</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">SGI0001</td>
			<td>Glass fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:156px;">Sigma Aldrich</td>
			<td style="width:119px;">32213-M</td>
			<td>&#8805; 99.8 %, Ph. Eur.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Non-vented filter</td>
			<td style="width:156px;">Merck</td>
			<td>SLMPL25SS</td>
			<td>Millex-MP filter 0.22 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PET/CT</td>
			<td style="width:156px;">Philips</td>
			<td style="width:119px;"></td>
			<td>Gemini TOF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH indicator strips</td>
			<td style="width:156px;">Merck</td>
			<td style="width:119px;">109584</td>
			<td>MColorpHast (pH2.0-9.0)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptic soy broth medium</td>
			<td style="width:156px;">Biotrading</td>
			<td>K111F010QK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vented filter</td>
			<td style="width:156px;">Merck</td>
			<td style="width:119px;">SLGV0250S&#160;</td>
			<td>Cathivex GV 0.22 &#181;m</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Well counter</td>
			<td style="width:156px;">Canberra (now Mirion)</td>
			<td style="width:119px;"></td>
			<td style="width:223px;">Osprey Digital Tube Base MCA 
			Detector 76 BP76/3M-X</td>
		</tr>
	</tbody>
</table>

a practical guide for the production and pet/ct imaging of 68ga-dotatate for neuroendocrine tumors in daily clinical practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

Making use of an automated labeling system, 357 batches of 68Ga-DOTATATE were produced between December 2014 and October 2018. Of the 357 produced, 17 batches failed and 340 batches were released, leading to an overall success rate of 95.2%. Of the failed batches, 11 were caused by a technical failure, whilst in six cases, the produced 68Ga-DOTATATE did not meet specifications. Figure 1 shows a flow chart of produced batches and the numb...

Watch this Scientific Journal Video about A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice at JoVE.com

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

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Research

JoVE Journal

Cancer Research

17.2K Views.  Netherlands Cancer Institute. Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Video: A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

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 ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...