We thank Bas J. van Brakel and Roxanna Haak for their help in performing IOARM. Klara A. Bouman-Zevenbergen, Ian Overduin, and Silvy L. Sabiran - Singoredjo for their assistance and supportive role in ensuring logistics, equipment, and availability of personnel at the pathology department.

This study was approved by the institutional Medical Ethics Committee (MEC-2015-150).
NOTE: All the patient and personnel information in the figures or examples are fictional (i.e., XXXXX and YYYYY).
1. Before surgery
<ol>
	<li>Surgery department: Request for IOARM during the planning of surgery.</li>
	<li>Pathology department: Ensure logistics/equipment (see Table of Materials) and the availability of personnel (pathologist/pathology resident and assistant).</li>
</ol>
2. During surgery
<ol>
	<li>Operation room (OR)
	<ol>
		<li>Ensure that all involved personnel are familiar with the relocation protocol12.</li>
		<li>Follow the relocation protocol.</li>
		<li>Submerge the tags in chlorhexidine for at least 30 min before the start of the surgery.</li>
		<li>Place the tags paired on either side of the intended line of resection (both superficial and deep), so that one tag is on the resection specimen and the other remains at the corresponding spot in the wound bed (Figure 1A) as described by Van Lanschot et al.12.</li>
		<li>Cut between each pair of tags.</li>
		<li>Remove the specimen with the tumor (one tag from each pair remains in the wound bed, Figure 1B).</li>
		<li>Fill out the pathology request form with a clear indication of the anatomical location of the tags (e.g., tag 1 = anterior, tag 2 = superior).</li>
		<li>Record the surgical procedure-related defects of the specimen and their location in relation to the tags, on the pathology request form. 
		NOTE: Procedure-related defects create false resection surfaces and can lead to incorrect allocation of inadequate margins during both IOARM and final pathology.</li>
		<li>Bring the specimen to the pathology department.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62446/62446fig01.jpg" /> 
Figure 1: Illustration of the relocation protocol. (A) Application of tags in a pair-wise manner. (B) Wound bed and specimen both with one tag of each pair. <a href="https://www.jove.com/files/ftp_upload/62446/62446fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>IOARM - Grossing room (GR), pathology department
	<ol>
		<li>Rinse the specimen with water and gently pat it dry with gauze or paper. 
		&#8203;NOTE: Register every next step with photographs and store them in the Electronic Patient File (EPF).</li>
		<li>Record the general information (date, patient id, pathology number, surgeon, pathologist, type of specimen, and tags used) on the anatomical template.</li>
		<li>Indicate the locations of the tags on the anatomical template (Figure 2).</li>
		<li>Place the specimen on the anatomical template.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62446/62446fig02.jpg" /> 
Figure 2: Example of anatomical template for IOARM. <a href="https://www.jove.com/files/ftp_upload/62446/62446fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Ink the resection surface according to standard protocol (e.g., superior blue and inferior green).</li>
	<li>Inspect the specimen visually and by palpation (pathologist and surgeon).</li>
	<li>Indicate the location of any suspicious region (i.e., margin &#60;5 mm) on the anatomical template and relate it to the numbered tags (section Result of IOARM, Figure 2).</li>
	<li>Perform an incision perpendicular to the resection surface at the suspicious region (Figure 3A). Depending on the size of the specimen and/or suspicious regions, make one or more incisions with a distance of about 5 mm. In case of more than one incision, number the incisions as IOA1, IOA2, etc.</li>
	<li>Measure the margins (i.e., the distance between resection surface and tumor border) on the tissue sections (Figure 3B) and record the exact values in mm on the anatomical template (section Result of IOARM, Figure 3C). 
	&#8203;NOTE: If the tumor border is macroscopically not distinguishable (e.g., the tumor cannot be distinguished from surrounding fibrotic or salivary gland tissue), microscopic analysis by frozen section is indicated.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62446/62446fig03.jpg" /> 
Figure 3: Illustration of IOARM. (A) Perpendicular incision performed after identification of suspicious region by palpation. (B) The margin is measured. (C) The result of IOARM and the recommendation are recorded. <a href="https://www.jove.com/files/ftp_upload/62446/62446fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="10">
	<li>Proceed with the completion of the operation, step 2.2.19. If an adequate margin is detected (i.e., additional resection is not needed).</li>
	<li>Indicate the exact location based on the tags if an inadequate margin is detected and record it on the template. Proceed with an additional resection if surgically/technically achievable, step 2.2.13.</li>
	<li>Annotate the reason on the template, if an additional resection is not achievable (section Additional comments, Figure 2).</li>
	<li>Recommend (pathologist/surgeon) the additional resection based on the exact location and indicate the thickness needed to achieve an adequate resection (Figure 3C). 
	NOTE: If the inadequate margin concerns a positive margin, a minimal thickness of 6 mm should be recommended for the additional resection.</li>
	<li>Keep (pathologist) the main resection specimen in the refrigerator until the additional resection is received.</li>
	<li>Relocate (surgeon) the area of additional resection in relation to the tags, in the wound bed, based on the record of IOARM (Figure 3C).</li>
	<li>Perform the additional resection.</li>
	<li>Send the additional resection to the GR.</li>
	<li>Verify (pathologist) the accuracy of the additional resection regarding its location (based on tags) and its size. 
	NOTE: The above steps are applicable in the case of a close margin. In the case of a positive margin, an IOARM of the additional resection is necessary (pathologist). The surgeon waits for the result of the second IOARM before completing the operation.</li>
	<li>Remove (surgeon) the remaining tags from the wound bed and complete the operation.</li>
	<li>Copy (pathologist) all data from the anatomical template to the EPF.</li>
</ol>
3. After IOARM - Grossing room (GR), pathology department
NOTE: To preserve the anatomical orientation and shape of the specimen the following steps are performed.
<ol>
	<li>Reassemble the specimen by the correct orientation of all tissue sections (cross-sections and the polar ends) based on the tags and the photographs recorded during IOARM. 
	&#8203;NOTE: Cross-sections are in the middle of the specimen and the polar ends are the outer parts of the specimen.</li>
	<li>Cut the pieces of cork slightly larger than the tissue sections.</li>
	<li>Place each tissue section on a piece of cork.</li>
	<li>Draw a line on the cork around the tissue section with a permanent marker and take a photograph (Figure 4A).</li>
	<li>Place another piece of cork on top of all tissue sections except the polar ends (Figure 4B).</li>
	<li>Keep the upper and lower cork together, with the tissue section in between, by placing pins through both corks next to the edge of the tissue section, but not through the tissue section (Figure 4B).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62446/62446fig04.jpg" /> 
Figure 4: Illustration of the method to preserve the anatomical orientation and shape of tissue sections. (A) Tissue sections are placed on a piece of cork with a line drawn on the cork around the tissue section with a permanent marker. (B) Pins are obliquely placed over the polar ends and another piece of cork is placed over the tissue section. (C) Illustration of a reassembled fresh specimen kept together with pins that puncture the adjacent corks. (D) Illustration of a reassembled fixed specimen kept together with pins that puncture adjacent corks. <a href="https://www.jove.com/files/ftp_upload/62446/62446fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>Place the polar ends on a separate piece of cork (Figure 4A).</li>
	<li>Keep the polar ends attached to the cork by obliquely placing pins over the tissue and piercing the cork just beside the edge of the tissue (Figure 4B). 
	CAUTION: Do not puncture the specimen with the pins.</li>
	<li>Reassemble the whole specimen: put all the tissue sections including the polar ends together in the correct anatomical orientation.</li>
	<li>Keep all the tissue sections together by puncturing the adjacent corks (Figure 4C).</li>
	<li>Position the specimen with the correct orientation on the anatomical template and take a photograph.</li>
	<li>Place the specimen in formaldehyde solution (formalin 4%). 
	NOTE: For proper fixation, pieces of paper can be placed on top of the specimen to keep it submerged in formalin.</li>
	<li>Make a clear and visible warning note on the container with the specimen (e.g., caution needles/pins), to avoid accidents.</li>
	<li>Store the container with the specimen for further processing, according to the standard pathology protocol.</li>
</ol>
4. Grossing of the fixed specimen after IOARM
NOTE: After formalin fixation, the specimen should be grossed preferably by the pathologist/resident/assistant, who performed the IOARM.
CAUTION: Be careful with the needles/pins when removing the specimen from the container.
<ol>
	<li>Follow the institutional grossing protocol. 
	&#8203;NOTE: Take additional measures to ensure the correct orientation and to facilitate the comparison of the margin status between IOARM and final pathologic assessment.
	<ol>
		<li>Consult the pictures of the IOARM.</li>
		<li>Take the specimen out of the container.</li>
		<li>Check whether all the tissue sections are present.</li>
		<li>Position the specimen with the correct orientation on the anatomical template and take a photograph (Figure 4D).</li>
		<li>Remove the pins.</li>
		<li>Separate the individual tissue sections with corresponding cork.</li>
		<li>Take photographs of each tissue section with their corresponding corks, focusing on the lines that were drawn around the tissue section to assess possible shrinkage of tissue after fixation (Figure 5).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62446/62446fig05.jpg" /> 
Figure 5: Illustration of a polar end with the cut surface facing the cork, held against the cork by tilted pins. (A) Fresh specimen. (B) After fixation. (C) The cut surface of the polar end is flat after fixation. <a href="https://www.jove.com/files/ftp_upload/62446/62446fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="8">
	<li>Detach all tissue sections systematically from the cork (e.g., start from anterior to posterior or left to right).</li>
	<li>Gross the IOARM-tissue sections to the standard final tissue sections (2-3 mm thick).</li>
	<li>Place all final tissue sections, in the correct anatomical orientation (e.g., from anterior to posterior), on a paper on the grossing table.</li>
	<li>Number all the final tissue sections consecutively with a permanent marker on the paper (Figure 6).</li>
	<li>Annotate the location of IOARM with a permanent marker (Figure 6).</li>
	<li>Take photographs, including all final tissue sections and store them in the EPF (Figure 6).</li>
	<li>Select the relevant final tissue sections and IOARM sections to be further processed for final pathologic assessment.</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62446/62446fig06.jpg" /> 
Figure 6: Grossed specimen with the location of the IOARM marked. Corresponding numbers 1-5 refer to tissue sections from left to right. A-E corresponds with tissue sections included for histopathologic evaluation. Note that the remaining piece of tissue that was evaluated by frozen section (FS) is indicated to enable direct comparison with the permanent HE-stained section. <a href="https://www.jove.com/files/ftp_upload/62446/62446fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. The final pathologic assessment - Impact of IOARM on final margin status
<ol>
	<li>Follow the local standardized protocol. The protocol followed here is the PALGA (Pathologisch-Anatomisch Landelijk Geautomatiseerd Archief, the nationwide network and registry of histo- and cytopathology in the Netherlands) national Head-Neck protocol for the final standardized structured pathology report. 
	NOTE: This protocol is based on the up-to-date standards of the American Joint Committee on Cancer (AJCC), Union for International Cancer Control (UICC), and the World Health Organisation (WHO).
	<ol>
		<li>Assess all the margins in millimeters, including mucosa, submucosa, and bone.</li>
		<li>If an inadequate margin is found, annotate its extent (e.g., submucosal margin anterior is 3.5 mm, extending over a trajectory of 6 mm).</li>
		<li>Assess the presence of dysplasia and its grade for mucosal resection margins.</li>
		<li>Indicate the final margin by adding the dimensions of the additional resection (if performed) to the margin measured on the main specimen.</li>
		<li>Record the unique pathology number of the additional resection in the pathology report of the main specimen (e.g., Margins: anterior 6 mm, posterior 8 mm, superior 6 mm (including 3mm of the additional resection, H20-2021), inferior 7 mm, medial 5.3 mm).</li>
		<li>Verify the margins found during IOARM.</li>
		<li>Annotate the result of this verification (e.g., NB Margins found during the intraoperative assessment are in concordance with margins based on the final pathology). 
		NOTE: The time needed for IOARM should be limited in order to not interfere with the surgical workflow. At the institute, the IOARM takes about 10 min. The surgeon and pathologist perform the IOARM together. For the relocation method (placing the tags during surgery) an additional time of 5 min is needed. This will differ for each institute depending on the logistics.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The goal of surgical treatment of OCSCC patients is the complete removal of the tumor with adequate margins. This is too often not achieved, which inspired to design an adjusted approach to oral cancer surgery with a focus on intraoperative assessment of resection margins. Aside from resection margins, other adverse tumor factors such as the pattern of invasion, perineural invasion, and lymphovascular invasion also affect the local recurrence. However, of all adverse tumor factors, surgeons and pathologists can only influence the resection margins7,8,11.
The specimen-driven IOARM method was implemented in 2013; this was eventually supported by the evidence that specimen-driven IOARM is superior to defect-driven IOARM7,13,14,15,16,17. This resulted in its recommendation by AJCC in 201718. Noteworthy, the specimen-driven IOARM method became the standard of care in the institute in 2015. From 2013 until 2020 the IOARM was performed in 304 cases with a steep increase from 2018.
It is important to realize that developing and implementing an IOARM method involves many personnel (pathologists/surgeons/assistants/trainees/researchers), in order to make it standard of care. Many professionals were involved, during many years, in the development of this protocol, which is actually the strength of the method. The development of this method started in 2013 and reached a consensus in 2015. This was achieved based on the two-weekly meetings during which discussions regarding all the patients treated with surgery, including IOARM, took place. In this way, it was possible to timely adjust and refine the procedure. Besides, the two-weekly meetings enabled prospective data collection, which provides the basis for the performance and follow-up studies9. Moreover, for every case, the team ensured that the final pathology was not compromised due to IOARM. Finally, it is important to realize that this kind of assessment is a dynamic process and will always undergo changes toward improvement.
With the specimen-driven IOARM method, the margins are assessed by inspection, palpation, and perpendicular incisions (grossing). This approach provides an as accurate as possible estimation of margins in millimeters and enables feedback on whether an additional resection is needed and what the dimensions should be. Kubik et al. described several reasons (e.g., additional resection at an incorrect location, the incorrect orientation of the additional resection, incorrect dimensions of the additional resection) for additional resections to be inadequate17. The IOARM is a valuable method but only when accompanied by an as accurate as possible relocation method of inadequate margins to enable the surgeon to perform an adequate additional resection. The spatial relationship between the additional resection and the main specimen is the key factor. Therefore, a simple but elegant relocation method as shown in Figure 1 was developed and described in detail by Van Lanschot et al.12. This method allows the surgeon to perform an additional resection based on the relocation of the inadequate margin defined by the tags in the wound bed. For example, a margin of 2 mm is found between tags 1-2-3, the surgeon performs an additional resection around tags 1-2-3 with a thickness of 4 mm. This relocation method is shown to be effective by the results of Smits et al.9.
This IOARM method is supported by frozen section procedure only if the tumor cannot be distinguished macroscopically from surrounding tissue (e.g., fibrosis of tissue after radiotherapy or scar formation after previous surgery, or salivary gland tissue). Some institutes use another approach, in which frozen sections are taken from the specimen from all quadrants13,19. This method enables a more standardized protocol. However, the comprehensiveness of this method might not be always efficient. Moreover, multiple frozen sections are needed which is costly, time-consuming, and not accessible for all institutes. The described method is more efficient because the region of interest is preselected (i.e., region of suspicious inadequate margin) and is therefore cheaper, faster, and available for every institute. This is in accordance with previous findings that frozen section analysis does not improve the accuracy of specimen-driven IOARM based on grossing in most cases and is not cost-effective20,21,22.
According to the literature &#62;93% of all inadequate margins are found at the submucosal resection margins23. This is in line with the findings of the institute. Mucosal alterations with high-grade dysplasia/CIS are often easy to detect during IOARM, only in a few cases, a frozen section is recommended. Until now in the IOARM cohort, any mucosal positive margins regarding cancer or high-grade dysplasia/CIS have not been encountered.
Even though specimen-driven IOARM significantly improves the rate of adequate resections in OCSCC patients and consequently improves patient outcome7,9,22,21, its wide implementation is lagging. The main cause of this is the fact that the grossing of fresh tissue is counter-intuitive for pathologists. The pathologists are fearful that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen, and therefore will affect the final histopathologic assessment24,25. However, the measures prescribed in the protocol prevent these possible negative effects. Since the implementation of this protocol, the anatomical orientation, shape, and size of the specimen have never been altered nor was the final pathologic assessment ever compromised (manuscript in preparation).
Although little additional time is required to perform IOARM, it is clear that no real obstacles exist to implement IOARM, but there must be a willingness to go through a learning curve, regarding the grossing of fresh tissue and identifying inadequate margins. The most important prerequisite is a dedicated and cooperative team of surgeons and pathologists. In this study, an IOARM method for head and neck cancer surgery has been described, that can easily be implemented in every institute and during any other cancer surgery. This protocol significantly improves the rate of adequate resections while concomitantly reducing the need for postoperative radiotherapy and improving the patient outcome. The specimen-driven IOARM method will help surgeons to achieve first-time-right surgery and patients will benefit.

Every year, around 350,000 new patients are diagnosed worldwide with cancer in the oral cavity; 90% of cases are squamous cell carcinoma1. The mortality rate is 175,000 worldwide per year and the 5-year survival is 50% to 64.8%1,2,3,4.
The primary treatment of oral cavity squamous cell carcinoma (OCSCC) is surgery5. The goal of the surgery is the complete removal of the tumor with adequate margins, according to the Royal College of Pathologists6. Margins &#62;5 mm (clear) are regarded as adequate, whereas margins from 0-5 mm are regarded as inadequate.
Adequate resection margins lead to higher survival and a reduction in local recurrence-rates of OCSCC7,8,9. Tumor resections with inadequate margins result in the need for adjuvant therapy (postoperative radiotherapy and/or chemotherapy). This brings an additional burden for the patient, increasing morbidity and reducing the quality of life10. The resection margin is the only oncological prognostic factor that the surgeon and pathologist can influence.
Recent reports have shown that adequate resections are only achieved in 15%-26% of cases7,8,11. These poor results are caused by the complex anatomy of the oral cavity and the lack of intraoperative guidance. During surgery, the surgeon can only rely on inspection, palpation, and preoperative imaging.
The final margin status follows only several days after the operation. If an inadequate margin is encountered at the final pathologic assessment, a second operation is usually not an option, because the wound bed reconstruction has usually healed by that time. Moreover, a second operation is mostly not effective, because the relocation of the inadequate margin is even more difficult in the postoperative setting.
To overcome the lack of intraoperative information about margin status, specimen-driven intraoperative assessment of resection margins (IOARM) was implemented in 20139. It became the standard of care in the institute in 2015. Described here is the IOARM method in detail to enable colleagues at other institutes to implement this protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anatomical templates</td>
			<td></td>
			<td></td>
			<td>https://www.palga.nl/assets/uploads/Protocollen/HoofdHalstumoren.pdf</td>
		</tr>
		<tr>
			<td>Anatomical tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td>to apply the inc to the tissue</td>
		</tr>
		<tr>
			<td>Bucket for formalin fixation</td>
			<td></td>
			<td></td>
			<td>Size of the container depends on the size fo the tissue</td>
		</tr>
		<tr>
			<td>Buffered formalin 4%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cork</td>
			<td></td>
			<td></td>
			<td>Thin plates of cork</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Examination gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze or Paper</td>
			<td></td>
			<td></td>
			<td>That wont leave particles on the specimen</td>
		</tr>
		<tr>
			<td>Grossing knife 15cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Grossing knife 30cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Grossing tabel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inc for tissue</td>
			<td></td>
			<td></td>
			<td>3 or more different colors</td>
		</tr>
		<tr>
			<td>Labcoat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Long pins/Sewing pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paper</td>
			<td></td>
			<td></td>
			<td>To place the tissue sections on during the grossing</td>
		</tr>
		<tr>
			<td>Permanent markers</td>
			<td></td>
			<td></td>
			<td>Different colors (black/blue/red/green)</td>
		</tr>
		<tr>
			<td>Relocation tags</td>
			<td>Premier Farnell Limited BV, Utrecht, The Netherlands</td>
			<td></td>
			<td>Numbered froimn 0-9, cut to a size of&#160; 5 mm x 7 mm x 2 mm</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon</td>
			<td>Ethilon 3.0</td>
			<td>To suture in the tags</td>
		</tr>
		<tr>
			<td>Tap water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transparant ruler 30 cm</td>
			<td></td>
			<td></td>
			<td>2 rulers needed</td>
		</tr>
	</tbody>
</table>

intraoperative assessment of resection margins in oral cavity cancer: this is the way

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and appearance. For oral cavity squamous cell carcinoma (OCSCC), different studies showed that only 15%-26% of all resections are adequate. A major reason for the low number of adequate resections is the lack of information during surgery; the margin status is only available after the final histopathologic assessment, days after surgery.
The surgeons and pathologists at the Erasmus MC University Medical Center in Rotterdam started the implementation of specimen-driven intraoperative assessment of resection margins (IOARM) in 2013, which became the standard of care in 2015. This method enables the surgeon to turn an inadequate resection into an adequate resection by performing an additional resection during the initial surgery. Intraoperative assessment is supported by a relocation method procedure that allows accurate identification of inadequate margins (found on the specimen) in the wound bed.
The implementation of this protocol resulted in an improvement of adequate resections from 15%-40%. However, the specimen-driven IOARM is not widely adopted because grossing fresh tissue is counter-intuitive for pathologists. The fear exists that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen and therefore will affect the final histopathologic assessment. These possible negative effects are countered by the described protocol. Here, the protocol for specimen-driven IOARM is presented in detail, as performed at the institute.

Example of IOARM resulting in an adequate resection 
The patient presents with a cT2N0M0 SCC of the left side of the tongue with no medical history. The patient undergoes hemiglossectomy supported by IOARM. The specimen is inspected and palpated; the mucosal margins are measured as &#62;5 mm. One area in the submucosal resection surface is suspicious for an inadequate margin, located around tag 5. The submucosal margin is 3-4 mm at tag 5. All the information is recorded on the template and copied to the EPF (Figure 7A).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62446/62446fig07.jpg" /> 
Figure 7: Examples of two different IOARMs recorded on the anatomical template. (A) IOARM resulting in an adequate resection. (B) IOARM not resulting in an adequate resection. <a href="https://www.jove.com/files/ftp_upload/62446/62446fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The surgeon returns to the OR and performs the additional resection. The pathologist verifies the accuracy and dimensions, including the thickness of the additional resection.
The final pathology report shows the presence of moderately differentiated pT2 squamous cell carcinoma on the left side of tongue. The tumor diameter is 2.5 cm and the depth of invasion is 6.0 mm. The worst pattern of invasion (WPOI) is category 3. Perineural invasion (PNI) is not present and the lymphovascular invasion (LVI) is present. The minimal margins (mucosal and submucosal) at the inferior, superior, anterior, and posterior location are 5.8 mm (including additional resection (PA number: XXXXX) of 3 mm thickness), 6.2 mm (including additional resection (PA number: XXXXX) of 3 mm thickness), 5.2 mm, and 5.5 mm, respectively. IOARM is in concordance with final pathology.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Margins (mm)</td>
		</tr>
		<tr>
			<td>Location</td>
			<td>Based on IOARM</td>
			<td>After additional resection</td>
			<td>Based on Final pathology</td>
		</tr>
		<tr>
			<td>Inferior</td>
			<td>3-4</td>
			<td>6-7</td>
			<td>5.8</td>
		</tr>
		<tr>
			<td>Superior</td>
			<td>3-4</td>
			<td>6-7</td>
			<td>6.2</td>
		</tr>
		<tr>
			<td>Anterior</td>
			<td>&#62;5</td>
			<td></td>
			<td>5.2</td>
		</tr>
		<tr>
			<td>Posterior</td>
			<td>&#62;5</td>
			<td></td>
			<td>5.5</td>
		</tr>
	</tbody>
</table>
Table 1: Example of resection margins during IOARM resulting in an adequate resection at final pathology, after additional resection.
Example of IOARM not resulting in an adequate resection 
The patient presents with a cT1N0M0 SCC of the right side of the tongue with no medical history. The patient underwent a resection supported by IOARM. The surgeon takes the specimen to the pathologist at the pathology department. The mucosa is visually inspected, and the mucosal margins are measured with a transparent ruler, all mucosal margins are &#62;5 mm. The submucosal margins are visually inspected and palpated and all margins seem &#62;5 mm. A suspicious area is found at tag 1 (anterior resection surface) and tag 3 (posterior resection surface). A grossing knife is placed perpendicular to the resection surface from anterior to posterior (tag 1 to tag 3) and an incision is made. The pathologist measures the margin on the cross-section and the margins are &#62;5 mm. All the information is recorded on the template and copied to the EPF (Figure 7B).
The final pathology report shows a well-differentiated pT1 squamous cell carcinoma on the right side of the tongue. The diameter of the tumor is 1.8 cm, and the depth of invasion is 3.8 mm. The worst pattern of invasion (WPOI) is category 2. Perineural invasion (PNI), lymphovascular invasion (LVI), and dysplasia are not present. The minimal margins (mucosal and submucosal) at the inferior, superior, anterior, and posterior locations are 4.0 mm, 6.1 mm, 6.4 mm, and 7.8 mm, respectively. IOARM is not in concordance with final pathology, margin inferior was missed.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Margins (mm)</td>
		</tr>
		<tr>
			<td>Location</td>
			<td>Based on IOARM</td>
			<td>After additional resection</td>
			<td>Based on Final pathology</td>
		</tr>
		<tr>
			<td>Inferior</td>
			<td>&#62;5</td>
			<td rowspan="4">Not recommended all margins &#62; 5mm</td>
			<td>4.0</td>
		</tr>
		<tr>
			<td>Superior</td>
			<td>&#62;5</td>
			<td>6.1</td>
		</tr>
		<tr>
			<td>Anterior</td>
			<td>6</td>
			<td>6.4</td>
		</tr>
		<tr>
			<td>Posterior</td>
			<td>8</td>
			<td>7.8</td>
		</tr>
	</tbody>
</table>
Table 2: Example of resection margins during IOARM not resulting in an adequate resection at final pathology.

Watch this Scientific Journal Video about Intraoperative Assessment of Resection Margins in Oral Cavity Cancer: This is the Way at JoVE.com

Intraoperative Assessment of Resection Margins in Oral Cavity Cancer: This is the Way

The goal of this protocol is to provide a clear overview of specimen-driven intraoperative assessment of resection margins. It is encouraged to implement this protocol to improve patient care at other institutes.

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and ...

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3.6K Views.  Erasmus MC University Medical Center. The goal of this protocol is to provide a clear overview of specimen-driven intraoperative assessment of resection margins. It is encouraged to implement this protocol to improve patient care at other institutes.

Video: Intraoperative Assessment of Resection Margins in Oral Cavity Cancer: This is the Way

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Oral Health Assessment by Lay Personnel for Older Adults

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral health, including type II diabetes, heart disease, and atherosclerosis. Thus, assessments of oral health, called oral screenings, have a significant role in assessing risk of disease, managing disease, and even improving disease by oral care. Here we present a method to assess oral health quickly and consistently across time. The protocol is simple enough for non-oral health professionals such as students, family, and caregivers. Useful for any age of patient, the method is particularly key for older individuals who are often at risk of inflammation and chronic disease. Components of the method include existing oral health assessment scales and inventories, which are combined to produce a comprehensive assessment of oral health. Thus, oral characteristics assessed include intraoral and extraoral structures, soft and hard tissues, natural and artificial teeth, plaque, oral functions such as swallowing, and the impact this oral health status has on the patient&#39;s quality of life. Advantages of this method include its inclusion of measures and perceptions of both the observer and patient, and its ability to track changes in oral health over time. Results acquired are quantitative totals of questionnaire and oral screening items, which can be summed for an oral health status score. The scores of successive oral screenings can be used to track the progression of oral health across time and guide recommendations for both oral and overall health care.

The goal of this protocol is to enable non-dental professionals to assess oral health status for research or health-screening purposes. Aspects assessed include lips, tongue, soft and hard tissues, natural and artificial teeth, oral cleanliness, plaque, swallowing, and impact of oral health on quality of life.

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral ...

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been implicated in impacting long-term cancer outcomes, possibly through modulation of adrenergic-inflammatory responses that impact cancer cell behavior and immune cell function. Emerging evidence suggests that propofol-based total intravenous anesthesia (TIVA) may be beneficial for long-term cancer outcomes when compared to inhaled volatile anesthesia. However, the available clinical findings are inconsistent. Preclinical studies that identify the underlying mechanisms involved are critically needed to guide the design of clinical studies that will expedite insight. Most preclinical models of anesthesia have been extrapolated from the use of anesthesia in in vivo research and are not optimally designed to study the impact of anesthesia itself as the primary endpoint. This paper describes a method for delivering propofol-TIVA anesthesia in a mouse model of breast cancer resection that replicates key aspects of clinical delivery in cancer patients. The model can be used to study mechanisms of action of anesthesia on cancer outcomes in diverse cancer types and can be extrapolated to other non-cancer areas of preclinical anesthesia research.

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

Orthopedic Robot-Assisted Femoral Neck System in the Treatment of Femoral Neck Fracture

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm fluoroscopy to place the screw freehand and requires several guide wire adjustments, which increases the operation time and radiation exposure. Repeated drilling can also cause damage to the blood supply and bone quality of the femoral neck, which can be followed by complications such as screw loosening, nonunion, and femoral head necrosis. In order to make fixation more precise and reduce the incidence of complications, our team applied robot-assisted orthopedic surgery for screw placement using the femoral neck system to modify the traditional procedure. This protocol introduces how to import a patient's X-ray information into the system, how to perform screw path planning in software, and how the robotic arm assists in screw placement. Using this method, the surgeons can place the screw successfully the first time, improve the accuracy of the procedure, and avoid radiation exposure. The whole protocol includes the diagnosis of femoral neck fracture; the collection of intraoperative X-ray images; screw path planning in the software; precise placement of the screw under the assistance of the robotic arm by the surgeon; and verification of the implant placement.

This article introduces a method of robot-assisted orthopedic surgery for screw placement during the treatment of femoral neck fracture using the femoral neck system, which allows for more accurate screw placement, improved surgical efficiency, and fewer complications.

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm ...

Application of Laparoscopic Hepatectomy Combined with Intraoperative Microwave Ablation in Colorectal Cancer Liver Metastasis

Laparoscopic hepatectomy is a common treatment for colorectal cancer liver metastasis. Previously, a sufficient number of functional liver masses had to be maintained during laparoscopic hepatectomy, with a residual liver volume of &gt;40% in cirrhotic patients and &gt;30% in non-cirrhotic patients. The high incidence of complications such as bleeding, bile leakage, or liver failure due to the exposure and difficulty of the resection of specific liver segments such as S2 and S7 reduces the success rate of liver resection. At present, microwave ablation is mainly applied in the treatment of liver metastasis using a percutaneous approach, which makes it difficult to identify hidden parts or small lesions. For some liver segments, the percutaneous puncture of liver segment 7 (S7) is likely to pass through the thoracic cavity, and the percutaneous puncture of liver segment 2 (S2) adjacent to the diaphragm is likely to injure the diaphragm and heart; these issues restrict the application of percutaneous ablation in colorectal cancer liver metastasis. Considering multiple lesions, laparoscopic microwave ablation combined with hepatectomy was performed in this study. The location of the lesions was determined by contrast-enhanced ultrasound under laparoscopy, and small lesions that were difficult to detect before the operation were identified. For the scattered lesions, which had diameters less than 3 cm and were difficult to resect, ablation was adopted to substitute hepatectomy. This technique helped to more explicitly locate the tumors, simplified the operation procedures, reduced the risk of complications such as bleeding and bile leakage, shortened the operation time, accelerated the postoperative recovery, significantly improved the success rate of operation, and enhanced the clinical prognosis of colorectal cancer liver metastasis by surgical resection.

This protocol describes the laparoscopic resection of colorectal cancer liver metastases combined with ultrasound-guided microwave ablation. This technique can safely, effectively, and accurately treat refractory liver metastases &lt;3 cm, reduce postoperative complications, and accelerate the postoperative rehabilitation of patients.

Laparoscopic hepatectomy is a common treatment for colorectal cancer liver metastasis. Previously, a sufficient number of functional liver masses had to ...

Clinical Application of Single-Surgeon, Three-Port, Laparoscopic Resection for Colorectal Cancer with Natural Orifice Specimen Extraction

Reduced-port laparoscopic surgery (RPLS) has been widely used for the radical resection of gastrointestinal tumors. Single-surgeon, three-port, laparoscopic radical resection for sigmoid colon or high rectal cancer with natural orifice specimen extraction surgery (NOSES) has the advantage of a small incision, quick postoperative recovery, and short hospital stay. Yet, there are still only a few reports on NOSES. This paper describes the indications, preoperative preparations, surgical steps, and precautions for single-surgeon, three-port, laparoscopic radical resection of the sigmoid colon and high rectal cancer, and intraoperative specimen collection through the natural orifice. 
The protocol focuses on the steps of radical dissection and the main technical points of resection and reconstruction. At the same time, a procedure for fixing an anvil seat by self-traction of extracorporeal silk thread, used for purse-string suture fixation after the proximal anvil was placed in the abdominal cavity, was creatively improved. This operation could effectively avoid problems such as an insufficient proximal intestinal tube, shaking off the anvil seat, and weak purse-string suture during a single operation. The surgical care had less variability and was easy to perform, effectively avoiding postoperative anastomotic leakage and bleeding due to excessive intraoperative anastomotic tissue. This surgery could be widely promoted in primary hospitals.

Here, we present a protocol to successfully perform single-surgeon, three-port, laparoscopic resection for colorectal cancer with natural orifice specimen extraction.

Reduced-port laparoscopic surgery (RPLS) has been widely used for the radical resection of gastrointestinal tumors. Single-surgeon, three-port, ...

Capturing Oral Submucous Fibrosis Tissue Samples for Spatial-Omics Analysis

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Author Spotlight: Unlocking the Mysteries of Oral Potential Malignancies 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...