The authors have no acknowledgements.

Data on all patients treated with EBUS-guided cisplatin were reviewed. The institutional review board at the University of Florida approved this study (#IRB201400823). All data was prospectively collected and retrospectively analyzed. Use the following enrollment criteria.
1. Patient Selection
<ol>
	<li>Select patients between the ages of 18 and 80 years who have a biopsy-confirmed non-small cell lung cancer (NSCLC) or small cell lung carcinoma (SCLC) and have a pathologically-confirmed recurrence. Enroll patients who have received at least 50 gy of radiation to the hilar and mediastinal structures for 6 months or longer. Include patients with limited recurrence to the hilar, mediastinal, and peribronchial structures (lymph nodes, nodules, and masses) accessible through EBUS and distant metastases.</li>
	<li>Present all such patients to a multi-disciplinary thoracic oncology tumor board and obtain consensus on proceeding with the intratumoral cisplatin due to a lack of other localized therapeutic options. The decision to proceed with EBUS-guided ITC should be a joint recommendation of the institutional thoracic oncology tumor board.</li>
</ol>
2. Endobronchial Ultrasound (EBUS)-guided Cisplatin Injection
<ol>
	<li>Use a convex-probe EBUS, which has a built-in ultrasound probe on a flexible bronchoscope and enables real-time visualization of hilar, mediastinal, and peribronchial structures for ITC.</li>
	<li>Ensure that the patients abstain from oral food and fluids for at least 6 h prior to the procedure.</li>
	<li>Create an aqueous cisplatin solution at a concentration of 1 mg/mL with a maximal dose of 40 mg per session, based on previously-published literature9,10. Dissolve lyophilized cisplatin powder in 0.9% NaCl solution just before use.</li>
	<li>Give 10 mg of dexamethasone and 8 mg of ondansetron intravenously at the beginning of the procedure to prevent nausea.</li>
	<li>Perform the procedure with conscious sedation using midazolam and fentanyl intravenously (IV). Use topical anesthesia in the form of 5 mL of 4% lidocaine nebulization prior to the procedure.</li>
	<li>Start out with an IV injection of 2 mg of midazolam and 50 &#181;g of fentanyl. Reassess the patient every 1-2 min and give an additional 1 mg midazolam and 25 &#181;g fentanyl bolus to achieve and maintain conscious sedation (a minimally depressed consciousness such that the patient is able to continuously and independently maintain a patent airway, retain protective reflexes, and remain responsive to verbal commands and physical stimulation). Do not exceed the maximal permissible doses of fentanyl and midazolam, 200 &#181;g and 10 mg, respectively.</li>
	<li>Use standard monitoring, including a continuous 3-lead electrocardiogram monitoring the heart rate, continuous pulse oximetry, and respiratory rate measurements. Provide local anesthesia and supplemental O2 as required.</li>
	<li>&#160;Advance the scope through the vocal cords in the airways.&#160;Identify and locate the target lesion.</li>
	<li>Turn on the EBUS processor and inflate the balloon at the tip of the scope using 1-2 mL of normal saline. Flex the scope across the airway wall and locate the lesion. The location of the lesion will depend on CT/PET findings, but some of the common sites include the subcarinal area, the right and left paratracheal areas, and the right and left hilar areas.</li>
	<li>Deploy a 22 G EBUS needle that is housed in a sheath through the working channel of the scope and lock it in position. 
	NOTE: Although there are no precise surgical coordinates for the injection site, some of the commonly-treated sites include the subcarinal (medially at the junction of the left and right main bronchi), the right paratracheal (laterally at the right main bronchus, just above the right upper lobe orifice), the left paratracheal (at the junction of the distal trachea and the left main between the arch of the aorta and the left pulmonary artery), the right hilar (laterally at the proximal bronchus intermedius, below the right upper lobe opening), and the left hilar (at the junction of the left upper lobe and the left lower lobe bronchi).</li>
	<li>Under real-time ultrasound guidance, puncture the tracheobronchial wall and place the needle in the target lesion. Remove the stylet within the needle and inject cisplatin into the lesion 10 mL (1 mg/mL of cisplatin) at a time. Retract the needle within the sheet.</li>
	<li>Treat each lesion with four punctures per session at different locations to facilitate the injection of the medication throughout. Inject 10 mg of cisplatin per puncture.</li>
	<li>Treat one to two lesions per session. If more than one lesion is treated per session, inject each site with 20 mg of cisplatin, with total dose per session not exceeding 40 mg.</li>
	<li>Remove the needle from the working channel of the scope.</li>
	<li>Suction any additional drug from the distal airways and remove the bronchoscope.</li>
	<li>Allow the patients to recover as per hospital protocol and discharge them on the same day.</li>
	<li>Repeat the cisplatin injection (steps 2.7-2.13) once a week for a total of 4 weeks (on days 1, 8, 15, and 22). 
	NOTE: Once treated, a particular lesion should never be considered for retreatment with ITC on subsequent encounters.</li>
</ol>
3. Post-injection
<ol>
	<li>Evaluate the response by a follow-up chest computed tomography (CT) or a positron emission tomography (PET/CT) scan 8-12 weeks after the last treatment session. Define the local recurrence as the recurrence at the site of treatment and the regional recurrence as the recurrence in the mediastinum, hilum, or supraclavicular fossa.</li>
</ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Agency for Research on Cancer. , (2012).</li><li>Siegel, R., Ma, J., Zou, Z., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2014.">Cancer statistics, 2014.</a> CA Cancer J Clin. 64 (1), 9-29 (2014).</li><li>Jeremic, B., Videtic, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chest+reirradiation+with+external+beam+radiotherapy+for+locally+recurrent+non-small-cell+lung+cancer:+a+review.">Chest reirradiation with external beam radiotherapy for locally recurrent non-small-cell lung cancer: a review.</a> Int J Radiat Oncol Biol Phys. 80 (4), 969-977 (2011).</li><li>Perez, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+irradiation+technique+and+tumor+extent+in+tumor+control+and+survival+of+patients+with+unresectable+non-oat+cell+carcinoma+of+the+lung:+report+by+the+Radiation+Therapy+Oncology+Group.">Impact of irradiation technique and tumor extent in tumor control and survival of patients with unresectable non-oat cell carcinoma of the lung: report by the Radiation Therapy Oncology Group.</a> Cancer. 50 (6), 1091-1099 (1982).</li><li>Hainsworth, J. D., 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=Weekly+docetaxel+with+either+gemcitabine+or+vinorelbine+as+second-line+treatment+in+patients+with+advanced+nonsmall+cell+lung+carcinoma:+Phase+II+trials+of+the+Minnie+Pearl+Cancer+Research+Network.">Weekly docetaxel with either gemcitabine or vinorelbine as second-line treatment in patients with advanced nonsmall cell lung carcinoma: Phase II trials of the Minnie Pearl Cancer Research Network.</a> Cancer. 92 (9), 2391-2398 (2001).</li><li>Grossi, F., 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=Phase+II+study+of+irinotecan+and+docetaxel+in+patients+with+previously+treated+non-small+cell+lung+cancer:+an+Alpe-Adria+Thoracic+Oncology+Multidisciplinary+group+study+(ATOM+007).">Phase II study of irinotecan and docetaxel in patients with previously treated non-small cell lung cancer: an Alpe-Adria Thoracic Oncology Multidisciplinary group study (ATOM 007).</a> Lung Cancer. 52 (1), 89-92 (2006).</li><li>Celikoglu, F., Celikoglu, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoural+chemotherapy+with+5-fluorouracil+for+palliation+of+bronchial+cancer+in+patients+with+severe+airway+obstruction.">Intratumoural chemotherapy with 5-fluorouracil for palliation of bronchial cancer in patients with severe airway obstruction.</a> J Pharm Pharmacol. 55 (10), 1441-1448 (2003).</li><li>Eisenhauer, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+response+evaluation+criteria+in+solid+tumours:+revised+RECIST+guideline+(version+1.1).">New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).</a> Eur J Cancer. 45 (2), 228-247 (2009).</li><li>Celikoglu, F., Celikoglu, S. I., York, A. M., Goldberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+administration+of+cisplatin+through+a+bronchoscope+followed+by+irradiation+for+treatment+of+inoperable+non-small+cell+obstructive+lung+cancer.">Intratumoral administration of cisplatin through a bronchoscope followed by irradiation for treatment of inoperable non-small cell obstructive lung cancer.</a> Lung Cancer. 51 (2), 225-236 (2006).</li><li>Celikoglu, S. I., Celikoglu, F., Goldberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endobronchial+intratumoral+chemotherapy+(EITC)+followed+by+surgery+in+early+non-small+cell+lung+cancer+with+polypoid+growth+causing+erroneous+impression+of+advanced+disease.">Endobronchial intratumoral chemotherapy (EITC) followed by surgery in early non-small cell lung cancer with polypoid growth causing erroneous impression of advanced disease.</a> Lung Cancer. 54 (3), 339-346 (2006).</li><li>Kilburn, J. 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=Management+of+mediastinal+relapse+after+treatment+with+stereotactic+body+radiotherapy+or+accelerated+hypofractionated+radiotherapy+for+stage+I/II+non-small-cell+lung+cancer.">Management of mediastinal relapse after treatment with stereotactic body radiotherapy or accelerated hypofractionated radiotherapy for stage I/II non-small-cell lung cancer.</a> J Thorac Oncol. 9 (4), 572-576 (2014).</li><li>Goldberg, E. P., Hadba, A. R., Almond, B. A., Marotta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+cancer+chemotherapy+and+immunotherapy:+opportunities+for+nonsystemic+preoperative+drug+delivery.">Intratumoral cancer chemotherapy and immunotherapy: opportunities for nonsystemic preoperative drug delivery.</a> J Pharm Pharmacol. 54 (2), 159-180 (2002).</li><li>Sandler, A. B., 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=Phase+III+trial+of+gemcitabine+plus+cisplatin+versus+cisplatin+alone+in+patients+with+locally+advanced+or+metastatic+non-small-cell+lung+cancer.">Phase III trial of gemcitabine plus cisplatin versus cisplatin alone in patients with locally advanced or metastatic non-small-cell lung cancer.</a> J Clin Oncol. 18 (1), 122-130 (2000).</li><li>Ciotti, R., 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=Prospective+evaluation+of+anthracycline-related+early+cardiac+damage:+how+do+we+monitor+it.">Prospective evaluation of anthracycline-related early cardiac damage: how do we monitor it.</a> J Clin Oncol. 19 (22), 4269-4270 (2001).</li><li>Burris, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+cisplatin/epinephrine-injectable+gel+as+a+palliative+treatment+for+accessible+solid+tumors:+a+multicenter+pilot+study.">Intratumoral cisplatin/epinephrine-injectable gel as a palliative treatment for accessible solid tumors: a multicenter pilot study.</a> Otolaryngol Head Neck Surg. 118 (4), 496-503 (1998).</li><li>Vogl, T. 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=CT-guided+intratumoural+administration+of+cisplatin/epinephrine+gel+for+treatment+of+malignant+liver+tumours.">CT-guided intratumoural administration of cisplatin/epinephrine gel for treatment of malignant liver tumours.</a> Br J Cancer. 86 (4), 524-529 (2002).</li><li>Monga, S. 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=Endoscopic+treatment+of+gastric+cancer+with+intratumoral+cisplatin/epinephrine+injectable+gel:+a+case+report.">Endoscopic treatment of gastric cancer with intratumoral cisplatin/epinephrine injectable gel: a case report.</a> Gastrointest Endosc. 48 (4), 415-417 (1998).</li><li>Monga, S. 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=Intratumoral+therapy+of+cisplatin/epinephrine+injectable+gel+for+palliation+in+patients+with+obstructive+esophageal+cancer.">Intratumoral therapy of cisplatin/epinephrine injectable gel for palliation in patients with obstructive esophageal cancer.</a> Am J Clin Oncol. 23 (4), 386-392 (2000).</li><li>Shepherd, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+randomized+trial+of+docetaxel+versus+best+supportive+care+in+patients+with+non-small-cell+lung+cancer+previously+treated+with+platinum-based+chemotherapy.">Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy.</a> J Clin Oncol. 18 (10), 2095-2103 (2000).</li><li>Hanna, N., 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=Randomized+phase+III+trial+of+pemetrexed+versus+docetaxel+in+patients+with+non-small-cell+lung+cancer+previously+treated+with+chemotherapy.">Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy.</a> J Clin Oncol. 22 (9), 1589-1597 (2004).</li></ol>

The authors have nothing to disclose.

Our manuscript focuses on the management of isolated mediastinal and hilar recurrences of lung cancer for patients in whom more radiation is not an option. The incidence of IMHR is approximately 9%11. Different treatment options have been tried in these patients, including palliative chemotherapy, more external beam radiation therapy, or supportive care and surveillance. At our institution, we treat these patients with intratumoral cisplatin guided by bronchoscopy.
There has been a growing interest in intratumoral chemotherapy and immunotherapy over the last several years. One of the potential advantages of injecting chemotherapeutic agents into the tumor mass is that a significantly higher concentration (6-10 times) of the medication can be achieved in the tumor mass12. Cisplatin is commonly used for the treatment of lung cancer as a part of combination systemic chemotherapy13,14,and it has also been used intralesionally for head, neck, and malignant liver cancers15,16. Additionally, it has been utilized endoscopically for palliation of obstructive esophageal cancers17 and injected into gastric tumors18. Based on available evidence and experience, we chose to use cisplatin as the chemotherapeutic agent of choice for bronchoscopic ITC.
The critical step in this protocol involves the proper patient selection. All patients should demonstrate in-field recurrence from a previous disease, should present isolated disease in the chest, and should be discussed at a multidisciplinary tumor board to confirm their limited therapeutic options. From a procedural perspective, the ability to identify the site of recurrent disease on EBUS and to advance the needle reliably into the diseased node is important. Cisplatin should be injected into the tumor in 10 mL aliquots. An attempt must be made to inject 40 mg in four separate passes at different areas on the node. After injecting the drug, excess cisplatin should be suctioned from the distal airways. Due to concerns for developing bronchopleural fistula, concurrent radiation therapy to the same site is not recommended. Up to 2 sites can be treated in the same session. If more than one site is treated, each site should be injected with 20 mg of cisplatin.
In patients with stage III or stage IV NSCLC previously treated with chemotherapy who are treated with second-line chemotherapy, the median survival time is 4-10 months19,20. However, no studies to date have looked into the survival among patients with isolated hilar and mediastinal recurrence of lung cancer following full-dose radiation therapy. The overall median survival of 8 months in our cohort is comparable to the number quoted in existing literature and would suggest a survival benefit compared to best supportive care only. Additionally, the localized cisplatin injection is tolerated much better and has fewer side effects as compared to systemic chemotherapy.
There are several limitations to our study, including retrospective design. Although we have a very well-structured protocol, its performance outside of our institution has not been studied. Serum and tumor concentration of the drug were not analyzed, and some patients were receiving systemic therapy, which could have confounded the results. However, we believe that the response seen in our cohort can largely be attributed to localized cisplatin injection7,12.
EBUS-guided intratumoral injection of cisplatin is feasible, well-tolerated, and potentially safe and effective in patients with isolated hilar and mediastinal recurrence of lung cancer for whom no localized therapeutic options are available. Multicenter randomized trials should be conducted to further validate our findings.

Lung cancer is the most prevalent cancer worldwide in both men and women and accounts for 1 in 5 of all cancer-related deaths. It has a case fatality rate of 0.871,2. Recurrence after initial treatment accounts for most deaths in patients with lung cancer. Therapeutic options for recurrent lung cancer are limited. Recurrent lung cancer also leads to significant impairment in quality of life and to significant caregiver burnout and requires additional supportive care, all of which decrease the likelihood of acceptance and tolerance to further interventions3.
Nearly 30% of patients with NSCLC have locoregional recurrence after radiation4. Treatment options for these patients are limited and include systemic chemotherapy, which has an objective response rate of only 10%5,6. Although repeat chest radiation is feasible, it has been studied mainly for palliative purposes and symptom relief and has not been used for disease remission3.
At our institution, all patients with isolated mediastinal and hilar recurrence (IMHR) accessible through bronchoscopy were treated with an endobronchial ultrasound (EBUS)-guided transbronchial intratumoral injection of cisplatin (ITC). Concurrent treatment with systemic chemotherapy and/or external beam radiation therapy (EBRT) was allowed as per the treating oncologists&#39; discretions. This manuscript highlights the safety, efficacy, feasibility, and detailed methods of the protocol used at our institution.

<table border="0" cellpadding="0" cellspacing="0" width="261">
	<tbody>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;width:261px;">Bronchoscope</td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">22 Guage olympus EBUS needle</td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">40 g of cisplatin in 40 mls of normal saline</td>
		</tr>
	</tbody>
</table>

endobronchial ultrasound-guided intratumoral injection of cisplatin for the treatment of isolated mediastinal recurrence of lung cancer

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients do not have many treatment options and are usually offered palliative chemotherapy or best supportive care. Endobronchial ultrasound (EBUS)-guided intratumoral injection of cisplatin (ITC) is a novel approach for these patients. The procedure is performed under conscious sedation. The lesion is located with a bronchoscopy using EBUS, and a 22-gauge EBUS needle is advanced through the working channel of the scope and locked in position. Under ultrasound guidance, the wall of the tracheobronchial tree is punctured and the needle is moved into the target lesion. The needle stylet is then removed and cisplatin (40 mg/40 mL) is injected into the lesion. One to two sites are treated per session. Details of the procedure are described in the protocol section of paper. At our center, 50 sites were treated in 36 patients (19 males, 17 females). The mean age of our cohort was 61.9 &#177;8.5 years. We performed final analyses on 35 patients and 41 sites. 24/35 (69%) had complete or partial response (responders), whereas 11/35 (31%) had stable or progressive disease (non-responders). Overall, survival in our group was 8 months (95% CI of 6-11 months), with patients who responded having significantly better survival than the ones who did not.

The response was measured by follow-up imaging 8-12 weeks after therapy. The response was classified as complete remission (CR), partial remission (PR), stable disease (SD), progressive disease (PD), or unable to assess response based on RECIST 1.1 criteria8. Patients with CR and PR were considered responders, and the others were classified as non-responders. Secondary outcomes included response based on tumor histology, size of recurrence, and concurrent systemic therapy. The side effects and feasibility of the protocol were also analyzed.
Kaplan-Meier curves were used to estimate the survival probabilities. The survival and PFS for responders versus non-responders was compared by the log-rank test. Associations between response rates and other factors were examined using Fisher&#39;s exact test.
Figure 1 shows a detailed distribution of patients, sites, and site-specific responses. The final analysis included 35 patients and 41 sites. 69% of the patients were classified as responders (24/35), and 31% were non-responders (11/35). Table 1 shows the outcomes of the treatment based on various sub-group analyses. The median survival for the group was 8 months (95% CI of 6-11 months): 10 months for responders (95% CI of 8-13 months) and 6 months for non-responders (95% CI of 3-10 months); this difference was statistically significant, with a p-value of 0.029 (Figure 2). Figure 3 shows the difference in PFS between responders and non-responders.
<img alt="Figure 1" src="/files/ftp_upload/54855/54855fig1.jpg" /> 
Figure 1: Number of Patients Analyzed and Their Outcomes. A detailed flow diagram of the number of patients and sites treated initially and the number of patients and sites presented here for final analysis. <a href="http://cloudflare.jove.com/files/ftp_upload/54855/54855fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54855/54855fig2.jpg" /> 
Figure 2: Kaplan-Meier Curves for Overall Survival in Months Amongst Responders and Non-responders. These curves show that patients who responded to therapy demonstrated statistically-significant overall survival as compared to non-responders. <a href="http://cloudflare.jove.com/files/ftp_upload/54855/54855fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54855/54855fig3.jpg" /> 
Figure 3: Kaplan-Meier Curves for Progression-free Survival in Months Amongst Responders and Non-responders. Patients who responded to treatment had statistically-significant improvements in progression-free survival. <a href="http://cloudflare.jove.com/files/ftp_upload/54855/54855fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td>Responders</td>
			<td>Non Responders</td>
			<td>p value</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Histology</td>
			<td></td>
			<td></td>
			<td>0.84</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Adenoca</td>
			<td>13 (72%)</td>
			<td>5 (28%)</td>
			<td></td>
		</tr>
		<tr>
			<td>-</td>
			<td>Squamous</td>
			<td>7 (58%)</td>
			<td>5 (42%)</td>
			<td></td>
		</tr>
		<tr>
			<td>-</td>
			<td>Small cell</td>
			<td>3 (75%)</td>
			<td>1 (25%)</td>
			<td></td>
		</tr>
		<tr>
			<td>-</td>
			<td>Large cell</td>
			<td>1 (100%)</td>
			<td>0</td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>Concurrent systemic Rx</td>
			<td></td>
			<td></td>
			<td>1</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Chemo</td>
			<td>5 (63%)</td>
			<td>3 (37%)</td>
			<td></td>
		</tr>
		<tr>
			<td>-</td>
			<td>Radiation</td>
			<td>2 (100%)</td>
			<td>0</td>
			<td></td>
		</tr>
		<tr>
			<td>-</td>
			<td>None</td>
			<td>17 (68%)</td>
			<td>8 (32%)</td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>Size of lesion</td>
			<td></td>
			<td></td>
			<td>0.066</td>
		</tr>
		<tr>
			<td>-</td>
			<td>&#60;=1cm</td>
			<td>8 (89%)</td>
			<td>1 (11%)</td>
			<td></td>
		</tr>
		<tr>
			<td>-</td>
			<td>1.1-2 cm</td>
			<td>10 (83%)</td>
			<td>2 (17%)</td>
			<td></td>
		</tr>
		<tr>
			<td>-</td>
			<td>2.1-3 cm</td>
			<td>3 (43%)</td>
			<td>4 (57%)</td>
			<td></td>
		</tr>
		<tr>
			<td>-</td>
			<td>&#62;3 cm</td>
			<td>3 (43%)</td>
			<td>4 (57%)</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5">Table 1: Response to ITC with cisplatin based on histology, concurrent systemic therapy and size</td>
		</tr>
	</tbody>
</table>
Table 1: Sub-group Analyses of Response to Cisplatin Based on a Few Select Variables. There was no statistically-significant difference in patient response based on tumor histology, size, or concurrent systemic therapy.

Watch this Scientific Journal Video about Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer at JoVE.com

Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer

The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field.

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients ...

endobronchial-ultrasound-guided-intratumoral-injection-cisplatin-for

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.3K Views.  University of Florida College of Medicine. The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field. 

Video: Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane permeabilization by means of &#34;electroporation&#34;. To achieve the &#34;electroporation&#34; of the cells, electric pulses are generated by a generator and delivered to the target tissue by the use of electrodes. Electroporation is a physical method which is used to introduce molecules, like cytostatic drugs, into the cells that could not pass the cell membrane on their own. In electrochemotherapy, currently, cisplatin and bleomycin are clinically used. Electrochemotherapy antitumor effectiveness is high, for example up to 100% complete response of canine mast cell tumors smaller than 2 cm3 was achieved. Additionally, electrochemotherapy can be used for the treatment of inoperable tumors. One of the important characteristics of electrochemotherapy is that it can be effective as a one-time treatment only. However, in the case of failure or partial tumor response it can be repeated several times with equal or improved effectiveness. Electrochemotherapy is already a standard treatment for cutaneous and subcutaneous tumors of various histologies in human and veterinary oncology. Furthermore, several clinical studies exploiting electrochemotherapy for deep-seated tumors are on-going.

Electrochemotherapy is the local treatment of solid tumors. The main mechanism is electroporation-mediated permeabilization of the tumor cells' membrane which enables increased internalization of cytostatics like bleomycin and cisplatin. Thus, antitumor effectiveness is potentiated at the site of the application of electric pulses to the tumors.

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% of patients. Surgery has been widely used in association with radiofrequency, cryotherapy, or microwaves to expand the number of treatments performed with a curative intent. Nevertheless, several limitations have been documented when using these techniques (i.e., a traumatic puncture of the parenchyma, a limited size of lesions, and an inability to monitor the treatment in real-time).High-intensity focused ultrasound (HIFU) technology can achieve precise ablations of biological tissues without incisions or radiation. Current HIFU devices are based on an extracorporeal approach with limited access to the liver. We have developed a HIFU device designed for intraoperative use. The use of a toroidal transducer enables an ablation rate (10 cm3&#183;min-1) higher than any other treatment and is independent of perfusion.
The feasibility, safety, and accuracy of intraoperative HIFU ablation were evaluated during an ablate-and-resect prospective study. This clinical phase I and IIa study was performed in patients undergoing hepatectomy for liver metastases. The HIFU treatment was performed on healthy tissue scheduled for resection.Liver metastases measuring less than 20 mm will be targeted during phase IIb (currently ongoing). This set-up allows the real-time evaluation of HIFU ablation while protecting participating patients from any adverse effects related to this new technique.
Fifteen patients were included in phase I&#8211;IIa and 30 HIFU ablations were safely created within 40 s and with a precision of 1&#8211;2 mm. The average dimensions of the HIFU ablations were 27.5 x 21.0 mm2, corresponding to a volume of approximately 7.5 cm3. The aim of the ongoing phase IIb is to ablate metastases of less than 20 mm in diameter with a 5 mm margin.

Here, we present an ablate-and-resect prospective study to evaluate the feasibility, safety, and accuracy of intraoperative high-intensity focused ultrasound ablation in patients undergoing hepatectomy for liver metastases.

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Perfusion and Inflation of the Mouse Lung for Tumor Histology

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform necropsies rapidly and efficiently from studies without sacrificing the quality of the tissues procured. The goal of this protocol is to present a method to rapidly perfuse, inflate, and fix mouse lungs for downstream histological analysis. This method does not standardize lung inflation; thus, it does not require any special procedures or equipment and instead simply instills fixative directly through the trachea following perfusion through the heart. This allows for sufficient estimation of tumor size, histology, and scoring. This also allows for the collection of frozen tissue prior to lung tissue fixation. This method is limited in that it does not allow for later morphometric quantification of the lung; however, it is more than sufficient for lung tumor analysis from genetically engineered mouse models (GEMMs), syngeneic models, as well as xenograft tumor and metastasis studies.

The purpose of this method is to present a simple and efficient method for the perfusion, inflation, and fixation of mouse lungs for the examination of lung tumor pathology and evaluation of metastases to the lung.

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...