The authors are grateful to Dr Liesbeth Boersma and colleagues at the Maastro Clinic for their advice on the voluntary breath-hold technique. The authors are also very grateful to the Pink Ribbon Foundation for funding the dissemination of the VBH technique. This article presents independent research funded by the National Institute for Health Research (NIHR) under its Research for Patient Benefit (RfPB) Programme (Grant Reference Number PB-PG-1010-23003). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The work was undertaken in The Royal Marsden NHS Foundation Trust which receives a proportion of its funding from the NHS Executive. We acknowledge NHS funding to the NIHR Biomedical Research Centre and the support of the NIHR, through the South London Cancer Research Network.

The study through which this protocol was implemented was approved by the Royal Marsden Committee for Clinical Research (Sutton, UK) and the Research Ethics Committee (London &#8211; Riverside, UK) (ISRCTN 53485935).
1. Radiotherapy Clinic
<ol>
	<li>Assess patient suitability for the voluntary breath-hold technique in the clinic: left breast or chest wall radiotherapy (without nodal irradiation) recommended by radiation oncologist.</li>
	<li>Review the patient&#8217;s performance status and comorbidities (especially lung-related).</li>
	<li>Ask the patient to practice holding their breath at home, while lying down, initially for 5 sec, and building up in 5 sec intervals to 20 sec.</li>
</ol>
2. Radiotherapy Planning-CT session
<ol>
	<li>Position the patient on the CT couch in the standard treatment position.</li>
	<li>Define the position of tattoos and place CT markers (crosses) on the patient midline in free breathing, approximately half way along the likely field edges. Add lateral markers to each side of the patient in free-breathing, in line with the midline marker.</li>
	<li>Ask the patient to practice taking a deep breath in and holding it, initially for 5 sec, before building up in 5 sec intervals to 20 sec. Instruct the patient to breathe in and breathe out twice before asking them to hold their breath for up to 20 sec. This relaxes the patient, helps them prepare for the breath-hold and helps breath-hold consistency.</li>
	<li>Record the maximum duration for which the patient can comfortably hold their breath.</li>
	<li>Repeat the breath-hold and mark the position of the anterior and lateral tattoos in relation to the lasers in breath-hold to help establish reproducibility. Record the height of the lateral tattoo above the couch top during breath-hold before proceeding with the CT scan.</li>
	<li>Give the standard breath-hold instructions to the patient and start the scan once satisfied the patient is in breath-hold.</li>
	<li>Once the CT scan has been completed, check and record the height of the lateral tattoos on the CT scan to confirm that a consistent breath-hold was performed. If the lateral couch height differs by greater than 3 mm from the initial couch height, remeasure the anterior and lateral reference points.</li>
</ol>
3. Radiotherapy Treatment Planning
NOTE: The radiotherapy treatment planning process is the same as for a standard breast patient.
<ol>
	<li>Apply tangential radiotherapy fields according to local protocol.</li>
	<li>Produce clinical radiotherapy treatment plan which fulfills ICRU criteria.</li>
	<li>Record the anterior beam source-to-skin distance (SSD) in addition to recording standard treatment planning data (according to local protocol). NOTE: The anterior beam SSD is used to check the anterior-posterior setup in the treatment room.</li>
</ol>
4. Radiotherapy Treatment Setup
<ol>
	<li>Align the tattoos in free-breathing. Mark the posterior and inferior measurements from the left lateral tattoo and the anterior midline tattoo on the patient&#8217;s skin (from information recorded at planning-CT session).</li>
	<li>Instruct the patient to breathe in and out twice before taking a deep breath in and holding. The reference mark on the patient&#8217;s skin should rise up to the level of the laser. Ask the patient to repeat the breath-hold procedure a couple of times to confirm reproducibility before proceeding with patient setup.</li>
	<li>Ask the patient to perform a breath-hold and align the midline tattoo to the isocenter position superior/inferior and set the focus-to-surface distance (FSD) at the midline.</li>
	<li>In free-breathing, move the bed laterally to the isocenter.</li>
	<li>Measure and mark the medial and lateral field borders in free-breathing.</li>
	<li>Set all other machine parameters (e.g., field size and gantry, collimator, and couch angles) for the first beam (anterior oblique). Ask the patient to perform a breath-hold and check the medial border aligns with mark made in Step 4.5.</li>
	<li>Mark the field edge (as defined by the light field) with a pen at every fraction: this aids visualization of the patient&#8217;s breath-hold.</li>
	<li>Repeat steps 4.6 and 4.7 for the posterior oblique beam, and treat using this beam first.</li>
	<li>If patient setup is out of tolerance (according to local tolerance levels for a standard breast radiotherapy patient), refer to the troubleshooting algorithm (Figure 2).</li>
	<li>If there are insufficient cameras to monitor the field edge as well as the position of the gantry relative to the couch from the control room, assess gantry rotation prior to leaving the treatment room in order to avoid collisions.</li>
</ol>
5. Radiotherapy Treatment Delivery
<ol>
	<li>Once in the control room, zoom the treatment room cameras so that the field borders marked on the patient&#8217;s skin are visible on the control room monitors.</li>
	<li>Once ready to commence treatment, ask the patient to perform a breath-hold (as detailed in 4.2) via the intercom system. Check the light field aligns satisfactorily with the marked field border and then commence treatment (Figure 3).</li>
	<li>Monitor the patient&#8217;s breath-hold during treatment delivery. Treatment should be interrupted if there is concern that there has been a change in breath-hold depth.</li>
</ol>
6. Radiotherapy Treatment Verification
<ol>
	<li>Perform imaging verification of patient position (such as with electronic portal imaging (EPI) or cone-beam CT), following local protocols for type/frequency of imaging and tolerance levels.</li>
	<li>Correct for systematic errors with isocenter moves according to local protocols for standard breast radiotherapy patients. Adjustment of marks on the patient&#8217;s skin should not be necessary.</li>
</ol>

<ol>
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Cancer. 107, 1195-1202 (2012).</li><li>Darby, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+radiotherapy+after+breast-conserving+surgery+on+10-year+recurrence+and+15-year+breast+cancer+death:+meta-analysis+of+individual+patient+data+for+10,801+women+in+17+randomised+trials.">Effect of radiotherapy after breast-conserving surgery on 10-year recurrence and 15-year breast cancer death: meta-analysis of individual patient data for 10,801 women in 17 randomised trials.</a> Lancet. 378, 1707-1716 (2011).</li><li>Clarke, 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=Effects+of+radiotherapy+and+of+differences+in+the+extent+of+surgery+for+early+breast+cancer+on+local+recurrence+and+15-year+survival:+an+overview+of+the+randomised+trials.">Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials.</a> Lancet. 366, 2087-2106 (2005).</li><li>Darby, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+ischemic+heart+disease+in+women+after+radiotherapy+for+breast+cancer.">Risk of ischemic heart disease in women after radiotherapy for breast cancer.</a> N. Engl. J. Med. 368, 987-998 (2013).</li><li>Team, D. o. H. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+Services+in+England+2012.">Radiotherapy Services in England 2012.</a> , (2012).</li><li>Zhang, F., Zheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosimetric+evaluation+of+conventional+radiotherapy,+3-D+conformal+radiotherapy+and+direct+machine+parameter+optimisation+intensity-modulated+radiotherapy+for+breast+cancer+after+conservative+surgery.">Dosimetric evaluation of conventional radiotherapy, 3-D conformal radiotherapy and direct machine parameter optimisation intensity-modulated radiotherapy for breast cancer after conservative surgery.</a> J Med Imaging Radiat Oncol. 55, 595-602 (2011).</li><li>Schubert, L. 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The authors have nothing to disclose.

Critical steps in the protocol include: 1) checking for breath-hold consistency at planning-CT and treatment setup; 2) checking the lateral couch height measured on CT is consistent with that measured pre-CT; 3) aligning tattoos in free-breathing but setting FSD in breath-hold; 4) ensuring light field aligns with marked field borders prior to commencing treatment.
The number of breath-holds required during treatment delivery varies from patient to patient, and is primarily dependent on the number of segments being delivered. Suitable interruption points during treatment delivery (to enable the patient to relax before repeating a breath-hold) should be determined on an individual basis depending on the method of delivery. We would strongly suggest that for initial implementation of VBH that a consistent team is used. This enables those involved to become competent more quickly and helps maintain treatment quality. Where problems are encountered during treatment setup, the patient may be asked to modify their breath-hold (deeper or shallower as required). If this fails to improve setup, the patient should be set up again. Vector couch moves should be employed as a last resort. A troubleshooting algorithm is shown in Figure 2.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51578/51578fig2highres.jpg" width="500" /> 
Figure 2.&#160;Troubleshooting algorithm for voluntary breath-hold technique treatment setup. This algorithm may be used to assist treatment setup where patients are not setting up within tolerance (according to local tolerance levels). As an example, our center uses a 5 mm tolerance level. The algorithm should be followed from top to bottom. In the majority of cases, setup can be brought within tolerance by asking the patient to modify their breath-hold depth (deeper or shallower as required).
Population systematic and random treatment setup errors are less than those seen in free-breathing tangential field breast radiotherapy24, and consistent with other published data on breath-holding techniques25,26. VBH reduces median normal tissue doses by 25-58% compared with standard free-breathing breast radiotherapy at our center (Table 1). Cardiac doses with VBH are lower than those seen in other published work on breath-holding techniques11,16,17,26,27, although methods of recording dose data vary between these studies.
As described in the introduction, all breath-hold techniques use a surrogate to measure inter- and intrafraction reproducibility. The VBH technique uses alignment of the light field with marked field borders to check for consistency before commencing treatment and during treatment delivery. Although not yet formally reported, we have found intrafraction reproducibility (measured using multiple intrafraction EPIs) to be extremely good, with little, if any, intrafraction movement. This is consistent with previously published work26. Given the consistent intrafraction reproducibility observed, radiotherapy systems in which light fields do not remain on during treatment delivery need not be a barrier to implementation of VBH. In-room lasers may be used as an alternative to light fields for checking that the breath-hold is maintained during treatment delivery. In addition, the reference point from which breath-hold reproducibility is monitored may be adapted; for example, centers using an asymmetric field technique may wish to use the superior tangential field border.
VBH offers significant advantages over other heart-sparing techniques, some of which have already been alluded to. It minimizes the trade-off between target and OAR compromise often required when using MLC, it reduces the low-dose irradiation of the heart and is much less resource-intensive than IMRT, and it is more reproducible than prone irradiation whilst benefiting women of all breast sizes. With respect to other breath-holding techniques, VBH gives comparable reproducibility and heart-sparing, while being less expensive to implement as no specialist equipment is required. The low cost of the technique means that there is a very real opportunity for it to benefit other healthcare systems, especially those with limited resources.
There is already published work demonstrating the feasibility of delivering nodal irradiation in addition to whole breast/chest wall irradiation using the infrared-reflecting markers17 and spirometry-based14 systems. Our center is now performing further work to confirm the feasibility of using VBH for nodal irradiation in breast cancer patients. Inverse-planned IMRT is likely to be of benefit in selected patients, especially when delivering a simultaneous integrated boost, and the feasibility of using VBH in these patients needs to be assessed. Finally, breath-holding techniques may be of benefit when treating other tumors, including lung28, liver29, and gastric30 cancers. Further work is needed to assess the suitability of using the VBH technique for treating sites other than breast.

Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths in 20081. Of all cancers, breast cancer is the most common with an incidence of over 13.8 million worldwide, and this incidence is increasing1. However, improvements in the diagnosis and treatment of breast cancer mean that the number of women surviving their breast cancer is also increasing, and is estimated to treble to 1.7 million by 2040 in the UK alone2. Breast radiotherapy forms an important part of many women&#8217;s breast cancer treatment, halving their risk of breast cancer recurrence and reducing the risk of breast cancer death by 3.8%3. With improvements in breast cancer survivorship, any long term side effects caused by breast cancer treatments are increasingly important. An innocent bystander in breast radiotherapy is the heart, which is exposed to unwanted radiation as a result of its proximity to radiation fields, especially during left breast irradiation. It is this unwanted dose to the heart that accounts for the 1% increase in non-breast cancer deaths associated with breast radiotherapy4. Recent evidence suggests that there is no threshold dose below which the late cardiac effects of breast radiotherapy do not occur5, making it critical for the oncology community to establish techniques which minimize cardiac doses without compromising breast tissue coverage. However, since breast radiotherapy accounts for approximately 30% of all radiotherapy treatments6, any new technique must be simple and inexpensive in order to be sustainable and avoid an unacceptable burden on healthcare resources.
There are a number of techniques which may be employed to reduce heart doses during breast radiotherapy. Multileaf collimation (MLC) is widely used in the UK [Royal College of Radiologists&#8217; (UK) audit 2012] and although effective at sparing heart tissue, it risks simultaneously shielding breast tissue. Inverse planned intensity modulated radiotherapy (IMRT) improves target tissue conformality7, but may also increase low-dose irradiation of the heart, lungs and contralateral breast7,8. An increase in low dose irradiation of the heart is undesirable, particularly in light of the data from Darby et al5. In addition, inverse-planned IMRT is more resource-intensive, requiring greater physics and quality assurance (QA) time and expertise. Treating women in the prone (face-down) position may reduce cardiac doses in larger-breasted women9, however, questions remain over the positional reproducibility of this technique10. Breath-holding techniques, in which patients hold their breath during radiotherapy delivery, result in the heart being pushed down and away from the radiotherapy fields and may minimize the need for a compromise between target tissue coverage and organ-at-risk (OAR) sparing (Figure 1)11.
There are currently two main breath-holding techniques in clinical use. The first consists of a digital spirometer attached to a balloon valve. Patients breathe through a mouthpiece and a clip is placed on their nose to avoid nasal respiration. The spirometry trace is visualized on a monitor, and inspiration interrupted and held at a predetermined lung volume. The second method was primarily designed for use as a respiratory gating system, although it also has an built-in breath-hold setting. This system uses a video camera to record the motion of an infrared-reflecting marker placed on the chest of the patient. The vertical movement of the marker is displayed in real-time on a monitor, and treatment delivery commences once the marker moves into a pre-specified threshold zone. Both systems markedly reduce cardiac doses in patients receiving left breast radiotherapy. The spirometry-based technique significantly reduces the volume of myocardium irradiated12-14, as well as demonstrating comparable intra- and inter-fraction reproducibility compared to standard supine free-breathing breast radiotherapy15. Similarly, treatment using the infrared-reflecting markers reduces the mean dose to the heart by over 50%11,16,17, whilst maintaining target tissue coverage11. Such dosimetric savings are projected to equate to a 10 fold reduction in cardiac deaths18.
A drawback of these systems, however, and a barrier to widespread implementation, is their cost. Both systems require investment in the devices themselves, however, in the case of the spirometry system there are also ongoing costs as the mouthpieces are disposable, requiring a new mouthpiece for planning-CT as well as for each fraction of treatment. Cost, coupled with a lack of staff training, explains why only 4% of UK breast treatments were performed using breath-holding techniques in 2012 [Royal College of Radiologists&#8217; (UK) audit]. Breath-holding techniques are in more widespread use in the rest of Europe, with 20% of centers using these techniques in 201019. One explanation for this is the development and implementation of a simple, inexpensive and equipment-free breath-holding technique, voluntary breath-hold (VBH). Until recently, however, data was lacking on the reproducibility of the VBH technique. A randomized study conducted at the Royal Marsden Hospital (Sutton, UK), The UK HeartSpare Study, has demonstrated that interfraction reproducibility with the VBH technique is comparable to that with the spirometry-based device. In addition, the VBH technique offers a time advantage at planning-CT and treatment setup and is preferred by patients and radiographers alike20. The VBH technique is currently being rolled out to ten UK radiotherapy centers to confirm that the technique is feasible in a multicenter setting and that heart-sparing is maintained (HeartSpare II). It is expected that this will pave the way for the UK-wide uptake of heart-sparing breast radiotherapy, and is likely to lead to a significant reduction in heart disease among UK breast cancer survivors.

<table border="0" cellpadding="0" cellspacing="0" width="604">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Brilliance CT big bore oncology</td>
			<td style="width:71px;">Philips</td>
			<td style="width:119px;"></td>
			<td>Other makes/models compatible with VBH technique</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MT350 breastboard</td>
			<td style="width:71px;">Med-Tec</td>
			<td style="width:119px;">MT-350-N</td>
			<td>Other makes/models compatible with VBH technique</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dorado virtual simulation laser system</td>
			<td style="width:71px;">LAP Laser</td>
			<td style="width:119px;">Dorado CT-1-3-Wall</td>
			<td>Other makes/models compatible with VBH technique</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:247px;">Pinnacle3radiation therapy planning system v9.2</td>
			<td style="width:71px;">Philips</td>
			<td style="width:119px;"></td>
			<td>Other makes/models compatible with VBH technique</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Synergy linear accelerator</td>
			<td style="width:71px;">Elekta</td>
			<td style="width:119px;"></td>
			<td>Other makes/models compatible with VBH technique</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Intuity XVI Release&#160;4.5.1&#160;</td>
			<td style="width:71px;">Elekta</td>
			<td style="width:119px;"></td>
			<td>Other makes/models compatible with VBH technique</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Iview Electronic Portal Imaging Release 3.4&#160;</td>
			<td style="width:71px;">Elekta</td>
			<td style="width:119px;"></td>
			<td>Other makes/models compatible with VBH technique</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Apollo room lasers</td>
			<td style="width:71px;">LAP Laser</td>
			<td style="width:119px;"></td>
			<td>Other makes/models compatible with VBH technique</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Active breathing coordinator (ABC)</td>
			<td style="width:71px;">Elekta</td>
			<td style="width:119px;"></td>
			<td>Commercially available breath-hold system, not required for VBH technique</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Real-time position management (RPM) system</td>
			<td style="width:71px;">Varian</td>
			<td style="width:119px;"></td>
			<td>Commercially available breath-hold system, not required for VBH technique</td>
		</tr>
	</tbody>
</table>

voluntary breath-hold technique for reducing heart dose in left breast radiotherapy

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.

Real-time electronic portal images (EPI) were matched on-line to digitally reconstructed radiographs (DRRs) for 23 patients (172 treatment fractions). EPI displacements were analyzed for the right anterior and left posterior oblique beams in the (u,v)-plane (v-direction parallel to craniocaudal axis and u-direction perpendicular to this)21, and setup errors for the VBH technique estimated. EPI-based population systematic error range (for each beam and in each plane) was 1.5-1.8 mm and random error range 1.7-2.5 mm.
Tabular dose-volume histogram (DVH) data was used to derive the NTDmean (a biologically weighted mean of total dose to tissue normalized to 2 Gy fractions using a standard linear quadratic model22, &#945;/&#946; = 3Gy) for heart, left anterior descending coronary artery (LAD), ipsilateral, and whole lungs. In addition, the maximum dose received by the LAD (LADmax) was estimated. Normal tissue doses are shown in Table 1.
Times for planning-CT session, treatment setup, treatment delivery and total treatment session were recorded, and are shown in Table 2. The data demonstrate that planning-CTs can be completed within a standard 30 min session. Treatment times include CBCT imaging, which was performed for every third fraction. Treatment setup and total session times are, therefore, expected to be shorter than reported here for centers in which CBCT imaging is not part of standard treatment. However, even with CBCT imaging, treatments may be completed within a 20 min treatment session.
Patients and radiographers were asked to complete validated questionnaires23 at their planning-CT session as well as twice during their treatment. Sixty-five patient questionnaires and 64 radiographer questionnaires were analyzed. The questionnaires were summarized as patient comfort scores (PCS) and radiographer satisfaction scores (RSS) (out of 9, higher score = more comfortable/satisfactory). Median PCS was 8 (interquartile range 8-9) and median RSS was 7 (interquartile range 6-8).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51578/51578fig1.jpg" /> 
Figure 1. The heart-sparing effect of VBH. Axial and sagittal CT slices from the same patient at the same chest wall level in free-breathing (A and C) and using the VBH technique (B and D). Note that the heart (outlined in yellow) has been pushed down and away from the radiotherapy fields using the VBH technique. <a href="https://www.jove.com/files/ftp_upload/51578/51578fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51578/51578fig3highres.jpg" /> 
Figure 3. Checking breath-hold consistency from the control room. Control room CCTV stills demonstrating the position of the light field relative to the marked field border for a right anterior oblique beam in free-breathing (A) and the aligning of the light field and marked field border once the patient is in breath-hold (B).
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Median dose (Gy)</td>
			<td colspan="2">Minimum dose (Gy)</td>
			<td colspan="2">Maximum dose (Gy)</td>
		</tr>
		<tr>
			<td></td>
			<td>VBH</td>
			<td>FB</td>
			<td>VBH</td>
			<td>FB</td>
			<td>VBH</td>
			<td>FB</td>
		</tr>
		<tr>
			<td>Heart NTDmean</td>
			<td>0.6</td>
			<td>0.8</td>
			<td>0.4</td>
			<td>0.4</td>
			<td>0.9</td>
			<td>2.1</td>
		</tr>
		<tr>
			<td>LAD NTDmean</td>
			<td>2.5</td>
			<td>6.0</td>
			<td>1.3</td>
			<td>1.2</td>
			<td>10.0</td>
			<td>22.6</td>
		</tr>
		<tr>
			<td>LADmax</td>
			<td>28.6</td>
			<td>43.7</td>
			<td>9.7</td>
			<td>4.6</td>
			<td>41.8</td>
			<td>51.3</td>
		</tr>
		<tr>
			<td>Ipsilateral lung NTDmean</td>
			<td>4.1</td>
			<td>-</td>
			<td>2.8</td>
			<td>-</td>
			<td>5.6</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Whole lungs NTDmean</td>
			<td>2.0</td>
			<td>-</td>
			<td>1.3</td>
			<td>-</td>
			<td>2.7</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 1. Normal tissue doses for the voluntary breath-hold (VBH) technique. Median, minimum and maximum NTDmean (Gy) for heart, LAD, ipsilateral and whole lungs are shown, as are median, minimum, and maximum LADmax (Gy). In addition, median, minimum, and maximum cardiac doses for standard free-breathing (FB) left breast radiotherapy at our center are shown for comparison.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Time (min)</td>
		</tr>
		<tr>
			<td>Event</td>
			<td>Median</td>
			<td>Minimum</td>
			<td>Maximum</td>
		</tr>
		<tr>
			<td>Planning-CT session</td>
			<td>22</td>
			<td>14</td>
			<td>44</td>
		</tr>
		<tr>
			<td>Treatment setup</td>
			<td>9</td>
			<td>6</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Treatment delivery</td>
			<td>7</td>
			<td>5</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Total treatment session</td>
			<td>18</td>
			<td>14</td>
			<td>27</td>
		</tr>
	</tbody>
</table>
Table 2. Planning-CT and treatment session times for the voluntary breath-hold technique. Median, minimum, and maximum planning-CT, treatment setup, treatment delivery, and total treatment session times are shown (min).

Watch this Scientific Journal Video about Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy at JoVE.com

Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

The current priority in breast cancer radiotherapy is to reduce cardiac doses without compromising target tissue coverage. The voluntary breath-hold technique described here is a simple, inexpensive solution to this problem and capable of being instituted widely without the need for specialized equipment.

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these ...

voluntary-breath-hold-technique-for-reducing-heart-dose-left-breast

Research

JoVE Journal

Medicine

46.4K Views.  Royal Marsden NHS Foundation Trust. The current priority in breast cancer radiotherapy is to reduce cardiac doses without compromising target tissue coverage. The voluntary breath-hold technique described here is a simple, inexpensive solution to this problem and capable of being instituted widely without the need for specialized equipment.

Video: Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

The WATCHMAN Left Atrial Appendage Closure Device for Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting an estimated 6 million people in the United States 1. Since AF affects primarily elderly people, its prevalence increases parallel with age. As such, it is expected that 15.9 million Americans will be affected by the year 2050 2. Ischemic stroke occurs in 5% of non-anticoagulated AF patients each year. Current treatments for AF include rate control, rhythm control and prevention of stroke 3.
The American College of Cardiology, American Heart Association, and European Society of Cardiology currently recommended rate control as the first course of therapy for AF 3. Rate control is achieved by administration of pharmacological agents, such as &beta;-blockers, that lower the heart rate until it reaches a less symptomatic state 3. Rhythm control aims to return the heart to its normal sinus rhythm and is typically achieved through administration of antiarrhythmic drugs such as amiodarone, electrical cardioversion or ablation therapy. Rhythm control methods, however, have not been demonstrated to be superior to rate-control methods 4-6. In fact, certain antiarrhythmic drugs have been shown to be associated with higher hospitalization rates, serious adverse effects 3, or even increases in mortality in patients with structural heart defects 7. Thus, treatment with antiarrhythmics is more often used when rate-control drugs are ineffective or contraindicated. Rate-control and antiarrhythmic agents relieve the symptoms of AF, including palpitations, shortness of breath, and fatigue 8, but don't reliably prevent thromboembolic events 6.
Treatment with the anticoagulant drug warfarin significantly reduces the rate of stroke or embolism 9,10. However, because of problems associated with its use, fewer than 50% of patients are treated with it. The therapeutic dose is affected by drug, dietary, and metabolic interactions, and thus requires detailed monitoring. In addition, warfarin has the potential to cause severe, sometimes lethal, bleeding 2. As an alternative, aspirin is commonly prescribed. While aspirin is typically well tolerated, it is far less effective at preventing stroke 10. Other alternatives to warfarin, such as dabigatran 11 or rivaroxaban 12 demonstrate non-inferiority to warfarin with respect to thromboembolic events (in fact, dabigatran given as a high dose of 150 mg twice a day has shown superiority). While these drugs have the advantage of eliminating dietary concerns and eliminating the need for regular blood monitoring, major bleeding and associated complications, while somewhat less so than with warfarin, remain an issue 13-15.
Since 90% of AF-associated strokes result from emboli that arise from the left atrial appendage (LAA) 2, one alternative approach to warfarin therapy has been to exclude the LAA using an implanted device to trap blood clots before they exit. Here, we demonstrate a procedure for implanting the WATCHMAN Left Atrial Appendage Closure Device. A transseptal cannula is inserted through the femoral vein, and under fluoroscopic guidance, inter-atrial septum is crossed. Once access to the left atrium has been achieved, a guidewire is placed in the upper pulmonary vein and the WATCHMAN Access Sheath and dilator are advanced over the wire into the left atrium. The guidewire is removed, and the access sheath is carefully advanced into the distal portion of the LAA over a pigtail catheter. The WATCHMAN Delivery System is prepped, inserted into the access sheath, and slowly advanced. The WATCHMAN device is then deployed into the LAA. The device release criteria are confirmed via fluoroscopy and transesophageal echocardiography (TEE) and the device is released.

The accompanying video describes a procedure for percutaneous placement of the WATCHMAN Left Atrial Appendage (LAA) Device. The WATCHMAN is a nitinol device designed to be permanently implanted at, or slightly distal to, the opening of the left atrial appendage (LAA) to trap blood clots before they exit the LAA, preventing thromboembolic stroke.

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting an estimated 6 million people in the United States 1. Since AF affects primarily ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...