Name: the clinical application of tumor treating fields therapy in glioblastoma
Uploaded: 2019-04-16T13:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma at JoVE.com

This research was supported in part by A Reason To Ride research fund. We thank Allison Diep for creating the 3-dimensional artwork in Figure 1C.

The presentation of this protocol follows ethical guidelines at Beth Israel Deaconess Medical Center and written authorization was obtained from the patient.
1. Application of the Second Generation TTFields Device
NOTE: The system consists of the portable electric field generator, transducer arrays, a connection cable and box, a rechargeable battery, charger for portable batteries, and a plug in power supply.
<ol>
	<li>Treatment planning procedure
	<ol>
		<li>Acquire MRI images of the patient&#8217;s brain. The MRI scan includes the margins of the scalp for treatment planning. Incomplete delineation of the full thickness of the scalp interferes with the electric field calculations.</li>
		<li>Using axial T1 sequence MRI scans and the tools on the DICOM image viewer, take baseline measurements of the front to back, right to left, and right to midline based on axial view head size (mm). Measure the superior to tentorium, right to left, and right to midline based on coronal view head size (mm).</li>
		<li>Focusing on the primary lesion, measure the front to back without nose, right to left, right to midline, right to close tumor margin, right to far tumor margin, front to close tumor margin, and front to far tumor margin based on axial view tumor size (mm). Measure the superior to tentorium, right to left, right to midline, right to close tumor margin, right to far tumor margin, superior to close tumor margin, and superior to far tumor margin based on coronal view tumor size (mm).</li>
		<li>Open treatment planning software, enter username and password, and select new patient transducer array.</li>
		<li>Enter in the measurements taken above, and click Generate Transducer Array Placement. Save the transducer array layout for future use on patient visit.</li>
	</ol>
	</li>
	<li>Applying transducer arrays to scalp
	<ol>
		<li>Prepare the scalp for transducer array placement by cutting hair and shaving hair stubbles with an electric razor down to the surface of the scalp until no hair remains. Avoid using a razor with blade(s) to prevent cuts on the scalp.</li>
		<li>Wipe the scalp with 70% isopropyl alcohol.</li>
		<li>Remove the transducer arrays from plastic packaging and begin planning placement upon the scalp according to the predetermined specific array layout scheme (see section 1.1). Locate the surgical scar and avoid placing transducer arrays upon scar.</li>
		<li>If the scar is located under a predetermined transducer array, then shift the four array placements 2 cm clockwise or counterclockwise.</li>
		<li>Determine the desired location of the connection wires as preferred by the patient (either right or left side of the body). Apply the transducer array that is nearest to the surgical scar first, while positioning the connection wire to the preferred side.</li>
		<li>Apply the next transducer array to either the right lateral or the left lateral in a clockwise or counterclockwise fashion, keeping the location of the connection wire consistent. Apply the third and fourth transducer arrays in the same clockwise or counterclockwise fashion.</li>
		<li>Place gauze strips underneath the metallic interface in between the array and the connection wire. Use silk tape to hold the gauze strip in place.</li>
		<li>Braid the four connection wires together and fasten with silk tape. Place fishnet retainer over the head in order to hold the arrays in place.</li>
	</ol>
	</li>
	<li>Assembling the TTFields device system
	<ol>
		<li>Connect each of the four white and black color-coded connection wires to a corresponding white or black port on the connection box, making sure each audibly snaps into place.</li>
		<li>If using the portable battery, connect the battery charger to a wall outlet and switch the power button on in order to initially charge the battery.</li>
		<li>Insert charged battery into the electric field generator by plugging it in through its connector to a socket labeled DC IN on the front panel of the device. Make sure the arrows on the battery connector face up towards the DC IN label.</li>
		<li>If not using the portable battery, plug the electric field generator into a wall outlet.</li>
		<li>Turn on the power button located at the bottom of electric field generator to start the device. Switch on the TTFields button located at the top of the electric field generator. The patient may experience a warm sensation.</li>
		<li>To achieve an optimal response, Have patient use TTFields therapy on a continuous basis for a minimum compliance of 75%, or 18 h a day. Treatment duration of less than 18 hour per day has been associated with suboptimal results.</li>
	</ol>
	</li>
	<li>Exchange of transducer arrays 
	NOTE: Array exchange procedures in this section are repeated every 3 to 4 days.
	<ol>
		<li>Use baby oil to remove adhesive from skin. Pull off arrays by applying slow and even tension with both hands.</li>
		<li>Wash scalp with gentle shampoo. Check scalp for dermatitis, erosions, ulcers, or infection. Apply anti-septic ointment as needed.
		<ol>
			<li>If ulcers or infections are present, discontinue treatment until ulcer heals or infection clears.</li>
		</ol>
		</li>
		<li>Shave off regrown hair.</li>
		<li>Clean scalp with 70% isopropyl alcohol. Reapply the transducer arrays (see section 1.2).</li>
	</ol>
	</li>
</ol>
2. Removal of Systemic Agents that May Interfere with Anti-tumor Iimmunity
<ol>
	<li>Lowering or discontinuation of dexamethasone 
	NOTE: Dexamethasone is a synthetic fluorinated glucocorticoid that has anti-inflammatory effects in humans by impairing cell-mediated immunity.
	<ol>
		<li>Wean dexamethasone in a stepwise fashion due to its hysteresis effect.</li>
		<li>Apply trimethopreme-sulfamethaxazole (400&#8211;80 mg single-strength tablet daily or 800&#8211;160 mg double-strength tablets three times per week) to prevent the development of pneumocystic pneumonia during the weaning process.</li>
		<li>Cut the dose half quickly every 7&#8211;10 days to achieve a daily dose of 4 mg/day. If the patient is already at 4 mg/day or lower the dose, cut the dosage slower, at a rate of every 10 to 14 days until discontinuation.</li>
		<li>Look for signs of adrenal suppression (i.e., lethargy, cold intolerance, weakness and hypersomnia). If signs of unacceptable neurologic deficits and/or adrenal suppression appear, the previous dose of dexamethasone is re-applied. 
		NOTE: Other means for reduction of dexamethasone are being sought (see concurrent bevacizumab administration).</li>
	</ol>
	</li>
	<li>Concurrent bevacizumab administration 
	NOTE: Bevacizumab is a humanized anti-vascular endothelial growth factor (VEGF) monoclonal IgG1 antibody. The antibody has a potent antiangiogenic effect by sequestering VEGF, rendering it unable to bind to the cognate receptors VEGFR1 and VEGFR2 and to exert its proangiogenic effect. Immature blood vessels also have high permeability and elimination of these newly generated vasculature within the glioblastoma microenvironment also helps to reduce cerebral edema. Bevacizumab has a long half-life of about 20 days28 and therefore it can be administered to patients every 2 to 3 weeks as an intravenous infusion. The indication for bevacizumab is to obviate the prolonged use of dexamethasone.
	<ol>
		<li>Exclude bevacizumab from patient who had recent hemorrhage (either intracranial or extracranial), myocardial infarction or stroke, major surgery (including craniotomy) within 4 weeks, uncontrolled hypertension, pregnancy or lactation. Exercise caution in patients with chronic kidney disease, proteinuria, bleeding disorder, uncontrolled angina, cardiac arrhythmia, congestive heart failure, prior chest wall irradiation, prior anthracycline exposure or other concurrent illness deemed unfit by the treating physician.</li>
		<li>Before treatment, make sure the patient has acceptable blood counts, kidney function, normal blood pressure and urine dipstick protein &#60;100 mg/dL.</li>
		<li>Once the patient is deemed to be an acceptable candidate, administer bevacizumab at a dose of 2.5, 5.0, or 10 mg/kg. There is class 2 evidence that bevacizumab at doses of &#60;10 mg/kg work as well as 10 mg/kg29,30. Start TTFields treatment either before or after initiation of bevacizumab.</li>
		<li>Infuse the initial dose of bevacizumab over 60 min in 100 cc of normal saline. If there is no adverse event, administer subsequent doses over 30 min.</li>
	</ol>
	</li>
	<li>Other systemic immunosuppressive agents to avoid 
	NOTE: There are a number of anti-cancer drugs that also have significant immunosuppressive properties. They are listed below.</li>
	<li>To avoid everolimus, which is an mTOR inhibitor. 
	NOTE: Everolimus is approved to treat subependymal giant cell astrocytoma, advanced hormone-receptor-positive, Her2-negative breast cancer, pancreatic neuroendocrine tumors and renal cell carcinoma. However, the addition of everolimus has been shown definitively in a randomized study to hasten the death of glioblastoma patients, most likely by impairing their anti-tumor cell-mediated immunity31. It is also used to prevent rejection of transplant organ recipients.</li>
	<li>Avoid Sirolimus, which is also known as rapamycin. 
	NOTE: Temsirolimus is a pro-drug that can be metabolized to sirolimus. It is an mTOR inhibitor with immunologic interference properties similar to everolimus. It is also used to prevent rejection of transplant organ recipients.</li>
</ol>

Drs. Kenneth Swanson and Eric Wong both received unrestricted research grants from Novocure, Ltd.

This article demonstrates the proper application of the second generation TTFields device to treat glioblastoma patients. The significance of TTFields therapy with respect to alternative treatments includes reduced toxicity, increased quality of life, and higher median overall survival especially when combined with temozolomide chemotherapy. Furthermore, we show in a step-by-step fashion the proper application of the transducer array onto the scalp, while avoiding pitfalls that may cause complications. In addition, we provide a detailed account of the cell biology effects of TTFields as well as electric field mapping as TTFields penetrate into the brain.
A few steps in the protocol are particularly critical for the successful implementation of the device. For proper treatment planning, the MRI images of the patient&#39;s brain must include the margins of the scalp. To ensure adequate contact between electrode and scalp, the hair stubbles must be shaved down to surface of the scalp until no hair remains. It is important to locate any surgical scars and avoid placing transducer arrays on the scar to obviate complications from scalp breakdown. During each exchange, check the scalp for dermatitis, erosions, ulcers or infection and, if needed, stop application of arrays until ulcers are healed and infections are resolved33,34.
The improvement of life expectancy depends most on high patient compliance of 18 hours per day or more. A post hoc analysis of the EF-11 phase III trial data showed significantly longer median overall survival in TTFields therapy patients with a compliance rate &#8805;75% (&#8805;18 h daily) versus those with a &#60;75% compliance rate (7.7 versus 4.5 months, p = 0.042)35. Patients who are less than 75% compliant appear to receive little benefit, while those who pass the 75% compliance cut-off exhibited significant benefit. Physician guidance and family support plays an important role in achieving higher patient compliance, and advice on application can be given so that the patient is more comfortable wearing the arrays for longer periods of time. Ambient temperature should remain in a comfortable range while wearing the arrays. Regular intervals of array changes, hair shaving of the scalp, and placement of a breathable net on the head to hold the arrays in place may also improve comfort leading to higher compliance.
There is accumulating evidence that TTFields treatment works better when combined with other therapies. TTFields were used as a monotherapy in the EF-11 pivotal phase III trial, and the median overall survival was 6.6 months for the TTFields arm compared to 6.0 months for the chemotherapy arm. Although these initial results showed no statistically significant improvement in overall survival over standard-of-care treatment, fewer severe adverse events and improved quality-of-life measures were noted in the TTFields arm which formed the basis for its approval for recurrent glioblastoma by the FDA27. The later EF-14 phase III trial on newly diagnosed glioblastoma showed a median overall survival of 20.9 months in the TTFields-temozolomide arm versus 16.0 months in the temozolomide-alone arm36,37. Another study on TTFields in clinical practice using the PRiDe registry showed a median overall survival of 9.6 months, which was significantly longer than the median overall survival in the control arm of EF-1135. Furthermore, preclinical data have shown that adding in alkylating agents like temozolomide improves tumor cell kill in tissue culture24. The PRiDe registry and EF-14 data support this concept because these patients had better outcomes when they received concurrent temozolomide and/or other treatments added to TTFields. Wong et al. showed similar results by comparing TTFields therapy and bevacizumab alone or in combination with a regimen consisting of 6-thioguanine, lomustine, capecitabine, and celecoxib (TCCC). The TCCC group exhibited prolonged overall survival, median 10.3 months versus 4.1 months for TTFields and bevacizumab alone38. Collectively, these data support the addition of adjuvant therapies to increase the effectiveness of the device in treating glioblastoma.
In the EF-14 trial, the patients that received TTFields in the experimental arm had a longer overall survival compared to the controls, but there was no difference between the experimental and control arms in the EF-11 trial. The EF-14 trial added a known therapeutic agent, temozolomide, which appears to combine synergistically with TTFields treatment. Another potential explanation for this difference may be due to the chemotherapy n&#228;ive status of newly diagnosed patients, which may enable them to mount a more effective anti-tumor immune response. Although the mechanism of an immune response from TTFields remains unclear, dexamethasone as an immunosuppressive agent may negate this benefit and has been shown to lower median survival when combined with TTFields39,40,41. In conclusion, lowering patients&#8217; dose of dexamethasone while on TTFields would increase the number of immune cells in the blood of glioblastoma patients and could lead to a stronger response and improved treatment result. TTFields may also sensitize tumor cells to the effects of ionizing radiation42,43. However, the selection of combination therapy should be individualized with respect to the neurologic and medical conditions of the patient.
The TTFields device was approved by the FDA for the treatment of adult patients with recurrent and newly diagnosed glioblastoma at age 22 years and older; the efficacy of this device for patients under 22 years is unknown. Furthermore, the side effects are unknown when the patient is using TTFields concurrently with an active implanted device, such as deep brain, spinal cord, or vagus nerve stimulators, defibrillators, and cardiac pacemakers, or patients with a metallic fragment (i.e., bullet) or apparatus (i.e., aneurysm clip) in the brain. Known allergic reaction to electrode gels, open wounds, skull defects, and pregnancy are also contraindicated. Patients with major skull defects, such as absence of a large segment of the calvarium from craniectomy, may have a higher penetration of TTFields44; however, craniectomy is not routinely performed on glioblastoma patients.
Poor patient compliance is a major limitation to this treatment modality. Factors that may decrease compliance include concurrent medical or psychiatric illness (i.e., depression)45,46,47, lack of support from caretakers, scalp breakdown due to erosions or infection, skin swelling, and dermatitis.
TTFields have an unequivocal anti-mitotic effect on dividing tumor cells. Quite possibly, this effect also extends to progenitor cells but preclinical or clinical data on normal tissue is lacking. Nevertheless, TTFields therapy shows promise in multiple solid tumor types, including some of the most aggressive forms of cancer. TTFields serve as an effective antimitotic treatment in preclinical pancreatic cancer models and have a long-term negative effect on the survival of these cancer cells. These results make TTFields an attractive treatment modality for testing in patients with pancreatic cancer48. TTFields have also shown encouraging preclinical results for the treatment of ovarian cancer49 and non-small cell lung cancer15,50. Therefore, TTFields are being applied in ongoing phase III clinical trials for primary (NCT02973789) and metastatic (NCT02831959) lung cancer, pancreatic cancer (NCT03377491), and mesothelioma (NCT02397928). Hopefully, TTFields will provide additional treatment options for these difficult-to-treat malignancies.

Glioblastoma 
Glioblastoma is the most common primary malignant brain tumor in adults. Due to its properties as a cytologically malignant, mitotically active, angiogenically proliferative and necrosis-prone neoplasm typically associated with rapid pre- and post-operative disease evolution and near-universal fatal outcome, the World Health Organization designated glioblastoma as a grade IV neoplasm1. Despite basic and translational research efforts, there is no curative treatment for glioblastoma. The 5 year survival rate of patients diagnosed with glioblastoma remains approximately 5%, highlighting the pressing need for more effective therapeutic interventions2.
Mechanisms of tumor treating fields: electric field 
TTFields are low-intensity, intermediate-frequency (100-300 kHz) alternating electric fields that permeate tumor-bearing tissues and are produced by insulated electrodes adhered externally to the patient&#39;s skin3. TTFields are thought to interfere with biological processes of tumor cells by exerting electromagnetic forces on intracellular molecules with high dipole moments during mitosis. TTFields exposure during mitosis resulted in aberrant mitotic exit leading to abnormal chromosome segregation, cellular multinucleation, and caspase dependent apoptosis of daughter cells4. These effects were frequency dependent and contingent on the incident direction of the field with relation to the mitotic plates of affected cells. The cells with mitotic plates perpendicular to the fields exhibited the greatest severity of damage. The intermediate frequency range is unique because it constitutes a transition region in which strength of the intracellular electric field, which is shielded at lower frequencies, increases significantly5. The threshold at which this increase occurs depends on the dielectric properties of the cell membrane5. For glioma cells, the optimal frequency of the TTFields with respect to both cell counts in culture and clonogenic assays is 200 kHz6.
Using patient-specific MRI measurements, a personalized mapping of electric fields can be developed by incorporating the volume, electric conductivity, and relative permittivity of different tissue structures in the brain7,8. Furthermore, an end-to-end, semi-automatic segmentation-based workflow can also be utilized to generate a personalized finite element model for the delineation of intracranial TTFields9. Electric fields maps demonstrating the distribution of electric fields within the patient brain may have utility for guiding optimal placement of transducer arrays to maximize field strength within the tumor.
Mechanisms of tumor treating fields: cell biology 
The precise mechanisms by which TTFields drive mitotic disruption are not completely understood, but two potential mechanisms by which electric fields may affect mitosis have been proposed. One involves the electric field&#39;s direct action on proteins with high dipole moments resulting in their functional perturbation; the second is dielectrophoresis of ions, causing a mislocalization of ions within the dividing cell that may interfere with cytokinetic furrow ingression3. Two proteins with high dipole moments have been proposed as targets, the &#945;/&#946;-tubulin monomer and the Septin 2, 6, 7 heterotrimer, with dipole moments of 1740 D10 and 2771 D11, respectively. It has been suggested that TTFields decrease the ratio between polymerized and total tubulin, preventing proper mitotic spindle assembly and perturbing the cells at the transition from metaphase to anaphase4. Cells exposed to TTFields show normal progression until metaphase, but then exhibit reduced septin localization to the anaphase spindle midline and cytokinetic furrow11. The cells undergo uncontrolled membrane blebbing that leads to aberrant mitotic exit12. The resulting post-mitotic cells exhibit abnormal nuclear architecture such as micronuclei, signs of cellular stress, and an overall decrease in cellular proliferation including G0 arrest followed by apoptosis11. Research has shown an up-regulation in calreticulin and secretion of HMGB1 in TTFields treated cells, both hallmarks of immunogenic cell death13,14. Kirson&#160;et al. showed treatment of tumors reduced the metastatic potential, and the metastases within TTFields-treated animals showed an increase in CD8+ cells15. Together, these data support a mechanism of action that extends beyond direct effects on mitosis, and likely initiates antitumor inflammatory responses.
TTFields device and treatment options 
Both first and second generation TTFields devices deliver alternating electric fields to the supratentorial brain for the treatment of glioblastoma. The device was first approved by the FDA in 2011 for the treatment of patients with recurrent glioblastoma and approved in 2015 for the treatment of patients with newly diagnosed glioblastoma16,17. Glioblastoma treatment should be undertaken in a multimodal fashion, with neurosurgical intervention, radiation oncology input, and chemotherapy administration. Since TTFields represent an additional anti-cancer treatment modality with few toxicities, neuro-oncologists should consider incorporating this therapy into current treatment regimens for both newly diagnosed and recurrent glioblastoma18,19.
In the newly diagnosed setting, standard treatment approach consists of concurrent radiation and temozolomide followed by maintenance temozolomide. In 2004, a randomized phase III trial showed improved median and 2-year survival for patients with glioblastoma treated with radiotherapy and concomitant and adjuvant temozolomide20. Benefits of adjuvant temozolomide with radiotherapy lasted throughout at least 5 years of follow-up21. However, patient 06-methylguanine-DNA methyltransferase (MGMT) methylation status identified those most likely to benefit from the addition of temozolomide22. In another randomized clinical trial of patients with glioblastoma who had received standard radiation and concomitant temozolomide chemotherapy, the addition of TTFields to maintenance temozolomide chemotherapy resulted in improved outcomes compared to those that received maintenance temozolomide alone23. Additionally, research has shown that TTFields work irrespective of patient MGMT promoter methylation status; therefore TTFields may constitute a clinical intervention that also works in patients with unmethylated MGMT status24. Taken together, these studies suggest broad implications on the effectiveness of TTFields for the treatment of glioblastomas. Specifically, after radiation, incorporating TTFields in combination with temozolomide provides an effective treatment option for newly diagnosed patients with glioblastoma.
In the recurrent setting, there exists no standard treatment approach. However, bevacizumab and TTFields therapy are the two FDA-approved treatment modalities25,26. The EF-11 phase III trial of TTFields monotherapy (with 20-24 h/day usage) versus active chemotherapy in patients with recurrent glioblastoma showed comparable overall survival, while toxicity and quality of life favored TTFields27. Therefore, bevacizumab alone, TTFields monotherapy, or a combination of both constitutes treatment options for those with recurrent glioblastoma.
Clinical application 
A previous JoVE publication demonstrated the application of the first generation device using a plastic model of a human head25. Here, we demonstrate the application of the second-generation device on a glioblastoma patient undergoing treatment. The protocol for using the device begins with configuring transducer array layout placement on the scalp using MRI measurements and a treatment planning system. The transducer array layout map delineates the orientation and location of each of the four arrays on the patient&#39;s head. The arrays are designed to adhere to the scalp to allow the transducers to deliver the 200 kHz frequency TTFields from an electric field generator. Patients receive treatment continuously and the arrays are typically exchanged every 3 to 4 days. In this paper we show the effects of TTFields on mitotic cells, the distribution of electric fields within the brain, and the step-by-step application method of the second-generation device on a human head to demonstrate treatment of a patient with glioblastoma.

<table><tbody><tr><td>Baby Oil</td><td>Johnson &amp;amp; Johnson</td><td>Product Code 473542</td><td></td></tr><tr><td>Bevacizumab</td><td>Genetech, Inc.</td><td>Not applicable</td><td></td></tr><tr><td>Elastic Net</td><td>Medline Industries</td><td>NET012</td><td></td></tr><tr><td>Gentle Shampoo</td><td>Johnson &amp;amp; Johnson</td><td>Product Code 108249</td><td></td></tr><tr><td>Isopropyl Alcohol 70%</td><td>The Betty Mills Company</td><td>MON 23222701</td><td></td></tr><tr><td>Medical Tape</td><td>The Betty Mills Company</td><td>MON 38202201</td><td></td></tr><tr><td>Sterile Gauze</td><td>The Betty Mills Company</td><td>MON 71392000</td><td></td></tr><tr><td>Trimethoprim-sulfamethoxazole</td><td>Pfizer, Inc.</td><td>Not applicable</td><td></td></tr><tr><td>TTFields Device (Optune)</td><td>Novocure, Ltd.</td><td>Not applicable</td><td>The system consists of the portable electric field generator, transducer arrays, a connection cable and box, a rechargeable battery, charger for portable batteries, and a plug in power supply.</td></tr></tbody></table>

the clinical application of tumor treating fields therapy in glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

TTFields cause disruption during mitosis leading to an asymmetric distribution of chromosomes and misalignment of metaphase plates during mitosis, (compare Figure 1A and Figure 1B). TTFields are thought to exert their effect by perturbing the function of high dipole moment possessing proteins such as &#945;/&#946;-tubulin or septin. One proposed model for TTFields action on mitotic cells is that they perturb septin function. Normally, septin acts to organize the cytokinetic furrow and to reinforce the structurally important interaction between the subcortical actin cytoskeleton and the overlying plasma membrane that is needed to resist intracellular hydrostatic forces produced during furrow ingression. This results in a loss of structural integrity within the dividing cells that is necessary for normal mitosis, resulting in the disruption of chromosomal segregation and cytokinetic furrow function leading to aberrant mitotic exit (Figure 1C).
Electric field intensity is not homogenous within the brain of patients undergoing TTFields treatment32. Electrical conductivity and relative permittivity of individual tissue types and their volume results in a variation of electric field intensity and distribution within the brain, shown in Figure 2A,B. Therefore, transducer array placement may have an effect on electric field strength in the region of the tumor. An example of this variability is shown in Figure 2C, which predicts the electric field strength within the patient&#39;s brain at adjacent axial, coronal, and sagittal slices.
Figure 3A shows the personalized output of the treatment planning software for the proper placement of the arrays on a patient, shown in Figure 3B. Scalp sensitivity to the arrays can be alleviated by topical application of corticosteroids and by shifting the arrays as described in Figure 4.
The above protocol was used to treat a 56-year-old woman who developed a hemorrhage in the left frontal brain. She underwent a gross total resection of the hemorrhagic mass and the pathology showed IDH-1 mutated glioblastoma with hypercellularity, cellular atypia, mitotic figures and necrosis. She subsequently received external beam radiotherapy and daily temozolomide. Dexamethasone was stopped early at the second week of radiation. She experienced pancytopenia due to temozolomide administered during the adjuvant phase of treatment, requiring growth factor support as well as platelet and blood transfusions. Increased gadolinium enhancement was noted on head MRI 5 months after diagnosis and bevacizumab was started. Eight months after diagnosis TTFields therapy was also added. She has been maintained on the regimen of bevacizumab and TTFields for 48+ months after the diagnosis of her glioblastoma. The MRI images of this patient revealed stable disease for 48 months after initial diagnosis of glioblalstoma, shown in Figure 5. She has survived thus far with a high Karnofsky score of 80.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58937/58937fig1.jpg" /> 
Figure 1: TTFields disrupt mitosis during cell division. (A) Phase contrast microscopy was used to observe HeLa cells during mitosis. DRAQ5 is a DNA stain and was used to monitor chromosomal behavior. Image taken from a video of cells undergoing normal mitosis, included as an additional supplement. The procedures for obtaining video images were described in previous work11. (B) Phase contrast and DRAQ5 under TTFields show cell blebbing and aberrant mitosis. Scale bar = 20 &#181;m. Image taken from a video of cells undergoing mitosis during TTFields treatment, included as an additional supplement. The procedures for obtaining video images were described in previous work11. (C) Proposed model for TTFields-induced mitotic disruption. TTFields perturb septin association with the cytokinetic furrow and the subcortical actin cytoskeleton. This creates insufficient furrow contractility and makes cells vulnerable to plasma membrane rupture from the underlying cytoskeleton, resulting in membrane blebbing. This leads to aberrant mitotic exit including mitotic slippage (failure to divide) and asymmetric cell division. <a href="https://www.jove.com/files/ftp_upload/58937/58937fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58937/58937fig2.jpg" /> 
Figure 2: In situ electric field intensities vary within tissues based on electric conductivity and relative permittivity of the tissues they pass through. (A) Electric Field-Volume Histogram (EVH) shows the magnitude of electric field strength. (B) Specific Absorption Rate-Volume histogram (SARVH) shows the rate of energy absorbed in different tissues. (C)&#160;Representative field mapping of a patient with a left frontal glioblastoma, showing field strength within distributions on axial, coronal and sagittal slices. Green arrows indicate location of tumor. Relative electric field intensity is arbitrary. <a href="https://www.jove.com/files/ftp_upload/58937/58937fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58937/58937fig3.jpg" /> 
Figure 3: Clinical application on a glioblastoma patient after surgery, radiation and temozolomide. (A) Treatment planning software output showing placement of the 4 arrays. (B) Array placement on the patient. <a href="https://www.jove.com/files/ftp_upload/58937/58937fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58937/58937fig4.jpg" /> 
Figure 4: Array placement variation during treatment. (A) The individual lateral arrays should be rotated in aggregate by 2 cm from their primary position in a clockwise fashion, and the frontal and posterior arrays moved forward by 2 cm from the (B) primary positions for array placement position, which are based on the output from the treatment planning software for the individual patient. (C) The individual electrodes in each array should be rotated in aggregate by 2 cm from the primary position in a counterclockwise fashion, and the anterior and posterior arrays moved in aggregate by 2 cm backwards. <a href="https://www.jove.com/files/ftp_upload/58937/58937fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58937/58937fig5.jpg" /> 
Figure 5: Patient MRI scans before and after TTFields treatment. MRI scans at diagnosis (left column), MRI scans after surgery, radiation, and temozolomide (middle column), and MRI scans after 43 months of TTFields treatment (right column). <a href="https://www.jove.com/files/ftp_upload/58937/58937fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma at JoVE.com

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

the-clinical-application-tumor-treating-fields-therapy

Research

JoVE Journal

Cancer Research

17.8K Views.  Harvard Medical School, Beth Israel Deaconess Medical Center. Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Video: The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (&#400; &#62; 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields TTFields

Tumor Treating Fields (TTFields) are an effective anti-tumor treatment modality delivered via the continuous, noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application to cell lines using a TTFields in vitro application system allows for the determination of the optimal frequency that leads to the highest reduction in cell counts.

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), ...

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 ...

Medicine

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Immunology and Infection

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Sonodynamic Therapy for the Treatment of Glioblastoma Multiforme in a Mouse Model Using a Portable Benchtop Focused Ultrasound System

Sonodynamic therapy (SDT) is an application of focused ultrasound (FUS) that enables a sonosensitizing agent to prime tumors for increased sensitivity during sonication. Unfortunately, current clinical treatments for glioblastoma (GBM) are lacking, leading to low long-term survival rates among patients. SDT is a promising method for treating GBM in an effective, noninvasive, and tumor-specific manner. Sonosensitizers preferentially enter tumor cells compared to the surrounding brain parenchyma. The application of FUS in the presence of a sonosensitizing agent generates reactive oxidative species resulting in apoptosis. Although this therapy has been shown previously to be effective in preclinical studies, there is a lack of established standardized parameters. Standardized methods are necessary to optimize this therapeutic strategy for preclinical and clinical use. In this paper, we detail the protocol to perform SDT in a preclinical GBM rodent model using magnetic resonance-guided FUS (MRgFUS). MRgFUS is an important feature of this protocol, as it allows for specific targeting of a brain tumor without the need for invasive surgeries (e.g., craniotomy). The benchtop device used here can focus on a specific location in three dimensions by clicking on a target on an MRI image, making target selection a straightforward process. This protocol will provide researchers with a standardized preclinical method for MRgFUS SDT, with the added flexibility to change and optimize parameters for translational research.

Here, we describe a protocol that details how to perform sonodynamic therapy in an in vivo mouse glioblastoma model using magnetic resonance-guided focused ultrasound.

Sonodynamic therapy (SDT) is an application of focused ultrasound (FUS) that enables a sonosensitizing agent to prime tumors for increased sensitivity ...