Chordoma Detection Using 5-ALA Tumor Paint: An In Vitro Study 1 - - PowerPoint PPT Presentation

Chordoma Detection Using 5-ALA Tumor Paint: An In Vitro Study

December 9th, 2017

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Kenan, S1; Nixon, R1; Liang, H2; Goodman, H1, Grande, D2; Levin, A3; Hofstra Northwell Health Medical Center, Department of Orthopaedic Surgery1 The Feinstein Institute for Medical Research2 Johns Hopkins University Department of Orthopaedic Surgery3 skenan1@northwell.edu

Disclosures: None

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Chordoma Detection Using 5-ALA Tumor Paint

Hypothesis: Five-aminolevulinic acid tumor paint will selectively induce fluorescence in chordoma cells Level of Evidence: V

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Chordoma

• A rare malignant primary bone tumor originating from embryonic

notochord remnants1

• Comprise 1% to 4% of all primary bone tumors2
• The most common primary bone tumor found in the mobile spine and

sacrum2

• Median age at diagnosis: 58.52

1. Garofalo F, di Summa PG, Christoforidis D, Pracht M, Laudato P, Cherix S, et al. Multidisciplinary approach of lumbo-sacral chordoma: From

• ncological treatment to reconstructive surgery. Journal of surgical oncology. 2015 Oct;112(5):544-54.

2. Sciubba DM, Cheng JJ, Petteys RJ, Weber KL, Frassica DA, Gokaslan ZL. Chordoma of the sacrum and vertebral bodies. The Journal of the American Academy of Orthopaedic Surgeons. 2009 Nov;17(11):708-17. 4

Management

• Goal: Wide resection with negative margins
• Prognosis in terms of overall survival and recurrence is highly dependent
• n initial surgical margins
• Poor prognosis (SEER database, 1973-19953):
• 5-year median survival of 6.29 years
• 5-year survival rate: 67.6%
• 10-year survival rate: 39.9%
• Postop complications4:
• Wound dehiscence/infections
• Bladder and bowel dysfunction
• Sexual dysfunction
• Motor deficits
• Lymphatic complications, hematomas and CSF fistulas

3. McMaster ML, Goldstein AM, Bromley CM, Ishibe N, Parry DM. Chordoma: incidence and survival patterns in the United States, 1973-1995. Cancer causes & control : CCC. 2001 Jan;12(1):1-11. 4. Fuchs B, Dickey ID, Yaszemski MJ, Inwards CY, Sim FH. Operative management of sacral chordoma. The Journal of bone and joint surgery American volume. 2005 Oct;87(10):2211-6. 5. Image: Varga, P.P., Bors, I. and Lazary, A., 2009. Sacral tumors and management. Orthopedic Clinics of North America, 40(1), pp.105-123. 5

Tumor Paint

• The use of an intraoperative optical imaging contrast agent to help differentiate cancer foci

from adjacent normal tissue6

• Pioneered by Stummer et. al. for GBM resections using 5-ALA in 20007
• A meta-analysis of 10 studies involving intraoperative 5-ALA fluorescence-guided resections of

gliomas suggested that 5-ALA–guided surgery is more effective than conventional surgery, enhancing quality of life and prolonging survival8

6. Mandana, et al. "Tumor paint: a chlorotoxin: Cy5. 5 bioconjugate for intraoperative visualization of cancer foci." Cancer Research 67.14 (2007): 6882-6888. 7. Stummer, Walter, et al. "Fluorescence-guided resection of glioblastoma multiforme utilizing 5-ALA-induced porphyrins: a prospective study in 52 consecutive patients." Journal of Neurosurgery 93.6 (2000): 1003-1013. 8. Zhao S, Wu J, Wang C, Liu H, Dong X, Shi C, et al. Intraoperative fluorescence-guided resection of high-grade malignant gliomas using 5- aminolevulinic acid-induced porphyrins: a systematic review and meta-analysis of prospective studies. PloS one. 2013;8(5):e63682. 6

5-Aminolevulinic Acid (5-ALA)

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5-ALA

• Safe to use on humans9
• FDA approved for use in various fields including

dermatology and urology10

• Approved in Europe for intravenous use in

neurosurgery11

9. Perez MH, Rodriguez BL, Shintani TT, Watanabe K, Miyanari S, Harrigan RC. 5-Aminolevulinic acid (5-ALA): analysis of preclinical and safety

• literature. Food and Nutrition Sciences. 2013;4(10):1009.

10. Krammer B, Plaetzer K. ALA and its clinical impact, from bench to bedside. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology. 2008 Mar;7(3):283-9. 11. Teixidor P, Arraez MA, Villalba G, Garcia R, Tardaguila M, Gonzalez JJ, et al. Safety and Efficacy of 5-Aminolevulinic Acid for High Grade Glioma in Usual Clinical Practice: A Prospective Cohort Study. PloS one. 2016;11(2):e0149244. 8

Prior Study: Myxofibrosarcoma Cell Detection Using 5-ALA: An In Vitro Study

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Conclusion: Myxofibrosarcoma cells retain cell fluorescence in response to 5-ALA exposure unlike control adipose cells

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Prior Study: Myxofibrosarcoma Cell Detection Using 5-ALA: An In Vitro Study

• 50,000 rat adipose derived stromal cells (ADS) carrying the GFP (green fluorescence

protein) gene were co-cultured with 50,000 human chordoma cells for 4 weeks

• Cells were exposed to 5-ALA (1000µg/mL) for 5 hours and then observed under

phase microscopy

• Microscope parameters:
• 10X magnification
• 509nm emission filter used to detect GFP cells, appears as green
• 635nm filter was used to detect 5-ALA fluorescence, appears as red

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Methods – Experiment I

• Two separate 3-dimensional micromass pellets consisting of 500,000 cells were

made:

• GFP-ADS rat cells
• Human chordoma cells
• Co-cultured for 3 weeks and then exposed to 5-ALA under same prior conditions
• Cells observed under phase microscopy with same magnification and emission filter

settings

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Methods – Experiment II

• 50,000 human adipose derived stromal cells and 50,000 chordoma cells were co-

cultured for 2 weeks and then exposed to 5-ALA as in prior experiments

• Cells observed under phase microscopy as in prior experiments

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Methods – Experiment III

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Results – Experiment I

Phase GFP 5-ALA

As a micromass, the distinction between chordoma cells (red color, center) and rat GFP adipose cells (green color, periphery) is clearly seen when both emission filters are combined

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Results – Experiment II

Phase Combined GFP 5-ALA

As in the first two experiments, the third experiment showed minimal fluorescence in response to 5-ALA when chordoma cells were visualized in less concentrated areas, whereas cells which were heavily clustered fluoresced greatly in response to 5-ALA

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Results – Experiment III

Phase Phase 5-ALA 5-ALA

Monolayer region Densely packed region

• The aim of this study was to analyze tumor paint application for fluorescence of

chordoma cells using the photosensitizer, 5-ALA, in vitro

• All of the experiments carried out in this study showed increased and selective

fluorescence of chordoma cells after exposure to 5-ALA when tumor cells were either heavily clustered or grown in micromass form

• As chordoma tumors are faced with increased cellular crowding, a decreased ability

to metabolize 5-ALA seems to ensue. This may be due to a loss of differentiation as the chordoma tumors increase in size, making it more difficult to expel 5-ALA

• The fluorescent by-product, protoporphyrin IX accumulates in these cells but not in

benign cells, leading to selective tumor fluorescence

• We believe visual detection of these complex tumors can help improve margin

identification, leading to clearer margins and decreased recurrence rates

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Conclusion

• Photodynamic therapy (PDT)
• In vivo rat model
• Other tumor types:
• Myxofibrosarcoma
• Osteosarcoma
• Breast cancer
• Other contrast agents
• Acridine Orange
• BLZ-100

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Future Steps

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Thank You