December 9 th , 2017 Kenan, S 1 ; Nixon, R 1 ; Liang, H 2 ; Goodman, H 1 , Grande, D 2 ; Levin, A 3 ; Hofstra Northwell Health Medical Center, Department of Orthopaedic Surgery 1 The Feinstein Institute for Medical Research 2 Johns Hopkins University Department of Orthopaedic Surgery 3 skenan1@northwell.edu Chordoma Detection Using 5-ALA Tumor Paint: An In Vitro Study 1
Disclosures: None 2
Chordoma Detection Using 5-ALA Tumor Paint Hypothesis: Five-aminolevulinic acid tumor paint will selectively induce fluorescence in chordoma cells Level of Evidence: V 3
Chordoma A rare malignant primary bone tumor originating from embryonic • notochord remnants 1 Comprise 1% to 4% of all primary bone tumors 2 • The most common primary bone tumor found in the mobile spine and • sacrum 2 Median age at diagnosis: 58.5 2 • 1. Garofalo F, di Summa PG, Christoforidis D, Pracht M, Laudato P, Cherix S, et al. Multidisciplinary approach of lumbo-sacral chordoma: From oncological 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 4 American Academy of Orthopaedic Surgeons. 2009 Nov;17(11):708-17.
Management Goal: Wide resection with negative margins • Prognosis in terms of overall survival and recurrence is highly dependent • on initial surgical margins Poor prognosis (SEER database, 1973-1995 3 ): • 5-year median survival of 6.29 years o 5-year survival rate: 67.6% o 10-year survival rate: 39.9% o Postop complications 4 : • Wound dehiscence/infections o Bladder and bowel dysfunction o Sexual dysfunction o Motor deficits o Lymphatic complications, hematomas and CSF fistulas o 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 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.
Tumor Paint The use of an intraoperative optical imaging contrast agent to help differentiate cancer foci • from adjacent normal tissue 6 Pioneered by Stummer et. al. for GBM resections using 5-ALA in 2000 7 • 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 survival 8 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- 6 aminolevulinic acid-induced porphyrins: a systematic review and meta-analysis of prospective studies. PloS one. 2013;8(5):e63682.
5-Aminolevulinic Acid (5-ALA) 7
5-ALA Safe to use on humans 9 • FDA approved for use in various fields including • dermatology and urology 10 Approved in Europe for intravenous use in • neurosurgery 11 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 8 Glioma in Usual Clinical Practice: A Prospective Cohort Study. PloS one. 2016;11(2):e0149244.
Prior Study: Myxofibrosarcoma Cell Detection Using 5-ALA: An In Vitro Study 9
Prior Study: Myxofibrosarcoma Cell Detection Using 5-ALA: An In Vitro Study Conclusion: Myxofibrosarcoma cells retain cell fluorescence in response to 5-ALA exposure unlike control adipose cells 10
Methods – Experiment I 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 o 509nm emission filter used to detect GFP cells, appears as green o 635nm filter was used to detect 5-ALA fluorescence, appears as red o 11
Methods – Experiment II Two separate 3-dimensional micromass pellets consisting of 500,000 cells were • made: GFP-ADS rat cells o Human chordoma cells o 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 12
Methods – Experiment III 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 • 13
Results – Experiment I 5-ALA GFP Phase 14
Results – Experiment II GFP Phase 5-ALA Combined 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 15
Results – Experiment III Monolayer region 5-ALA Phase Densely packed region Phase 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 16
Conclusion 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 17
Future Steps Photodynamic therapy (PDT) • In vivo rat model • Other tumor types: • o Myxofibrosarcoma o Osteosarcoma o Breast cancer Other contrast agents • o Acridine Orange o BLZ-100 18
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