BIOM-12. DEVELOPMENT OF 5-ALA BASED LIQUID BIOPSY FOR THE NON-INVASIVE DIAGNOSIS OF GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi12-vi12
Author(s):  
Anudeep Yekula ◽  
Tiffaney Hsia ◽  
Leonora Balaj ◽  
Bob Carter
Keyword(s):  
Liquid Biopsy ◽  
Aminolevulinic Acid ◽  
Expression Patterns ◽  
Protoporphyrin Ix ◽  
Digital Pcr ◽  
Sequencing Analysis ◽  
Guided Surgery ◽  
Non Invasive ◽  
Fluorescence Guided Surgery ◽  
Healthy Control

Abstract INTRODUCTION Tumor specificity of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence is widely applied for fluorescence-guided surgery (FGS) in gliomas. We recently showed the feasibility of detecting tumour-specific fluorescent PpIX extracellular vesicles (EVs) derived from the plasma of glioblastoma (GBM) patients undergoing 5-ALA based fluorescence-guided surgery. Here, we further develop methods to characterize, sort and study fluorescent PpIX EVs in plasma of patients with glioma. METHODS We used imaging flow cytometry and Astrios EQ nanoFACS to characterize and sort PpIX EVs, respectively. Downstream RNA analysis utilized transcriptome sequencing analysis and droplet digital PCR (EGFRvIII mRNA). RESULTS All GBM cell lines (Gli36vIII, U87, Gli36 WT) dosed with 5-ALA demonstrated PpIX fluorescence, and released PpIX positive EVs. There was a high correlation between fluorescence in cells and the number of PpIX EVs released (r2=0.9). We sorted 100,000 PpIX EVs from Gli36vIII cells dosed with 5-ALA and detected 65 copies and 24 copies of mutant EGFRvIII mRNA and wildtype EGFR mRNA per 100,000 EVs, respectively. RNAseq analysis of the sorted PpIX EVs showed expression patterns reflective of parent cells. Furthermore, 100,000 sorted PpIX EVs from the plasma of a patient with EGFRvIII glioma yielded 22 copies of EGFRvIII mRNA while < 5 copies were detected in 1ml of plasma and healthy control plasma, demonstrating the tumor-specific nature of PpIX EVs. Finally, we performed transcriptome analysis on 250,000 PpIX EVs each from 8 patients undergoing 5-ALA based FGS. We identified several mRNAs including Gli3, STAG2, ELF3, PHLPP1 which play an important role in cancer. CONCLUSION The ability to sort and characterize tumor specific PpIX EVs following 5-ALA administration opens new avenues for liquid biopsy-based glioma diagnosis.

scholarly journals 5-Aminolevulinic Acid–Protoporphyrin IX Fluorescence-Guided Surgery for CNS Tumors

2018 ◽  
Vol 27 (1) ◽  
pp. 13-19
Author(s):  
Ricardo Ramina ◽  
Erasmo Barros Da Silva Júnior ◽  
Maurício Coelho Neto ◽  
Leonardo Gilmone Ruschel ◽  
Felipe Andrés Constanzo Navarrette
Keyword(s):  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
High Grade Glioma ◽  
Awake Surgery ◽  
Histopathological Diagnosis ◽  
Low Grade ◽  
High Grade ◽  
Guided Surgery ◽  
Fluorescence Guided Surgery ◽  
High Grade Gliomas

Introduction: In the last two decades the 5-aminolevulinic acid (5-ALA) has been utilized in primary brain lesions and metastases surgery to aid the identification of tumor limits and infiltration. Objectives: In this retrospective study, we demonstrate our experience with the first 41 cases Latin America of surgical resection of central nervous system (CNS) lesions with 5-ALA. Methods: In 41 consecutive patients, we recorded age, sex, histopathological diagnosis, intraoperative 5-ALA fluorescence tumor response, 5-ALA post-resection resection grade through magnetic resonance image (MRI) and other concomitantintra-operative techniques utilized (transoperative imaging, awake surgery, electrophysiological stimulation and monitoring). Results: Twenty seven high-grade gliomas and 4 non-glial lesions were 5-ALA fluorescence positive; 6 low-grade gliomas, 1 high-grade glioma and a hippocampal gliosis were 5-ALA fluorescence negative. In one case of a low-grade glioma, the patient developed a cardiac arrhythmia, probably not related to 5-ALA administration, but the surgery was suspended. Conclusions: 5-ALA fluorescence-guided surgery is a safe and easy technique to be used, increasing tumor total gross resection in glioma cases, proving to be an invaluable neurosurgical tool for intracranial tumor surgery. There was no serious side effect in this series. This dye should be utilized in all cases of high-grade gliomas. 

Influence of dexamethasone on visible 5-ALA fluorescence and quantitative protoporphyrin IX accumulation measured by fluorescence lifetime imaging in glioblastomas: is pretreatment obligatory before fluorescence-guided surgery?

2021 ◽  
pp. 1-9
Author(s):  
Lisa I. Wadiura ◽  
David Reichert ◽  
Veronika Sperl ◽  
Alexandra Lang ◽  
Barbara Kiesel ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Aminolevulinic Acid ◽  
Ex Vivo ◽  
Protoporphyrin Ix ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Guided Surgery ◽  
Fluorescence Guided Surgery ◽  
Significant Difference ◽  
The Mean

OBJECTIVE Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) is nowadays widely applied for improved resection of glioblastomas (GBMs). Initially, pretreatment with dexamethasone was considered to be essential for optimal fluorescence effect. However, recent studies reported comparably high rates of visible fluorescence in GBMs despite absence of dexamethasone pretreatment. Recently, the authors proposed fluorescence lifetime imaging (FLIM) for the quantitative analysis of 5-ALA–induced protoporphyrin IX (PpIX) accumulation. The aim of this study was thus to investigate the influence of dexamethasone on visible fluorescence and quantitative PpIX accumulation. METHODS The authors prospectively analyzed the presence of visible fluorescence during surgery in a cohort of patients with GBMs. In this study, patients received dexamethasone preoperatively only if clinically indicated. One representative tumor sample was collected from each GBM, and PpIX accumulation was analyzed ex vivo by FLIM. The visible fluorescence status and mean FLIM values were correlated with preoperative intake of dexamethasone. RESULTS In total, two subgroups with (n = 27) and without (n = 20) pretreatment with dexamethasone were analyzed. All patients showed visible fluorescence independent from preoperative dexamethasone intake. Furthermore, the authors did not find a statistically significant difference in the mean FLIM values between patients with and without dexamethasone pretreatment (p = 0.097). CONCLUSIONS In this first study to date, the authors found no significant influence of dexamethasone pretreatment on either visible 5-ALA fluorescence during GBM surgery or PpIX accumulation based on FLIM. According to these preliminary data, the authors recommend administering dexamethasone prior to fluorescence-guided surgery of GBMs only when clinically indicated.

scholarly journals Fluorescence-Guided Surgery in the Surgical Treatment of Gliomas: Past, Present and Future

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3508
Author(s):  
Rosa Sun ◽  
Hadleigh Cuthbert ◽  
Colin Watts
Keyword(s):  
Surgical Treatment ◽  
Surgical Resection ◽  
Patient Outcomes ◽  
Aminolevulinic Acid ◽  
Guided Surgery ◽  
Improved Outcomes ◽  
Fluorescence Guided Surgery ◽  
Central Nervous Systems ◽  
Extent Of Surgical Resection ◽  
Disease Free

Gliomas are central nervous systems tumours which are diffusely infiltrative and difficult to treat. The extent of surgical resection is correlated with improved outcomes, including survival and disease-free progression. Cancerous tissue can be directly visualised intra-operatively under fluorescence by administration of 5-aminolevulinic acid to the patient. The adoption of this technique has allowed surgeons worldwide to achieve greater extents of resection, with implications for improved prognosis. However, there are practical limitations to use of 5-aminolevulinic acid. New adjuncts in the field of fluorescence-guided surgery aim to improve recognition of the interface between tumour and brain with the objective of improving resection and patient outcomes.

scholarly journals Characterizing the heterogeneity in 5-aminolevulinic acid–induced fluorescence in glioblastoma

2020 ◽  
Vol 132 (6) ◽  
pp. 1706-1714 ◽  
Author(s):  
Damian A. Almiron Bonnin ◽  
Matthew C. Havrda ◽  
Myung Chang Lee ◽  
Linton Evans ◽  
Cong Ran ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Tissue ◽  
Aminolevulinic Acid ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Protoporphyrin Ix ◽  
Progression Free Survival ◽  
The Cancer Genome Atlas ◽  
Diagnostic Challenge ◽  
Biological Basis

OBJECTIVE5-aminolevulinic acid (5-ALA)–induced protoporphyrin IX (PpIX) fluorescence is an effective surgical adjunct for the intraoperative identification of tumor tissue during resection of high-grade gliomas. The use of 5-ALA-induced PpIX fluorescence in glioblastoma (GBM) has been shown to double the extent of gross-total resection and 6-month progression-free survival. The heterogeneity of 5-ALA-induced PpIX fluorescence observed during surgery presents a technical and diagnostic challenge when utilizing this tool intraoperatively. While some regions show bright fluorescence after 5-ALA administration, other regions do not, despite that both regions of the tumor may be histopathologically indistinguishable. The authors examined the biological basis of this heterogeneity using computational methods.METHODSThe authors collected both fluorescent and nonfluorescent GBM specimens from a total of 14 patients undergoing surgery and examined their gene expression profiles.RESULTSIn this study, the authors found that the gene expression patterns characterizing fluorescent and nonfluorescent GBM surgical specimens were profoundly different and were associated with distinct cellular functions and different biological pathways. Nonfluorescent tumor tissue tended to resemble the neural subtype of GBM; meanwhile, fluorescent tumor tissue did not exhibit a prominent pattern corresponding to known subtypes of GBM. Consistent with this observation, neural GBM samples from The Cancer Genome Atlas database exhibited a significantly lower fluorescence score than nonneural GBM samples as determined by a fluorescence gene signature developed by the authors.CONCLUSIONSThese results provide a greater understanding regarding the biological basis of differential fluorescence observed intraoperatively and can provide a basis to identify novel strategies to maximize the effectiveness of fluorescence agents.

scholarly journals A spectrally constrained dual-band normalization technique for protoporphyrin IX quantification in fluorescence-guided surgery

2012 ◽  
Vol 37 (11) ◽  
pp. 1817 ◽  
Author(s):  
P. A. Valdés ◽  
F. Leblond ◽  
A. Kim ◽  
B. C. Wilson ◽  
K. D. Paulsen ◽  
...  
Keyword(s):  
Protoporphyrin Ix ◽  
Dual Band ◽  
Guided Surgery ◽  
Fluorescence Guided Surgery

scholarly journals Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3297
Author(s):  
Sybren L. N. Maas ◽  
Thomas S. van Solinge ◽  
Rosalie Schnoor ◽  
Anudeep Yekula ◽  
Joeky T. Senders ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Aminolevulinic Acid ◽  
Culture Media ◽  
Protoporphyrin Ix ◽  
Liquid Biopsies ◽  
Guided Surgery ◽  
Tumor Visualization ◽  
Isolation And Characterization ◽  
Droplet Pcr

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

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