The Search for Molecular Markers in a Gene-Orphan Case Study of a Pediatric Spinal Cord Pilocytic Astrocytoma

Abstract

Background/Aim: We herein presented a case of pediatric spinal cord pilocytic astrocytoma diagnosed on the basis of histopathological and clinical findings. Materials and Methods: Given the paucity of data on genetic features for this tumor, we performed exome, array CGH and RNA sequencing analysis from nucleic acids isolated from a unique and not repeatable very small amount of a formalinfixed, paraffin-embedded (FFPE) specimen. Results: DNA mutation analysis, comparing tumor and normal lymphocyte peripheral DNA, evidenced few tumor-specific single nucleotide variants in DEFB119, MUC5B, NUDT1, LTBP3 and CPSF3L genes. Differently, tumor DNA was not characterized by for the main pilocytic astrocytoma gene variations, including BRAFV600E. An inframe trinucleotides insertion involving DLX6 or lnc DLX6-AS1 genes was scored in 44.9% of sequenced reads; the temporal profile of this variation on the expression of DLX-AS1 was investigated in patient`s urine-derived exosomes, reporting no significant variation in the one-year molecular follow-up. Array CGH identified a tumor microdeletion at the 6q25.3 chromosomal region, spanning 1,01 Mb and comprising ZDHHC14, SNX9, TULP4 and SYTL3 genes. The expression of these genes did not change in urine-derived exosomes during the one-year investigation period. Finally, RNAseq did not reveal any of the common pilocytic BRAF-KIAA1549 genes fusion events. Conclusion: To our knowledge, the present report is one of the first described gene-orphan case studies of a pediatric spinal cord pilocytic astrocytoma.

Enhanced Delivery of Rose Bengal by Amino Acids Starvation and Exosomes Inhibition in Human Astrocytoma Cells to Potentiate Anticancer Photodynamic Therapy Effects

“…In detail, culture media were collected, and one 0.5 volume of total exosome isolation reagent was added to the media and incubated at 4 °C for 20 h. Then, EVs were pelleted at 12,000 g for 60 min at 4 °C and finally resuspended into 500 μL of sterile ice-cold D-PBS (Thermofisher) and next filtered (0.22 μm). The qNano Gold instrument (Izon Science, Christchurch, New Zeeland) was employed to measure the size distribution and concentration of the isolated nanoparticles using the Tunable Resistive Pulse Sensing (TRPS) principle [ 29 , 30 ]. Briefly, 35 μL of purified particles were analyzed with a qNano Gold instrument using a NP200 Nanopore (Izon Science) and applying 49 mm stretch, 0.1 V, and 20 mBar parametric conditions.…”

Development of Artificial Plasma Membranes Derived Nanovesicles Suitable for Drugs Encapsulation

“…The qNano Gold instrument (Izon Science, Christchurch, New Zeeland) was employed to measure the size distribution, concentration and z-charge of the isolated nanoparticles (EVs, exosomes and M-NVs) using the Tunable Resistive Pulse Sensing (TRPS) principle [ 35 ]. Briefly, 35 μL of purified particles were analyzed with qNano Gold instrument using a NP200 Nanopore (Izon Science) and applying 46 mm stretch, 0.34 V, and 8 mBar parametric conditions as described [ 36 ]. The calibration particles (CPC100, Izon Science) were assayed before the experimental samples under identical conditions.…”

Development of Berberine-Loaded Nanoparticles for Astrocytoma Cells Administration and Photodynamic Therapy Stimulation

“…Drug loading (DL%) was calculated as follows: The qNano Gold instrument (Izon Science, Christchurch, New Zeeland) was employed to measure the size distribution and concentration of the isolated NPs using the tunable resistive pulse sensing (TRPS) principle as already reported [53,54]. Briefly, 35 µL of purified particles were analyzed with a qNano Gold instrument using a NP200 Nanopore (Izon Science) and applying 49 mm stretch, 0.1 V, and 20 mBar parametric conditions.…”