Rigorous Comparison of Extracellular Vesicle Processing to Enhance Downstream Analysis for Glioblastoma Characterization.

Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike-in into healthy donor plasma samples is performed to further validate biofluid-derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC-derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA-isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest.

The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring.

The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays.

Extrinsic and intrinsic preanalytical variables affecting liquid biopsy in cancer.

Liquid biopsy, through isolation and analysis of disease-specific analytes, has evolved as a promising tool for safe and minimally invasive diagnosis and monitoring of tumors. It also has tremendous utility as a companion diagnostic allowing detection of biomarkers in a range of cancers (lung, breast, colon, ovarian, brain). However, clinical implementation and validation remains a challenge. Among other stages of development, preanalytical variables are critical in influencing the downstream cellular and molecular analysis of different analytes. Although considerable progress has been made to address these challenges, a comprehensive assessment of the impact on diagnostic parameters and consensus on standardized and optimized protocols is still lacking. Here, we summarize and critically evaluate key variables in the preanalytical stage, including study population selection, choice of biofluid, sample handling and collection, processing, and storage. There is an unmet need to develop and implement comprehensive preanalytical guidelines on the optimal practices and methodologies.

Decoding vesicle-based precision oncology in gliomas.

Extracellular vesicles (EVs) represent a valuable tool in liquid biopsy with tremendous clinical potential in diagnosis, prognosis, and therapeutic monitoring of gliomas. Compared to tissue biopsy, EV-based liquid biopsy is a low-cost, minimally invasive method that can provide information on tumor dynamics before, during, and after treatment. Tumor-derived EVs circulating in biofluids carry a complex cargo of molecular biomarkers, including DNA, RNA, and proteins, which can be indicative of tumor growth and progression. Here, we briefly review current commercial and noncommercial methods for the isolation, quantification, and biochemical characterization of plasma EVs from patients with glioma, touching on whole EV analysis, mutation detection techniques, and genomic and proteomic profiling. We review notable advantages and disadvantages of plasma EV isolation and analytical methods, and we conclude with a discussion on clinical translational opportunities and key challenges associated with the future implementation of EV-based liquid biopsy for glioma treatment.

Highly Sensitive EGFRvIII Detection in Circulating Extracellular Vesicle RNA of Glioma Patients.

PURPOSE: Liquid biopsy offers an attractive platform for noninvasive tumor diagnosis, prognostication, and prediction of glioblastoma clinical outcomes. Prior studies report that 30% to 50% of GBM lesions characterized by EGFR amplification also harbor the EGFRvIII mutation. EXPERIMENTAL DESIGN: A novel digital droplet PCR (ddPCR) assay for high GC content amplicons was developed and optimized for sensitive detection of EGFRvIII in tumor tissue and circulating extracellular vesicle RNA (EV RNA) isolated from the plasma of patients with glioma. RESULTS: Our optimized qPCR assay detected EGFRvIII mRNA in 81% [95% confidence interval (CI), 68%-94%] of EGFR-amplified glioma tumor tissue, indicating a higher than previously reported prevalence of EGFRvIII in glioma. Using the optimized ddPCR assay in discovery and blinded validation cohorts, we detected EGFRvIII mutation in 73% (95% CI, 64%-82%) of patients with a specificity of 98% (95% CI, 87%-100%), compared with qPCR tumor tissue analysis. In addition, upon longitudinal monitoring in 4 patients, we report detection of EGFRvIII in the plasma of patients with different clinical outcomes, rising with tumor progression, and decreasing in response to treatment. CONCLUSIONS: This study demonstrates the feasibility of detecting EGFRvIII mutation in plasma using a highly sensitive and specific ddPCR assay. We also show a higher than previously reported EGFRvIII prevalence in glioma tumor tissue. Several features of the assay are favorable for clinical implementation for detection and monitoring of EGFRvIII-positive tumors.

Chronic hypertrophic malunion of C2 Fracture causing cervical quadriparesis; Case report and focused literature review.

BACKGROUND: Pseudarthrosis of Type II C2 odontoid fractures typically leads to displacement and subluxation resulting in canal compression/cervical myelopathy. CASE DESCRIPTION: Here, we present a 43-year-old male who sustained cervical trauma 28 years ago. He now presented with an acute 10-day onset of quadriparesis attributed to a chronic malunion of an unstable type II odontoid fracture. He successfully underwent a circumferential decompression and fusion (e.g., warranting a trans-oral odontoidectomy followed by C1-C3 posterior fusion). CONCLUSION: Progressive cervical myelopathy attributed to a chronic malunion of a type II odontoid fracture may require circumferential decompression/stabilization (e.g., an anterior decompression with osteophyte resection and posterior C1-C3 spinal stabilization).

Need of Physical and Chemical Restraints: Experiences at Inpatient Psychiatric Ward in a Tertiary Care Hospital in Karachi, Pakistan.

In psychiatry, agitated / aggressive patients are often treated with de-escalation techniques. If this does not work, physical or chemical restrains are required; but in the event of resistance, seclusion is applied. We report the findings of baseline study of experiences of physical and chemical restraints in a tertiary care hospital in Karachi, where 104 files were evaluated retrospectively. The mean age of patients was 32.5 ±14.3 years with 54.8% men, while the average length of stay was 11.5 ±9.3 days. Agitation, violent behaviour, and aggression were the most common indications for restraints. In total, 94.5% of patients had both physical and chemical restraints with the latter being used as the first choice in 70 patients; whereas, 67.1% of patients' families were not informed before application of restraints. The seclusion need assessment was conducted in 4.1% of patients.

Diffusion-weighted magnetic resonance imaging may be useful in differentiating fungal abscess from malignant intracranial lesion: Case report.

BACKGROUND: Diffusion-weighted magnetic resonance has a well-defined role in differentiating between important intracranial lesions. Sometimes, the surgeon is faced with a dilemma of how to diagnose an infectious versus malignant lesion. CASE DESCRIPTION: A 28-year-old male presented to the neurosurgery clinic with complaints of headache and left-sided weakness for 2 weeks. Neurological examination was intact. Magnetic resonance imaging (MRI) scan showed a large infiltrating heterogeneous mass involving the right parietal lobe. On further reviewing, there was homogenous diffusion restriction in the center of lesion. In addition, its aggressive behavior confirmed it to be a fungal abscess. CONCLUSIONS: Correctly identifying an infectious versus tumor etiology is important. Research has been carried out to employ diffusion-weighted imaging (DWI) in differentiating the variable radiological findings. The role of DWI in diagnosing bacterial abscess is more commonly seen in comparison to fungal abscess. DWI has a high diagnostic potential, but more works need to be done.