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.

O-Arm Accuracy and Radiation Exposure in Adult Deformity Surgery.

OBJECTIVE: In long thoracolumbar deformity surgery, accurate screw positioning is critical for spinal stability. We assessed pedicle and pelvic screw accuracy and radiation exposure in patients undergoing long thoracolumbar deformity fusion surgery (≥4 levels) involving 3-dimensional fluoroscopy (O-Arm/Stealth) navigation. METHODS: In this retrospective single-center cohort study, all patients aged >18 years who underwent fusion in 2016-2018 were reviewed. O-Arm images were assessed for screw accuracy. Effective radiation doses were calculated. The primary outcome was pedicle screw accuracy (Heary grade). Secondary outcomes were pelvic fixation screw accuracy, radiation exposure, and screw-related perioperative and postoperative complications or revision surgery within 3 years. RESULTS: Of 1477 pedicle screws placed in 91 patients (mean 16.41 ± 5.6 screws/patient), 1208 pedicle screws (81.8%) could be evaluated by 3-dimensional imaging after placement. Heary Grade I placement was achieved in 1150 screws (95.2%), Grade II in 47 (3.9%), Grade III in 10 (0.82%), Grade IV in 1 (0.08%), and Grade V in 0; Grade III-V were replaced intraoperatively. One of 60 (1.6%) sacroiliac screws placed showed medial cortical breach and was replaced. The average O-Arm-related effective dose was 29.54 ± 14.29 mSv and effective dose/spin was 8.25 ± 2.65 mSv. No postoperative neurological worsening, vascular injuries, or revision surgeries for screw misplacement were recorded. CONCLUSIONS: With effective radiation doses similar to those in interventional neuroendovascular procedures, the use of O-Arm in multilevel complex deformity surgery resulted in high screw accuracy, no need for surgical revision because of screw malposition, less additional imaging, and no radiation exposure for the surgical team.

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.

The Effect of Hospital Transfer on Patient Outcomes After Rehabilitation for Spinal Injury.

OBJECTIVE: Spine fractures, including associated spinal cord injury, account for 3%-6% of all skeletal fractures annually in the United States. Patients who undergo interhospital transfer after injury may have a greater likelihood of nonroutine disposition, longer hospital stay, and higher cost. We evaluated the effects of patient transfer on functional outcomes after spine trauma. METHODS: Patients were treated after acute traumatic spine injury at a rehabilitation hospital in 2011-2017. Compared patients were those directly admitted to the tertiary hospital or transferred from a community hospital. RESULTS: A total of 188 patients (mean age 46.1 ± 18.6 years, 77.1% men) were evaluated, including 130 (69.1%) directly admitted and 58 (30.9%) transferred patients. The most common levels of injury were at C5 (19.1%) and C6 (12.2%), and most injuries were American Spinal Injury Association injury severity score grade D (33.2%) or grade A (32.1%). No statistical difference in age, injury pattern, timing from injury to surgery, or rehabilitation length of stay was seen between admitted and transferred patients. A significant improvement in ambulation distances was seen at discharge for directly admitted compared with transferred patients (447.7 ± 724.9 vs. 159.9 ± 359.5 feet; P = 0.005). However, no significant difference primary outcomes, namely American Spinal Injury Association injury severity score distribution (P = 0.2) or Functional Independence Measures (Δ30.9 ± 15.9 vs. 30.1 ± 17.1; P = 0.7), were seen between admitted and transferred patients at time of rehabilitation discharge. CONCLUSIONS: Interhospital transfer status did not diminish time to rehabilitation after injury or reduce functional recovery, suggesting early surgical treatment in community settings may have merit prior to transfer.