LBC Protocols/Projects

563

Establishing an assay pipeline for comprehensive molecular profiling of extracellular vesicles

Among the consortium members, the Lee and the Balaj teams have exploited EVs as cancer biomarkers, focusing on different molecular targets: EV proteins (Lee)4 and RNA (Balaj)5. Combining these complementary sets of expertise, we propose to establish a streamlined workflow for comprehensive molecular EV phenotyping from various bio-samples (Fig. 2). Specifically, we will focus on i) optimizing EV isolation protocol compatible with downstream EV protein and RNA analyses and ii) performing multifaceted EV profiling using GBM as a model system.</p>

Aim 1. Standardize method for sample processing Aim 2. Profile EV protein and RNA signatures for GBM diagnostics and resistance monitoring

564

Early detection of pancreatic cancer utilizing the liquid biopsy

The primary goal of this work is to develop a blood based liquid biopsy that could be used as a primary test for pancreatic cancer in symptomatic patients that are otherwise low-risk. The second goal is to determine whether the liquid biopsy can be exploited t o both provide higher specificity and predict whether a patient will progress to metastatic disease.

To determine the cancer specific liquid biopsy signal, we require non-malignant disease controls for comparison. Dr. Nissen of Cedars Sinai Medical Center will provide portal vein and peripheral blood from patients with non-malignant pancreatic disease to complement the pancreatic cancer samples being collected in parallel. To precisely measure the state of the disease the use of disease-specific controls allows for a comparison with a group of individuals who share similar characteristics except for the malignant nature of the lesion. This helps ensure that observed outcomes are directly attributable to the intervention rather than other confounding factors. Including disease-specific controls also helps minimize bias in study results. We have 254 samples from 62 pancreatic cancer patients and over 200 normal (healthy) samples already accrued and cryobanked (current Aim 2). A group of 50 patients with non-malignant pancreatic disorders will now be added to this cohort. The blood is separated into cellular and acellular fractions. We will perform rare event analysis (Kuhn), cfDNA methylation (Zhou), and exosome characterization (Pandol).

565

Multi-analyte Approach for Earlier Detection of Cancers in Non-Plasma Biofluids

Primary glial tumors confer the greatest numbers of years of life lost for both men and women, as evidenced by earlier onset and low survivability as compared to other tumor types. Overall, gliomas are one of the most expensive and morbid neoplasms with average survival still reported to be between 15 and 30 months. Clinical management and the affiliated neurological complications pose potential intellectual and motor impairment and effectuate clinical costs and care obligations. Compared with other malignancies, there have only been small improvements in the prognosis of glioma and particularly glioblastoma patients over recent decades. Currently, most diagnoses of gliomas occur via direct biopsy of tissue, an invasive procedure, with large studies demonstrating a modest risk (up to 5%) of intracranial and symptomatic hemorrhage, particularly for brainstem tumors, as well as a risk of inaccurate tumor grading due to sampling and/or intratumoral heterogeneity. Prior to biopsy, MRI is the mainstay diagnostic platform used to develop a differential diagnosis of a patient with a suspected brain tumor. Although MRI remains a powerful, noninvasive tool that positively improves management of patients with brain tumors, it has many limitations, such as the inability to determine the genetic landscape (genome/transcriptome) of the tumor or to determine pseudoprogression from an actual tumor regression in monitoring treatment response.

Task 1. Validation of the performance of ddPCR assays (EGFRvIII, IDH1.R132H, TERTp) in plasma and compare to performance by Methyl-SaferSeqS Task 2. Validation of the performance of ddPCR assays (EGFRvIII, IDH1, TERTp) in CSF samples

566

Methylated cfDNA for Malignancy Assessment in Indeterminate Pulmonary Nodules (IPN)

cfDNA methylome emerging as promising biomarkers for detection of cancers4. Identifying the differentially methylated regions and tissue of origins based on epigenetic modifications, is immensely advancing Liquid Biopsy (LB) for cancer detection2, 5. Methylation signatures offer insights into cfDNA origins and association with different biological processes6. At present, methylation analysis of cfDNA is based on fragments measuring about ~167 bps, mncfDNA. With identification of shorter fragments of cfDNA, opens newer avenues and aspects for cfDNA-based LB assays development

Aim 1: Establishing techniques for analysis of methylated uscfDNA Aim 2: BRcfDNAmethyl-Seq for early assessment of malignancy in IPN Aim 3: Integration of validated BRcfDNAmethyl-Seq metrics with parent Lung Cancer Prediction algorithm (IPN.CA)