Evaluating Circulating Tumor DNA From Cerebrospinal Fluid

34 structural variants. While many labs are beginning to use targeted NGS panels to detect such events, we considered whole transcriptome sequencing (RNA-Seq) to be the ultimate solution as it samples all expressed loci including those only relevant to rare pediatric tumors. Clinical RNA-Seq, although an attractive option, presents several unique challenges including i) How to avoid an informatics bottleneck within a time-sensitive workflow ii) How to minimally burden those reviewing the results and iii) How to standardize and share complex computational analysis methods. Here we present a pilot study of clinical RNASeq analyzed in the cloud. Using a highly secure platform, we developed our Rapid RNA-Seq pipeline centered on an extensively validated fusion gene detection algorithm “CICERO.” We initially detected fusion transcripts and structural variants in 78 diverse but well-characterized samples and showed that Rapid RNA-Seq detected all abnormalities found previously. We next ran Rapid RNA-Seq in parallel with standard testing in real time, again showing concordance, but also detecting a large cache of previously undetected events—some of which were targetable. Run times averaged approximately four hours from raw data acquisition to reporting and were not affected by caseload. To aid in clinical review, we developed a rich graphical interface based on our ProteinPaint software (https://pecan.stjude.org/#/home) that allowed pathologists to easily assess the supporting evidence and functional impact of any given event. We make our tools and workflows securely available at www.stjude.cloud.

7 Optimization of Blood-Based Liquid Biopsy Assays for the Rapid Identification of 19 Actionable RNA Fusion Variants in NSCLC Kristin E. Alexander a, Leisa P. Jackson a, Hestia S. Mellert a, Gary A. Pestano a a Biodesix, Boulder, CO, USA

Background: Non-small-cell lung cancer (NSCLC) remains a diagnostic challenge for physicians. Considering the lethality of this disease, rapid diagnostic turnaround times are a critical factor in patient care. While the highest risk factor for NSCLC is smoking history, nonsmokers who develop the disease more commonly have chromosomal rearrangements, including fusions in anaplastic lymphoma kinase (ALK), c-rosoncogene 1, receptor tyrosine kinase (ROS1), and rearranged during transfection (RET) proto-oncogene. The incidence of these variants in the total NSCLC population is estimated to be 5%, 2%, and 1%, respectively. Biodesix has developed commercially available blood-based tests for circulating tumor RNA that detects the presence of actionable RNA fusion variants in NSCLC for use in its CLIA certified laboratory. Methods: Starting with up to 4 mL of patient plasma, circulating RNA is isolated and converted into cDNA in a single reaction. The cDNA produced is tested with multiplexed ALK, ROS1, and RET ddPCR assays covering 78%, 87.5% and 99% of the prevalent fusion variants, respectively. Results are provided to physicians within 72 hours of sample receipt. Results: 19 fusion variants were detected with an analytic sensitivity of 0.2% or lower and 100% analytic specificity. With EML4-ALK variants, a clinical concordance of 92% was achieved. Conclusion: We have developed novel blood-based tests for the most common NSCLC RNA fusion variants that result from ALK, ROS1, and RET rearrangements. The rapid turn-around offered by these blood-based tests

Abstracts has the potential to significantly shorten time to treatment for patients.

8 Evaluating Circulating Tumor DNA From Cerebrospinal Fluid Leomar Ballester a,b, Dzifa Douse c, Chieh Lan c, Lauren Haydu d, Jason Huse b,c, Sinchita Roy-Chowdhuri b, Rajyalakshmi Luthra e, Isabella Glitza-Oliva d, Ignacio Wistuba c, Michael Davies d a Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, USA; bDepartment of Pathology, UT-MD Anderson Cancer Center, Houston, TX, USA; cDepartment of Translational Molecular Pathology, UT-MD Anderson Cancer Center, Houston, TX, USA; dDepartment of Melanoma Medical Oncology, UT-MD Anderson Cancer Center, Houston, TX, USA; e Department of Hematopathology, UT-MD Anderson Cancer Center, Houston, TX, USA

Circulating tumor DNA (ctDNA) refers to cell-free DNA that circulates in body fluids as a result of tumor cell death. Fluid samples are easier to collect than tumor tissue, and are amenable to serial collection at multiple time points during the course a patient’s illness. Studies have demonstrated the feasibility of performing cancer mutation profiling from blood samples in patients with a variety of tumor types. However, the detection of ctDNA in the blood of patients with brain tumors appears to be suboptimal. Cerebrospinal fluid (CSF) can be obtained via lumbar puncture or implanted intraventricular catheter, and may be a suitable fluid to assess ctDNA in patients with primary and metastatic brain tumors. We evaluated the feasibility of obtaining ctDNA from CSF and performed droplet-digital PCR and next generation sequencing (NGS) in CSF-derived ctDNA from melanoma patients with brain metastasis and leptomeningeal disease. We compared the NGS results from CSF-derived ctDNA with formalin-fixed, paraffinembedded tumor tissue from intracranial and extracranial lesions. Our results demonstrate that CSF is a suitable sample for evaluating ctDNA in patients with brain metastasis. Interestingly, in the setting of genetic heterogeneity among intracranial and extracranial lesions, the mutation profile of CSF-derived ctDNA recapitulates the intracranial tumor.

9 Circulating Tumor DNA Genotyping in Endometrial Cancer Ana Bolivar a, Rajyalakshmi Luthra b, Meenakshi Mehrotra b, Wei Chen b, Bedia Barkoh b, Peter Hu a, Rajesh Singh b, Russel Broaddus c a School of Health Professions, Houston, TX, USA; bDepartment of Hematopathology, MD Anderson Cancer Center, Houston, TX, USA; cDepartment of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Extra-vaginal recurrence of endometrial endometrioid adenocarcinoma (EEA) is incurable; currently, there is no method to identify at-risk patients presenting with early stage disease. This proof-of-principal study was designed to detect mutations in circulating tumor DNA (ctDNA) of early stage EEA patients. We have previously shown that 98% of EEA have mutations in CTNNB1, KRAS, PTEN, and/or PIK3CA. Using a custom panel (Swift Biosciences) targeting 30 amplicons in these 4 genes, MiSeq NGS was performed on ctDNA extracted from frozen plasma and the matching tumor DNA (tDNA) extracted from FFPE slides from 33