DNA derived from NSCLC tumors can be detected with high sensitivity in urine and plasma, enabling diagnostic detection and monitoring of therapeutic response from these noninvasive "liquid biopsy" samples.

Identification of marker genes expressed in specific cell types is essential for the genetic dissection of neural circuits. Here we report a new strategy for classifying heterogeneous populations of neurons into functionally distinct types and for identifying associated marker genes. Quantitative single-cell expression profiling of genes related to neurotransmitters and ion channels enables functional classification of neurons; transcript profiles for marker gene candidates identify molecular handles for manipulating each cell type. We apply this strategy to the mouse medial vestibular nucleus (MVN), which comprises several types of neurons subserving cerebellar-dependent learning in the vestibulo-ocular reflex. Ion channel gene expression differed both qualitatively and quantitatively across cell types and could distinguish subtle differences in intrinsic electrophysiology. Single-cell transcript profiling of MVN neurons established 6 functionally distinct cell types and associated marker genes. This strategy is applicable throughout the nervous system and could facilitate the use of molecular genetic tools to examine the behavioral roles of distinct neuronal populations.

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Background: Non-invasive detection and monitoring of circulating tumor DNA (ctDNA) mutations for personalized treatment of cancer patients can be realized by combining the practical advantages of urine as a ctDNA sample source with high throughput of next-generation sequencing (NGS). Methods: Our platform couples an extraction process capable of isolating ctDNA from the entire void volume of a urine sample (∼100ml) with an ultra-sensitive NGS-integrated mutation enrichment method with single copy detection sensitivity. Assays have been developed and validated to interrogate clinically actionable mutations/deletions in the KRAS, BRAF and EGFR (Exons 19, 20, 21) oncogenes in both urine and plasma samples. For mutation detection with high sensitivity, a novel allele-specific competitive cycling (ASCC) method was used prior to NGS to amplify ultra-short target DNA (31-45 bp) using kinetically-favorable binding conditions for a wild type (WT) blocking oligonucleotide. Enriched amplicons were sequenced and a proprietary algorithm was used to quantify the mutant ctDNA input level in analytical and clinical samples. Results: An extraction method optimized for enrichment of fragmented urine ctDNA enabled isolation of large amounts of DNA (mean ∼2 μg) from advanced stage patients with different cancer types. Analytical performance characterization demonstrated sensitivity of 0.0047 - 0.01% mutant copies in WT/mutant DNA blends. To show that our assays have a true single copy detection threshold, we used DNA blends with defined mutant spike-in levels of 2 - 60 copies distributed over 20 wells (to obtain 0 - 3 mutant copies/well). The observed distribution of positive and negative hits matched the theoretical hit rate of an ideal Poisson distribution for these replicates, confirming single copy sensitivity of our assays. Using the KRAS ASCC assay, 1 to 17,555 mutant fragments were detected per 1 mL of urine collected from KRAS tissue biopsy-positive advanced cancer patients. Analysis of serial patient-matched urine and plasma longitudinal samples from KRAS, BRAF, or EGFR tissue positive patients demonstrated a high level of concordance between urine and plasma samples and feasibility of monitoring mutation load in a variety of clinical settings, including monitoring post-surgery and responsiveness to targeted therapy or chemotherapy. Conclusion: The analytical characterization and clinical feasibility studies demonstrate that this methodology can successfully detect and quantitate mutational load in urinary ctDNA, thus enabling for the dynamic monitoring of therapy response, drug resistance, and minimal residual disease in cancer patients from a truly non-invasive sample. Citation Format: Karena Kosco, Jason C. Poole, Saege Hancock, Errin Samuelsz, Timothy T. Lu, Erin Clark, Latifa Hassaine, Shiloh Guerrero, Cecile Rose T. Vibat, Vlada Melnikova, Mark G. Erlander. Methodology for single copy detection and quantitative monitoring of clinically actionable circulating tumor DNA mutations in urine from cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5238. doi:10.1158/1538-7445.AM2015-5238