BackgroundA major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients.Patients and methodsMatched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing.ResultsFor exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection.ConclusionsCombining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone.Clinical TrialsNCT01526928

Tumor tissue biopsies carry risks and are not always possible, making liquid biopsies an attractive way to acquire molecular information from cancer patients. The EGFR T790M mutation is a critical biomarker in non-small cell lung cancer and has introduced new challenges in how these patients are managed. With the approval of osimertinib for patients failing first generation EGFR inhibitor therapy, the use of a liquid biopsy to detect EGFR T790M would reduce the number of unnecessary repeat biopsies. Detection of EGFR T790M using cfDNA has proved to be challenging due to low abundance in blood. Here we present a novel EGFR T790M assay based on a platform validated in a CLIA laboratory that simultaneously monitors the mutation on exosomal RNA/DNA and cfDNA from plasma that achieves 92% sensitivity and 89% specificity.Research. AbstractPurpose: About 60% of non-small cell lung cancer (NSCLC) patients develop resistance to targeted epidermal growth factor receptor (EGFR) inhibitor therapy through the EGFR T790M mutation. Patients with this mutation respond well to third generation tyrosine kinase inhibitors, but obtaining a tissue biopsy to confirm the mutation poses risks and is often not feasible. Liquid biopsies using circulating free tumor DNA (cfDNA) have emerged as a non-invasive option to detect the mutation, however sensitivity is low as many patients have too few detectable copies in circulation. Here we have developed and validated a novel test that overcomes the limited abundance of the mutation by simultaneously capturing and interrogating exosomal RNA/DNA and cfDNA (exoNA) in a single step followed by a sensitive allele specific qPCR.Experimental design: ExoNA was extracted from the plasma of NSCLC patients with biopsy-confirmed T790M-positive (N = 102) and T790M-negative (N = 108) samples.The T790M mutation status was determined using an analytically validated allelespecific qPCR assay in a CLIA laboratory.Results: Detection of the T790M mutation on exoNA achieved 92% sensitivity and 89% specificity using tumor biopsy results as gold standard. We also obtained high sensitivity (88%) in patients with intrathoracic disease (M0/M1a), for whom detection by liquid biopsy has been particularly challenging.Research.on March 26,

interfering ribonucleic acid; siRNA1-siRNA22, 22 distinct siRNAs used in the in vitro and in vivo studies with data presented in this work; STC, standard template chemistry; t 1/2 , half-life; T max , time at which maximal concentration was observed.

NA interference (RNAi) therapeutics use an endogenous mechanism whereby short interfering RNAs (siRNAs) direct the RNA-induced silencing complex (RISC) to sequence matched target transcripts for knockdown 1 . Both lipid nanoparticles and N-acetylgalactosamine (GalNAc) conjugates are clinically validated and approved delivery strategies for liver targets [2][3][4][5][6][7][8] . Building on nearly 2 decades of siRNA design and chemistry optimization [9][10][11][12] , we demonstrate here that, with suitable delivery solutions, the RNAi pathway can be harnessed in extrahepatic tissues, such as the central nervous system (CNS), eye and lung. Multiple CNS diseases, representing some of the highest unmet medical needs and greatest therapeutic challenges, have been associated with dominant gain-of-function mutations, making them suitable candidates for an RNAi-based silencing approach. As such, chemically modified siRNAs have demonstrated potent and sustained silencing in rodents and non-human primates (NHPs); however, using an invasive intracerebroventricular (ICV) administration approach 13 that is not suitable for repeated dosing in humans. Furthermore, technologies enabling siRNA delivery across the blood-brain barrier following a less challenging systemic administration are similarly being explored [14][15][16][17] , which are, however, still in early stages of discovery. In the eye, intravitreal (IVT) dosing of siRNAs has been evaluated in late-stage clinical studies, with few safety concerns, but did not advance further due to lack of efficacy 18 . Recently, the Coronavirus Disease 2019 (COVID-19) pandemic has highlighted the importance of optimizing siRNA delivery to the lung for the treatment of emergent viral respiratory diseases. Although earlier programs have already shown potential clinical benefits of siRNA-based therapeutics in the lung 19 , 2′-O-hexadecyl (C16) conjugates demonstrate enhanced delivery and siRNA uptake into the alveolar and bronchiolar epithelium. Taken together, this work highlights that the combination of a C16 lipophilic modification with our fully chemically modified, metabolically stable siRNAs achieves efficient delivery to the CNS, eye and lung, resulting in a robust and durable gene silencing in rodents and NHPs, with a favorable safety profile. We think that these advances have the potential to generate multiple candidates for investigating clinical safety and efficacy in humans. ResultsOptimization of the siRNA conjugate design. Lipophilic moieties represent one of the earliest approaches to improve cellular uptake and delivery of antisense oligonucleotides (ASOs) and siRNAs to the liver and various other organ systems 20 , including the CNS [21][22][23] . We reasoned that, by carefully optimizing the lipophilicity of chemically modified siRNAs, we could enhance intracellular delivery without compromising broad biodistribution, potency and safety. We used the 2′ position of the ribose sugar backbone to introduce

BRAFV600E mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAFV600E inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAFV600E selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAFV600E-PTC, intrathyroidal BRAFV600E-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAFV600E-PTC orthotopic mouse. We find that metastatic BRAFV600E-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAFWT-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAFV600E-PTC cells but lesser in metastatic BRAFV600E-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment–associated pro-metastatic molecules, with no effects in BRAFWT-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAFWT/V600E-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAFV600E-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAFV600E-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAFV600E-positive PTC.