Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform.

BACKGROUND: With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids. METHODS: Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes. RESULTS: Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02-2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92-1.00) and 0.90 (CI = 0.74-1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease. CONCLUSIONS: The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.

Laboratory operations, specimen processing, and handling for viral load testing and surveillance.

RNA remains the most informative and accurate biomarker for human immunodeficiency virus type 1 load diagnostics and for surveillance of drug resistance markers. Viral load testing by nucleic acid amplification currently is a complex and expensive test that is restricted to centralized laboratory testing. Successful extension of centralized viral load testing to rural or remote settings is a major challenge. Emerging nucleic acid-based technologies are progressing rapidly toward platforms appropriate for field use in low-resource settings, leaving a growing gap for sample processing technologies that complement them. One area in which new technologies could be applied to improve access is clinical specimen preservation and processing. Novel technologies that extract nucleic acid from clinical specimens and stabilize it at the point of specimen collection could fill this gap. In addition, these technologies may provide alternative viral load detection and surveillance solutions to the current centralized laboratory testing paradigm.

A small molecule-kinase interaction map for clinical kinase inhibitors.

Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.