Technical Reports in 2014

Aaron Newman and his colleagues introduce a next-generation sequencing–based approach for the cost-effective detection and quantitation of tumor-derived circulating DNA in both early- and advanced-stage tumors and with high levels of sensitivity and specificity. CAPP-Seq (cancer personalized profiling by deep sequencing) can simultaneously detect multiple mutations and mutation types, including rearrangements. Here, utility is demonstrated for non–small-cell lung cancer.

A new technique that allows high-resolution in vivo imaging of myelinated fibers without the use of a fluorescent marker is described by Aaron Schain and colleagues. This label-free approach, which does not require histological or immunocytochemical staining, uses spectral confocal reflectance microscopy, can be performed on a conventional confocal microscope and can be used for deep-tissue transcranial imaging up to 400 μm deep, longitudinally tracking fine changes in axonal myelination. It has potential for the in vivo analysis of normal myelin development, as well as demyelinating diseases of the CNS and peripheral nervous system.

Alain Thierry and his colleagues offer a new allele-specific, quantitative PCR–based method designed for the detection of point mutations and determination of mutation load using circulating cell-free DNA isolated from blood. In a blinded study of patients with colorectal cancer, the method exhibited 98% specificity and 92% sensitivity for the seven KRAS point mutations tested and compared favorably with other routinely used detection methods. The approach should help in patients selection for anti-EGFR treatments and for monitoring resistance.

The work of Michael Angelo and colleagues uses multiplexed ion beam imaging (MIBI) to localize and visualize protein expression in a manner analogous to immunohistochemistry (IHC) while circumventing some of the limitations of conventional IHC with clinical samples. MIBI uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters, expanding the number of targets that can be analyzed simultaneously to about 100. The approach, used here to image breast tumor tissue sections, offers over a five-log dynamic range and compatibility with standard formalin-fixed, paraffin-embedded tissue sections.

The use of antibodies against tumour necrosis factor (TNF) has markedly improved the treatment of Crohn's disease, but only certain patients respond to therapy. Here, Raja Atreya and colleagues have developed an approach using topical fluorescent antibodies to TNF and confocal laser endomicroscopy to evaluate the expression of transmembrane TNF (mTNF) in the intestinal mucosa of patients with active Crohn's disease in order to identify patients likely to respond to subsequent treatment with the anti-TNF therapy, adalimumab.

Relapses in malaria are caused by hypnozoites, the latent hepatic stage formed by species such as Plasmodium vivax and Plasmodium ovale. Drug discovery programs have been severely hampered by a lack of in vitro cultivation methods for malarial hypnozoites. Only one drug, primaquine, is currently available, but its use is limited in people with glucose-6-phosphate dehydrogenase deficiency. Here, Laurent Dembélé and colleagues offer a system that can be used to monitor the growth and development of Plasmodium cynomologi liver-stage forms, a model for P. vivax, for up to 40 d.

Diagnosis of bacterial infections is largely based on nonspecific criteria, with definitive diagnoses made only after biopsy or culture. Frank Hernandez and his colleagues demonstrate noninvasive imaging of Staphylococcus aureus infections in mice with an activatable fluorescent molecular imaging probe. The approach exploits the properties of micrococcal nuclease and uses short, synthetic oligonucleotides that are highly sensitive to micrococcal nuclease but are rendered resistant to mammalian serum nucleases by chemical modifications.

PET imaging using 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) has been widely used for the detection of plaque inflammation. Here, however, Nobuhiro Tahara and colleagues explore the possibility of using ¹⁸F-labeled mannose (2-deoxy-2-[¹⁸F]fluoro-d-mannose, [¹⁸F]FDM), a structural analog to FDG, as an alternative and potentially more specific PET tracer than FDG for assessing the risk of acute vascular events in patients. The approach targets the mannose receptor–bearing macrophages that are abundant in high-risk atherosclerotic plaques, with feasibility demonstrated in a rabbit model of atherosclerosis.

The measurement of metabolites such as creatine, which are involved in the tissue creatine kinase reaction, enable the study of the effects of energy deprivation on the heart. Haris and colleagues introduce a new magnetic resonance imaging technique that maps the distribution of creatine in the heart that does not use radiation or exogenous contrast agents and offers higher sensitivity compared to proton (¹H) magnetic resonance spectroscopy methods. Feasibility is demonstrated in vivo in infarcted swine myocardium using a standard clinical MRI scanner.