GeoMx™ RNA Assay: High Multiplex, Digital, Spatial Analysis of RNA in FFPE Tissue.

RNA in situ hybridization (ISH) is a widely used technique for the localization of mRNA in tissues. Limitations to traditional ISH include the number of targets that can be analyzed concurrently and the ability for many of these assays to be used in formalin-fixed, paraffin-embedded tissues (FFPE). Here, we describe the GeoMx™ RNA assay that is capable of the highly multiplexed detection of mRNA targets in FFPE tissues. This assay utilizes ISH probes linked to indexing oligo barcodes via a photocleavable linker and the GeoMx Digital Spatial Profiler (DSP) Instrument to enable profiling of RNA targets in a region-of-interest-based method. In brief, 5 µm FFPE sections are dewaxed, target retrieved, digested with proteinase K, post-fixed, and then incubated overnight with GeoMx RNA detection probes. Stringent washes are performed followed by the addition of fluorescently labeled antibodies for use as morphology markers. User-defined regions of interest are then profiled on the GeoMx DSP through region-specific cleaving and collecting the photocleaved indexing oligos. Cleaved indices are then quantified using NanoString nCounter® Technology generating digital quantification of RNA expression with spatial context.

Multiplex digital spatial profiling of proteins and RNA in fixed tissue.

Digital Spatial Profiling (DSP) is a method for highly multiplex spatial profiling of proteins or RNAs suitable for use on formalin-fixed, paraffin-embedded (FFPE) samples. The approach relies on (1) multiplexed readout of proteins or RNAs using oligonucleotide tags; (2) oligonucleotide tags attached to affinity reagents (antibodies or RNA probes) through a photocleavable (PC) linker; and (3) photocleaving light projected onto the tissue sample to release PC oligonucleotides in any spatial pattern across a region of interest (ROI) covering 1 to ~5,000 cells. DSP is capable of single-cell sensitivity within an ROI using the antibody readout, with RNA detection feasible down to ~600 individual mRNA transcripts. We show spatial profiling of up to 44 proteins and 96 genes (928 RNA probes) in lymphoid, colorectal tumor and autoimmune tissues by using the nCounter system and 1,412 genes (4,998 RNA probes) by using next-generation sequencing (NGS). DSP may be used to profile not only proteins and RNAs in biobanked samples but also immune markers in patient samples, with potential prognostic and predictive potential for clinical decision-making.

High-Plex Predictive Marker Discovery for Melanoma Immunotherapy-Treated Patients Using Digital Spatial Profiling.

PURPOSE: Protein expression in formalin-fixed, paraffin-embedded tissue is routinely measured by IHC or quantitative fluorescence (QIF) on a handful of markers on a single section. Digital spatial profiling (DSP) allows spatially informed simultaneous assessment of multiple biomarkers. Here we demonstrate the DSP technology using a 44-plex antibody cocktail to find protein expression that could potentially be used to predict response to immune therapy in melanoma.Experimental Design: The NanoString GeoMx DSP technology is compared with automated QIF (AQUA) for immune marker compartment-specific measurement and prognostic value in non-small cell lung cancer (NSCLC). Then we use this tool to search for novel predictive markers in a cohort of 60 patients with immunotherapy-treated melanoma on a tissue microarray using a 44-plex immune marker panel measured in three compartments (macrophage, leukocyte, and melanocyte) generating 132 quantitative variables. RESULTS: The spatially informed variable assessment by DSP validates by both regression and variable prognostication compared with QIF for stromal CD3, CD4, CD8, CD20, and PD-L1 in NSCLC. From the 132 variables, 11 and 15 immune markers were associated with prolonged progression-free survival (PFS) and overall survival (OS). Notably, we find PD-L1 expression in CD68-positive cells (macrophages) and not in tumor cells was a predictive marker for PFS, OS, and response. CONCLUSIONS: DSP technology shows high concordance with QIF and validates based on both regression and outcome assessment. Using the high-plex capacity, we found a series of expression patterns associated with outcome, including that the expression of PD-L1 in macrophages is associated with response.

Role of oxygen-containing functional groups in forest fire-generated and pyrolytic chars for immobilization of copper and nickel.

Char as a carbon-rich material, can be produced under pyrolytic conditions, wildfires or prescribed burn offs for fire management. The objective of this study was to elucidate mechanistic interactions of copper (Cu2+) and nickel (Ni2+) with different chars produced by pyrolysis (green waste, GW; blue-Mallee, BM) and forest fires (fresh-burnt by prescribed fire, FC; aged char produced by wild fire, AC). The pyrolytic chars were more effective sorbents of Cu2+ (∼11 times) and Ni2+ (∼5 times) compared with the forest fire chars. Both cross-polarization (CPMAS-NMR) and Bloch decay (BDMAS-NMR) 13C NMR spectroscopies showed that forest fire chars have higher woody components (aromatic functional groups) and lower polar groups (e.g. O-alkyl C) compared with the pyrolytic chars. The polarity index was greater in the pyrolytic chars (0.99-1.34) than in the fire-generated chars (0.98-1.15), while aromaticity was lower in the former than in the latter. Fourier transform infrared (FTIR) and Raman spectroscopies indicated the binding of carbonate and phosphate with both Cu2+ and Ni2+ in all chars, but with a greater extent in pyrolytic than forest fire-generated chars. These findings have demonstrated the key role of char's oxygen-containing functional groups in determining their sorption capacity for the Cu2+ and Ni2+ in contaminated lands.

The risk of exposure to diagnostic ultrasound in postnatal subjects: thermal effects.

This review evaluates the thermal mechanism for ultrasound-induced biological effects in postnatal subjects. The focus is the evaluation of damage versus temperature increase. A view of ultrasound-induced temperature increase is presented, based on thermodynamic Arrhenius analyses. The hyperthermia and other literature revealed data that allowed for an estimate of a temperature increase threshold of tissue damage for very short exposure times. This evaluation yielded an exposure time extension of the 1997 American Institute of Ultrasound in Medicine Conclusions Regarding Heat statement (American Institute of Ultrasound in Medicine, Laurel, MD) to 0.1 second for nonfetal tissue, where, at this exposure time, the temperature increase threshold of tissue damage was estimated to be about 18 degrees C. The output display standard was also evaluated for soft tissue and bone cases, and it was concluded that the current thermal indices could be improved to reduce the deviations and scatter of computed maximum temperature rises.

Does hands-on obstetric US experience improve performance on the radiology oral board examination?

PURPOSE: To investigate whether the inclusion of hands-on obstetric ultrasonography (US) experience as a formal part of radiology residency affects performance on the US section of the oral board examination. MATERIALS AND METHODS: An electronic survey was sent to all radiology residency programs to assess (a) the time assigned to US rotation, (b) the inclusion of obstetrics in US rotation, and (c) the opportunity for hands-on scanning of obstetric patients. Blinded results from the 2002 Diagnostic Radiology Oral Board Examination were provided by the American Board of Radiology. We recorded the overall examination score, US section score, and individual score for all obstetric cases for each resident from programs that responded to the survey. A Student t test and stratified statistical analyses were performed. This study was determined to be exempt from institutional review board approval. Residency directors who consented to participate were informed of pertinent information. RESULTS: Of the 159 programs, 64 (40.3%) responded and 63 (98%) of these had scores available. US section scores were provided for 280 residents, with 869 obstetric case scores. Fifty (79%) of 63 programs provided the opportunity for hands-on obstetric scanning. After adjusting for covariates, there was no significant difference in individual resident performance between residents with hands-on scanning experience and those without hands-on scanning experience (P = .61). When evaluated according to program, there was no significant difference in performance between programs with and those without hands-on training (P = .39). CONCLUSION: Radiology resident performance in obstetric US on the American Board of Radiology Oral Board Examination is similar for programs that provide the opportunity for hands-on obstetric scanning compared with those that do not.

In vivo elastographic investigation of ethanol-induced hepatic lesions.

Ethanol-induced hepatic lesions were investigated in swine for in vivo use as a strain imaging animal model. Lesions (n = 25) were induced by injecting ethanol (doses 0.33 to 2.0 mL) directly into the surgically exposed liver at depths of 12, 15 or 25 mm. Lesions were imaged with a modified HDI 1000 scanner (Philips Medical Systems, Bothell, WA, USA). The elastograms (n = 91) characterized lesions as being areas harder than the surrounding soft hepatic tissue. Elastographic lesion sizes and the corresponding injected ethanol dose used to induce the lesions were shown to be statistically significant (r(2) = 0.22; p = 0.029) using a linear regression analysis. Additionally, lesion depth was shown to be statistically insignificant (r(2) < 0.12; p > 0.10) when regressed against elastographic lesion size. An analysis of elastographic and gross pathology lesion sizes indicated no correlation (r(2) < 0.01; p = 0.973). Subsequently, lesion types were sorted by size and regression lines were computed from quasilinear regions of the corresponding run charts. Trend lines indicate a four-to-three size relationship between the selected elastographic and pathology lesion sizes. Comparison of elastogram lesion sizes from two independent observers using a paired t-test resulted in no statistically significant difference (p = 0.14). In conclusion, ethanol-induced hepatic lesions in swine is a suitable animal model for evaluation of strain-based imaging systems, due to the ease of generation and repeatability.

On the usefulness of the mechanical index displayed on clinical ultrasound scanners for predicting contrast microbubble destruction.

OBJECTIVE: The purpose of this study was to evaluate the mechanical index (MI) displayed on clinical ultrasound scanners as a predictor of exposure conditions related to the destruction of sonographic microbubble contrast agents. METHODS: Sonazoid (GE Healthcare, Oslo, Norway) and Optison (GE Healthcare, Princeton, NJ) microbubbles were injected into a tissue-mimicking flow phantom. Gray scale imaging was performed with 4 different scanners and 3 different transducers (3.5 MHz curved linear, 2.5 MHz convex, and 7.5 MHz linear array), and the MI displayed by the scanner was varied from 0.2 to 1.5 by changing the system output power. All other scanning parameters were kept constant. Downstream changes in echogenicity were monitored with a PowerVision 7000 scanner (Toshiba America Medical Systems, Tustin, CA) as an indirect measure of bubble destruction. Video intensity changes within the flow tube were determined as a function of MI for the different scanner/transducer combinations, and the best linear fit was determined. RESULTS: At a displayed MI of 0.7, different scanner/transducer combinations exhibited a range in video intensity from +16% to -3% of baseline for Sonazoid and from +8% to -71% for Optison. At an MI of 0.3, reductions in video intensity of up to 32% were produced. These results indicate a wide range in bubble destruction at identical MI values. Likewise, regression analysis found no linear fits for all scanner/transducer combinations (r2 < 0.046). CONCLUSIONS: The MI displayed on clinical ultrasound scanners does not predict the degree of microbubble destruction and should not be used by itself to define exposure conditions for destruction of microbubble contrast agents.

Diagnosing breast lesions with contrast-enhanced 3-dimensional power Doppler imaging.

OBJECTIVE: To compare mammography with contrast-enhanced 2- and 3-dimensional power Doppler imaging for the diagnosis of breast cancer. METHODS: Fifty-five patients, who underwent breast biopsies with histopathologic assessment, participated in a study of mammography and contrast-enhanced sonography. Levovist (Berlex Laboratories, Montville, NJ) and Optison (Mallinckrodt, St Louis, MO) were administrated to 22 and 33 patients, respectively. Precontrast and postcontrast 2-dimensional power Doppler data of the lesion were obtained with an HDI 3000 system (Philips Medical Systems, Bothell, WA), and 3-dimensional data were acquired with an LIS 6000A system (Life Imaging Systems Inc, London, Ontario, Canada). Two independent and blinded readers assessed diagnosis. Receiver operating characteristic curves were computed individually and in combination for mammography and 2- and 3-dimensional sonography (before and after contrast). Histopathologic and imaging parameters were compared by Mann-Whitney statistics. RESULTS: Mammographic findings were available for 50 patients, biopsy for 54, and 2- and 3-dimensional sonographic images for 53 and 52, respectively. Of the 50 patients who had all 4 measures, 15 (30%) had malignancies. The areas under the receiver operating characteristic curve for the diagnosis of breast cancer were 0.51 for 2-dimensional contrast-enhanced imaging, 0.60 for 3-dimensional power Doppler imaging, and 0.76 for 3-dimensional contrast-enhanced imaging (P < .01). Mammography produced an area of 0.86, which increased when combined with 3-dimensional contrast-enhanced imaging to 0.90 and with all sonographic modalities to 0.96 (P < .001). The histopathologic diagnosis of benign or malignant correlated with the presence or absence of anastomoses and with the degree of vascularity assessed with contrast-enhanced 3-dimensional power Doppler imaging (P = .007 and .02). CONCLUSIONS: Contrast-enhanced 3-dimensional power Doppler imaging increases the ability to diagnose breast cancer relative to conventional 2- and 3-dimensional sonographic imaging.

Advantages of real-time spatial compound sonography of the musculoskeletal system versus conventional sonography.

OBJECTIVE: Spatial compound sonography is a method that obtains sonographic information from several different angles of insonation and combines them to produce a single image. By reducing speckle and improving definition of tissue planes, this method can potentially improve image quality in musculoskeletal sonography. The purpose of our study was to compare real-time spatial compound sonography with conventional high-resolution musculoskeletal sonography. MATERIALS AND METHODS: Thirty-four patients underwent sonography of the musculoskeletal system for a variety of indications. All patients were evaluated using conventional high-resolution sonography and real-time spatial compound sonography performed with a 12-5-MHz multifrequency linear array transducer. Conventional images and compound images depicting the same musculoskeletal structure were obtained in pairs. A total of 118 images (59 image pairs) were randomly assorted and reviewed on a computer monitor by three experienced sonologists working independently. The reviewers were unaware of the type of images they were evaluating. Image quality was rated using a 5-point scale. The image parameters evaluated were definition of tissue planes, speckle, other noise, and image detail. RESULTS: Analysis of variance revealed that real-time spatial compound sonography significantly improved definition of soft-tissue planes, reduced speckle and other noise, and improved image detail when compared with conventional high-resolution sonography (p < 0.0001 for all evaluated parameters). CONCLUSION: Real-time spatial compound sonography significantly improved sonographic image quality in the musculoskeletal system when compared with conventional high-resolution sonography. Because musculoskeletal sonography is highly dependent on image quality and tissue-plane definition, spatial compound sonography represents an important development.

Management of suspected hemodialysis graft dysfunction: usefulness of diagnostic US.

PURPOSE: To evaluate the role and usefulness of diagnostic ultrasonography (US) in the assessment of hemodialysis access grafts with an intermediate probability of graft malfunction. MATERIALS AND METHODS: One hundred forty-seven complete US examinations were performed in 91 patients. Average volume of flow, peak systolic velocity, and diameter of residual lumen were recorded. Patients were referred by the nephrology department when clinical findings were suggestive, but not obviously, of graft malfunction. Study results were deemed normal if flow volume exceeded 1,300 mL/min without significant visualized stenosis of 50% of the diameter or greater or if flow approached 1,300 mL/min without peak systolic velocity greater than 400 cm/sec. RESULTS: Of the 147 examinations, 49 (33%) had normal results, seven (5%) showed thrombosis at examination, and 91 (62%) had evidence of at least one significant visualized stenosis or diffuse notable degree of thrombus. Three patients with normal results required fistulography within 90 days, one for thrombosis. In the 91 studies with abnormal results, 69 patients underwent fistulography; results in 63 showed agreement, and three showed false-positive results. More central venous stenoses were found at fistulography than at US. CONCLUSION: US is a useful and reliable first step in managing clinically suspected hemodialysis graft stenosis. One-third of the studies showed no significant stenosis and did not require angiographic evaluation. US should be the initial study in patients suspected of having hemodialysis access dysfunction without exceptional evidence of stenosis.

Elastography: Imaging the elastic properties of soft tissues with ultrasound.

Elastography is a method that can ultimately generate several new kinds of images, called elastograms. As such, all the properties of elastograms are different from the familiar properties of sonograms. While sonograms convey information related to the local acoustic backscatter energy from tissue components, elastograms relate to its local strains, Young's moduli or Poisson's ratios. In general, these elasticity parameters are not directly correlated with sonographic parameters, i.e. elastography conveys new information about internal tissue structure and behavior under load that is not otherwise obtainable. In this paper we summarize our work in the field of elastography over the past decade. We present some relevant background material from the field of biomechanics. We then discuss the basic principles and limitations that are involved in the production of elastograms of biological tissues. Results from biological tissues in vitro and in vivo are shown to demonstrate this point. We conclude with some observations regarding the potential of elastography for medical diagnosis.