The Performance of Flash Replenishment Contrast-Enhanced Ultrasound for the Qualitative Assessment of Kidney Lesions in Patients with Chronic Kidney Disease.

We investigated the accuracy of CEUS for characterizing cystic and solid kidney lesions in patients with chronic kidney disease (CKD). Cystic lesions are assessed using Bosniak criteria for computed tomography (CT) and magnetic resonance imaging (MRI); however, in patients with moderate to severe kidney disease, CT and MRI contrast agents may be contraindicated. Contrast-enhanced ultrasound (CEUS) is a safe alternative for characterizing these lesions, but data on its performance among CKD patients are limited. We performed flash replenishment CEUS in 60 CKD patients (73 lesions). Final analysis included 53 patients (63 lesions). Four readers, blinded to true diagnosis, interpreted each lesion. Reader evaluations were compared to true lesion classifications. Performance metrics were calculated to assess malignant and benign diagnoses. Reader agreement was evaluated using Bowker's symmetry test. Combined reader sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for diagnosing malignant lesions were 71%, 75%, 45%, and 90%, respectively. Sensitivity (81%) and specificity (83%) were highest in CKD IV/V patients when grouped by CKD stage. Combined reader sensitivity, specificity, PPV, and NPV for diagnosing benign lesions were 70%, 86%, 91%, and 61%, respectively. Again, in CKD IV/V patients, sensitivity (81%), specificity (95%), and PPV (98%) were highest. Inter-reader diagnostic agreement varied from 72% to 90%. In CKD patients, CEUS is a potential low-risk option for screening kidney lesions. CEUS may be particularly beneficial for CKD IV/V patients, where kidney preservation techniques are highly relevant.

Association of Contrast-Enhanced Ultrasound-Derived Kidney Cortical Microvascular Perfusion with Kidney Function.

Background: Individuals with chronic kidney disease (CKD) have decreased kidney cortical microvascular perfusion, which may lead to worsening kidney function over time, but methods to quantify kidney cortical microvascular perfusion are not feasible to incorporate into clinical practice. Contrast-enhanced ultrasound (CEUS) may quantify kidney cortical microvascular perfusion, which requires further investigation in individuals across the spectrum of kidney function. Methods: We performed CEUS on a native kidney of 83 individuals across the spectrum of kidney function and calculated quantitative CEUS-derived kidney cortical microvascular perfusion biomarkers. Participants had a continuous infusion of the microbubble contrast agent (Definity) with a flash-replenishment sequence during their CEUS scan. Lower values of the microbubble velocity (ß) and perfusion index (ß×A) may represent lower kidney cortical microvascular perfusion. Multivariable linear regression models tested the associations of the microbubble velocity (ß) and perfusion index (ß×A) with estimated glomerular filtration rate (eGFR). Results: Thirty-eight individuals with CKD (mean age±SD 65.2±12.6 years, median [IQR] eGFR 31.5 [18.9-41.5] ml/min per 1.73 m2), 37 individuals with end stage kidney disease (ESKD; age 54.8±12.3 years), and eight healthy volunteers (age 44.1±15.0 years, eGFR 117 [106-120] ml/min per 1.73 m2) underwent CEUS without side effects. Individuals with ESKD had the lowest microbubble velocity (ß) and perfusion index (ß×A) compared with individuals with CKD and healthy volunteers. The microbubble velocity (ß) and perfusion index (ß×A) had moderate positive correlations with eGFR (ß: rs=0.44, P<0.001; ß×A: rs=0.50, P<0.001). After multivariable adjustment, microbubble velocity (ß) and perfusion index (ß×A) remained significantly associated with eGFR (change in natural log transformed eGFR per 1 unit increase in natural log transformed biomarker: ß, 0.38 [95%, CI 0.17 to 0.59]; ß×A, 0.79 [95% CI, 0.45 to 1.13]). Conclusions: CEUS-derived kidney cortical microvascular perfusion biomarkers are associated with eGFR. Future studies are needed to determine if CEUS-derived kidney cortical microvascular perfusion biomarkers have prognostic value.

Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors.

Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.

Conventional dose rate spatially-fractionated radiation therapy (SFRT) treatment response and its association with dosimetric parameters-A preclinical study in a Fischer 344 rat model.

PURPOSE: To identify key dosimetric parameters that have close associations with tumor treatment response and body weight change in SFRT treatments with a large range of spatial-fractionation scale at dose rates of several Gy/min. METHODS: Six study arms using uniform tumor radiation, half-tumor radiation, 2mm beam array radiation, 0.3mm minibeam radiation, and an untreated arm were used. All treatments were delivered on a 320kV x-ray irradiator. Forty-two female Fischer 344 rats with fibrosarcoma tumor allografts were used. Dosimetric parameters studied are peak dose and width, valley dose and width, peak-to-valley-dose-ratio (PVDR), volumetric average dose, percentage volume directly irradiated, and tumor- and normal-tissue EUD. Animal survival, tumor volume change, and body weight change (indicative of treatment toxicity) are tested for association with the dosimetric parameters using linear regression and Cox Proportional Hazards models. RESULTS: The dosimetric parameters most closely associated with tumor response are tumor EUD (R2 = 0.7923, F-stat = 15.26*; z-test = -4.07***), valley (minimum) dose (R2 = 0.7636, F-stat = 12.92*; z-test = -4.338***), and percentage tumor directly irradiated (R2 = 0.7153, F-stat = 10.05*; z-test = -3.837***) per the linear regression and Cox Proportional Hazards models, respectively. Tumor response is linearly proportional to valley (minimum) doses and tumor EUD. Average dose (R2 = 0.2745, F-stat = 1.514 (no sig.); z-test = -2.811**) and peak dose (R2 = 0.04472, F-stat = 0.6874 (not sig.); z-test = -0.786 (not sig.)) show the weakest associations to tumor response. Only the uniform radiation arm did not gain body weight post-radiation, indicative of treatment toxicity; however, body weight change in general shows weak association with all dosimetric parameters except for valley (minimum) dose (R2 = 0.3814, F-stat = 13.56**), valley width (R2 = 0.2853, F-stat = 8.783**), and peak width (R2 = 0.2759, F-stat = 8.382**). CONCLUSIONS: For a single-fraction SFRT at conventional dose rates, valley, not peak, dose is closely associated with tumor treatment response and thus should be used for treatment prescription. Tumor EUD, valley (minimum) dose, and percentage tumor directly irradiated are the top three dosimetric parameters that exhibited close associations with tumor response.

Erratum: Early Assessment of Tumor Response to Radiation Therapy using High-Resolution Quantitative Microvascular Ultrasound Imaging: Erratum.

[This corrects the article DOI: 10.7150/thno.19703.].

Cavitation Enhancement Increases the Efficiency and Consistency of Chromatin Fragmentation from Fixed Cells for Downstream Quantitative Applications.

One of the most sensitive, time-consuming, and variable steps of chromatin immunoprecipitation (ChIP) is chromatin sonication. Traditionally, this process can take hours to properly sonicate enough chromatin for multiple ChIP assays. Further, the length of sheared DNA is often inconsistent. In order to faithfully measure chemical and structural changes at the chromatin level, sonication needs to be reliable. Thus, chromatin fragmentation by sonication represents a significant bottleneck to downstream quantitative analysis. To improve the consistency and efficiency of chromatin sonication, we developed and tested a cavitation enhancing reagent based on sonically active nanodroplets. Here, we show that nanodroplets increase sonication efficiency by 16-fold and provide more consistent levels of chromatin fragmentation. Using the previously characterized chromatin in vivo assay (CiA) platform, we generated two distinct chromatin states in order to test nanodroplet-assisted sonication sensitivity in measuring post-translational chromatin marks. By comparing euchromatin to chemically induced heterochromatin at the same CiA:Oct4 locus, we quantitatively measure the capability of our new sonication technique to resolve differences in chromatin structure. We confirm that nanodroplet-assisted sonication results are indistinguishable from those of samples processed with traditional sonication in downstream applications. While the processing time for each sample was reduced from 38.4 to 2.3 min, DNA fragment distribution sizes were significantly more consistent with a coefficient of variation 2.7 times lower for samples sonicated in the presence of nanodroplets. In conclusion, sonication utilizing the nanodroplet cavitation enhancement reagent drastically reduces the amount of processing time and provides consistently fragmented chromatin of high quality for downstream applications.

Oxygen microbubbles improve radiotherapy tumor control in a rat fibrosarcoma model - A preliminary study.

Cancer affects 39.6% of Americans at some point during their lifetime. Solid tumor microenvironments are characterized by a disorganized, leaky vasculature that promotes regions of low oxygenation (hypoxia). Tumor hypoxia is a key predictor of poor treatment outcome for all radiotherapy (RT), chemotherapy and surgery procedures, and is a hallmark of metastatic potential. In particular, the radiation therapy dose needed to achieve the same tumor control probability in hypoxic tissue as in normoxic tissue can be up to 3 times higher. Even very small tumors (<2-3 mm3) comprise 10-30% of hypoxic regions in the form of chronic and/or transient hypoxia fluctuating over the course of seconds to days. We investigate the potential of recently developed lipid-stabilized oxygen microbubbles (OMBs) to improve the therapeutic ratio of RT. OMBs, but not nitrogen microbubbles (NMBs), are shown to significantly increase dissolved oxygen content when added to water in vitro and increase tumor oxygen levels in vivo in a rat fibrosarcoma model. Tumor control is significantly improved with OMB but not NMB intra-tumoral injections immediately prior to RT treatment and effect size is shown to depend on initial tumor volume on RT treatment day, as expected.

Early Assessment of Tumor Response to Radiation Therapy using High-Resolution Quantitative Microvascular Ultrasound Imaging.

Measuring changes in tumor volume using anatomical imaging weeks to months post radiation therapy (RT) is currently the clinical standard for indicating treatment response to RT. For patients whose tumors do not respond successfully to treatment, this approach is suboptimal as timely modification of the treatment approach may lead to better clinical outcomes. We propose to use tumor microvasculature as a biomarker for early assessment of tumor response to RT. Acoustic angiography is a novel contrast ultrasound imaging technique that enables high-resolution microvascular imaging and has been shown to detect changes in microvascular structure due to cancer growth. Data suggest that acoustic angiography can detect longitudinal changes in the tumor microvascular environment that correlate with RT response. Methods: Three cohorts of Fisher 344 rats were implanted with rat fibrosarcoma tumors and were treated with a single fraction of RT at three dose levels (15 Gy, 20 Gy, and 25 Gy) at a dose rate of 300 MU/min. A simple treatment condition was chosen for testing the feasibility of our imaging technique. All tumors were longitudinally imaged immediately prior to and after treatment and then every 3 days after treatment for a total of 30 days. Both acoustic angiography (using in-house produced microbubble contrast agents) and standard b-mode imaging was performed at each imaging time point using a pre-clinical Vevo770 scanner and a custom modified dual-frequency transducer. Results: Results show that all treated tumors in each dose group initially responded to treatment between days 3-15 as indicated by decreased tumor growth accompanied with decreased vascular density. Untreated tumors continued to increase in both volume and vascular density until they reached the maximum allowable size of 2 cm in diameter. Tumors that displayed complete control (no tumor recurrence) continued to decrease in size and vascular density, while tumors that progressed after the initial response presented an increase in tumor volume and volumetric vascular density. The increase in tumor volumetric vascular density in recurring tumors can be detected 10.25 ± 1.5 days, 6 ± 0 days, and 4 ± 1.4 days earlier than the measurable increase in tumor volume in the 15, 20, and 25 Gy dose groups, respectively. A dose-dependent growth rate for tumor recurrence was also observed. Conclusions: In this feasibility study we have demonstrated the ability of acoustic angiography to detect longitudinal changes in vascular density, which was shown to be a potential biomarker for tumor response to RT.

A Pilot Clinical Study in Characterization of Malignant Renal-cell Carcinoma Subtype with Contrast-enhanced Ultrasound.

Malignant renal cell carcinoma (RCC) is a diverse set of diseases, which are independently difficult to characterize using conventional MRI and CT protocols due to low temporal resolution to study perfusion characteristics. Because different disease subtypes have different prognoses and involve varying treatment regimens, the ability to determine RCC subtype non-invasively is a clinical need. Contrast-enhanced ultrasound (CEUS) has been assessed as a tool to characterize kidney lesions based on qualitative and quantitative assessment of perfusion patterns, and we hypothesize that this technique might help differentiate disease subtypes. Twelve patients with RCC confirmed pathologically were imaged using contrast-enhanced ultrasound. Time intensity curves were generated and analyzed quantitatively using 10 characteristic metrics. Results showed that peak intensity ( p = 0.001) and time-to-80% on wash-out ( p = 0.004) provided significant differences between clear cell, papillary, and chromophobe RCC subtypes. These results suggest that CEUS may be a feasible test for characterizing RCC subtypes.

Management of Indeterminate Cystic Kidney Lesions: Review of Contrast-enhanced Ultrasound as a Diagnostic Tool.

Indeterminate cystic kidney lesions found incidentally are an increasingly prevalent diagnostic challenge. Standard workup includes Bosniak classification with contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI). However, these tests are costly and not without risks. Contrast-enhanced ultrasound (CEUS) is a relatively new technique with lower risk of adverse events than iodine-containing contrast or gadolinium. In our review of the evidence for characterization of cystic kidney lesions with CEUS, CEUS displayed sensitivity (89%-100%) and negative predictive value (86%-100%) comparable to contrast-enhanced CT or MRI, with no decrease in specificity compared with CT and only a slight decrease compared with MRI.

Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath.

A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation.