Christopher T. Barry

SHEAR WAVE DISPERSION MEASURES LIVER STEATOSIS

Crawling waves, which are interfering shear wave patterns, can be generated in liver tissue over a range of frequencies. Some important biomechanical properties of the liver can be determined by imaging the crawling waves using Doppler techniques and analyzing the patterns. We report that the dispersion of shear wave velocity and attenuation, that is, the frequency dependence of these parameters, are strongly correlated with the degree of steatosis in a mouse liver model, ex vivo. The results demonstrate the possibility of assessing liver steatosis using noninvasive imaging methods that are compatible with color Doppler scanners and, furthermore, suggest that liver steatosis can be separated from fibrosis by assessing the dispersion or frequency dependence of shear wave propagations.

Micro RNA Expression Profiles as Adjunctive Data to Assess the Risk of Hepatocellular Carcinoma Recurrence After Liver Transplantation

Donor livers are precious resources and it is, therefore, ethically imperative that we employ optimally sensitive and specific transplant selection criteria. Current selection criteria, the Milan criteria, for liver transplant candidates with hepatocellular carcinoma (HCC) are primarily based on radiographic characteristics of the tumor. Although the Milan criteria result in reasonably high survival and low‐recurrence rates, they do not assess an individual patients tumor biology and recurrence risk. Consequently, it is difficult to predict on an individual basis the risk for recurrent disease. To address this, we employed microarray profiling of microRNA (miRNA) expression from formalin fixed paraffin embedded tissues to define a biomarker that distinguishes between patients with and without HCC recurrence after liver transplant. In our cohort of 64 patients, this biomarker outperforms the Milan criteria in that it identifies patients outside of Milan who did not have recurrent disease and patients within Milan who had recurrence. We also describe a method to account for multifocal tumors in biomarker signature discovery.

Integration of Crawling Waves in an Ultrasound Imaging System. Part 2: Signal Processing and Applications

This paper introduces methods to generate crawling wave interference patterns from the displacement fields generated from radiation force pushes on a GE Logiq 9 scanner. The same transducer and system provides both the pushing pulses to generate the shear waves and the tracking pulses to measure the displacements. Acoustic power and system limitations result in largely impulsive displacement fields. Measured displacements from pushes on either side of a region-of-interest (ROI) are used to calculate continuously varying interference patterns. This technique is explained along with a brief discussion of the conventional mechanical source-driven crawling waves for comparison. We demonstrate the method on three example cases: a gelatin-based phantom with a cylindrical inclusion, an oil-gelatin phantom and mouse livers. The oil-gelatin phantom and the mouse livers demonstrate not only shear speed estimation, but the frequency dependence of the shear wave speeds.

Mouse liver dispersion for the diagnosis of early-stage Fatty liver disease: a 70-sample study.

The accumulation of fat droplets within the liver is an important marker of liver disease. This study assesses gradations of steatosis in mouse livers using crawling waves, which are interfering patterns of shear waves introduced into the liver by external sources. The crawling waves are detected by Doppler ultrasound imaging techniques, and these are analyzed to estimate the shear wave speed as a function of frequency between 200 and 360 Hz. In a study of 70 mice with progressive increases in steatosis from 0% to >60%, increases in steatosis are found to increase the dispersion, or frequency dependence, of shear wave speed. This finding confirms an earlier, smaller study and points to the potential of a scoring system for steatosis based on shear wave dispersion.

MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors.

Vascular invasion provides a direct route for tumor metastasis. The degree to which microRNA (miRNA) expression plays a role in tumor vascular invasion is unclear. Here, we report that miR‐494 is up‐regulated in human hepatocellular carcinoma (HCC) tumors with vascular invasion and can promote HCC cell invasiveness by gene inactivation of multiple invasion‐suppressor miRNAs. Our results show that ten eleven translocation (TET) methylcytosine dioxygenase, predominantly TET1 in HCC cells, is a direct target of miR‐494. The reduced 5′‐hydroxymethylcytosine levels observed in the proximal cytosine‐phosphate‐guanine (CpG) regions of multiple invasion‐suppressor miRNA genes are strongly associated with their transcriptional repression upon miR‐494 overexpression, whereas enforced DNA demethylation can abolish the repression. Furthermore, TET1 knockdown shows a similar effect as miR‐494 overexpression. Conversely, miR‐494 inhibition or enforced TET1 expression is able to restore invasion‐suppressor miRNAs and inhibit miR‐494‐mediated HCC cell invasion. Conclusions: miR‐494 can trigger gene silencing of multiple invasion‐suppressor miRNAs by inhibiting genomic DNA demethylation by direct targeting of TET1, thereby leading to tumor vascular invasion. (Hepatology 2015;62:466–480

ELASTICITY ESTIMATES FROM IMAGES OF CRAWLING WAVES GENERATED BY MINIATURE SURFACE SOURCES

We describe a surface-based approach to the generation of shear wave interference patterns, called crawling waves (CrW), within a medium and derive local estimates of biomechanical properties of tissue. In previous experiments, elongated bars operating as vibration sources were used to generate CrW propagation in samples. In the present study, however, a pair of miniature circular vibration sources was applied to the overlying skin to generate the CrW within the medium. The shape and position of the miniature sources make this configuration more applicable for in vivo implementation. A modified ultrasound imaging system is used to display the CrW propagation. A shear speed mapping algorithm is developed using a detailed analysis of the CrW. The proposed setup is applied to several biomaterials including a homogeneous phantom, an inhomogeneous phantom and an ex vivo human liver. The data are analyzed using the mapping algorithm to reveal the biomechanical properties of the biomaterials.

Functionally Distinct Subpopulations of CpG-Activated Memory B Cells

During the human B cell (Bc) recall response, rapid cell division results in multiple Bc subpopulations. The TLR-9 agonist CpG oligodeoxynucleotide, combined with cytokines, causes Bc activation and division in vitro and increased CD27 surface expression in a sub-population of Bc. We hypothesized that the proliferating CD27lo subpopulation, which has a lower frequency of antibody-secreting cells (ASC) than CD27hi plasmablasts, provides alternative functions such as cytokine secretion, costimulation, or antigen presentation. We performed genome-wide transcriptional analysis of CpG activated Bc sorted into undivided, proliferating CD27lo and proliferating CD27hi subpopulations. Our data supported an alternative hypothesis, that CD27lo cells are a transient pre-plasmablast population, expressing genes associated with Bc receptor editing. Undivided cells had an active transcriptional program of non-ASC B cell functions, including cytokine secretion and costimulation, suggesting a link between innate and adaptive Bc responses. Transcriptome analysis suggested a gene regulatory network for CD27lo and CD27hi Bc differentiation.

Gene pathway analysis of hepatocellular carcinoma genomic expression datasets.

BACKGROUND Genomic analyses of cancer rarely show significant overlap in reported significant genes from one study to the next. We posit that viewing transcriptomic data from the broader view of gene pathways and biologic processes will yield a more coherent and meaningful understanding compared with analyzing lists of individual genes. MATERIALS AND METHODS To this end, we collected publicly available data from hepatocellular carcinoma (HCC) gene expression studies and collectively analyzed them using ANOVA, Gene Set Enrichment Analysis, and gene pathway analyses. RESULTS The degree of pathway and function overlap was very high between datasets compared with individual gene overlap. Analysis of pathways shared among all the datasets showed that processes such as cell proliferation, cell cycle control, and apoptosis were highly represented in the HCC samples, and liver-specific processes such as lipid synthesis, coagulation protein synthesis, and drug metabolism were present in normal liver cells. Specific gene networks known to be important in HCC, such as WNT, PCNA, TGF, and TP53, are present in the study. CONCLUSIONS We describe a generalizable method to combine multiple genomic datasets generated from diverse experimental platforms and study populations into an intuitive and biologically meaningful format. This approach allows the delineation of biologic processes of clinical significance that can predict important endpoints such as survival and tumor recurrence.

Shear Wave Dispersion in Lean Versus Steatotic Rat Livers

The precise measurement of fat accumulation in the liver, or steatosis, is an important clinical goal. Our previous studies in phantoms and mouse livers support the hypothesis that, starting with a normal liver, increasing accumulations of microsteatosis and macrosteatosis will increase the lossy viscoelastic properties of shear waves in a medium. This increase results in an increased dispersion (or slope) of the shear wave speed in the steatotic livers.

Accuracy of vascular invasion reporting in hepatocellular carcinoma before and after implementation of subspecialty surgical pathology sign-out

Context: Liver cancers (including hepatocellular carcinoma [HCC] and cholangiocarcinoma) are the fifth most common cause of cancer death. The most powerful independent histologic predictor of overall survival after transplantation for HCC is the presence of microscopic vascular invasion (VI). Aims: Given that VI is known to have somewhat high interobserver variability in both HCC and other tumors, we hypothesized that pathologists with special interest and training in liver pathology would be more likely to identify and report VI in HCC than would general surgical pathologists. Settings and Design: We searched our departmental surgical pathology archives for transplant hepatectomies performed for HCC. Subjects and Methods: We identified 143 such cases with available sign-out reports and hematoxylin and eosin-stained slides. Statistical Analysis Used: Kappa results (level of agreement) were calculated. Results: Before surgical pathology subspecialty sign-out (SSSO) implementation, 49 of 88 HCC cases were reported as negative for VI; on rereview, 20 of these had VI. After SSSO implementation, 39 of 55 cases were reported as negative for VI; on our review, 8 of these had VI. Kappa (agreement) between general SO and subspecialty rereview was 0.562 (95% confidence interval [CI] = 0.411–0.714) “weak agreement.” Kappa (agreement) between SSSO and rereview by select liver pathologists was 0.693 (95% CI = 0.505–0.880) “moderate agreement.” Conclusions: Our study is one of only a few so far that have suggested improved accuracy of certain parameters under SSSO.