Development and preliminary evaluation of the VPS ReplaySuite: a virtual double-headed microscope for pathology

BACKGROUND: Advances in computing and telecommunications have resulted in the availability of a range of online tools for use in pathology training and quality assurance. The majority focus on either enabling pathologists to examine and diagnose cases, or providing image archives that serve as reference material. Limited emphasis has been placed on analysing the diagnostic process used by pathologists to reach a diagnosis and using this as a resource for improving diagnostic performance. METHODS: The ReplaySuite is an online pathology software tool that presents archived virtual slide examinations to pathologists in an accessible video-like format, similar to observing examinations with a double-headed microscope. Delivered through a customised web browser, it utilises PHP (Hypertext PreProcessor) to interact with a remote database and retrieve data describing virtual slide examinations, performed using the Virtual Pathology Slide (VPS). To demonstrate the technology and conduct a preliminary evaluation of pathologists opinions on its potential application in pathology training and quality assurance, 70 pathologists were invited to use the application to review their own and other pathologists examinations of 10 needle-core breast biopsies and complete an electronic survey. 9 pathologists participated, and all subsequently completed an exit survey. RESULTS: Of those who replayed an examination by another pathologist, 83.3% (5/6) agreed that replays provided an insight into the examining pathologists diagnosis and 33.3% (2/6) reconsidered their own diagnosis for at least one case. Of those who reconsidered their original diagnosis, all re-classified either concordant with group consensus or original glass slide diagnosis. 77.7% (7/9) of all participants, and all 3 participants who replayed more than 10 examinations stated the ReplaySuite to be of some or great benefit in pathology training and quality assurance. CONCLUSION: Participants conclude the ReplaySuite to be of some or of great potential benefit to pathology training and quality assurance and consider the ReplaySuite to be beneficial in evaluating the diagnostic trace of an examination. The ReplaySuite removes temporal and spatial issues that surround the use of double-headed microscopes by allowing examinations to be reviewed at different times and in different locations to the original examination. While the evaluation set was limited and potentially subject to bias, the response of participants was favourable. Further work is planned to determine whether use of the ReplaySuite can result in improved diagnostic ability

In vivo characterization of [18F]AVT-011 as a radiotracer for PET imaging of multidrug resistance

Multidrug resistance (MDR) impedes cancer treatment. Two efflux transporters from the ATP-binding cassette (ABC) family, ABCB1 and ABCG2, may contribute to MDR by restricting the entry of therapeutic drugs into tumor cells. Although a higher expression of these transporters has been correlated with an unfavorable response to chemotherapy, transporter expression does not necessarily correlate with function. In this study, we characterized the pharmacological properties of [18F]AVT-011, a new PET radiotracer for imaging transporter-mediated MDR in tumors.AVT-011 was radiolabeled with 18F and evaluated with PET imaging in preclinical models. Transport of [18F]AVT-011 by ABCB1 and/or ABCG2 was assessed by measuring its uptake in the brains of wild-type, Abcb1a/b-/-, and Abcg2-/- mice at baseline and after administration of the ABCB1 inhibitor tariquidar (n = 5/group). Metabolism and biodistribution of [18F]AVT-011 were also measured. To measure ABCB1 function in tumors, we performed PET experiments using both [18F]AVT-011 and [18F]FDG in mice bearing orthotopic breast tumors (n = 7-10/group) expressing clinically relevant levels of ABCB1.At baseline, brain uptake was highest in Abcb1a/b-/- mice. After tariquidar administration, brain uptake increased 3-fold and 8-fold in wild-type and Abcg2-/- mice, respectively, but did not increase further in Abcb1a/b-/- mice. At 30 min after injection, the radiotracer was > 90% in its parent form and had highest uptake in organs of the hepatobiliary system. Compared with that in drug-sensitive tumors, uptake of [18F]AVT-011 was 32% lower in doxorubicin-resistant tumors with highest ABCB1 expression and increased by 40% with tariquidar administration. Tumor uptake of [18F]FDG did not significantly differ among groups.[18F]AVT-011 is a dual ABCB1/ABCG2 substrate radiotracer that can quantify transporter function at the blood-brain barrier and in ABCB1-expressing tumors, making it potentially suitable for clinical imaging of ABCB1-mediated MDR in tumors

Morphometrical analysis of transbronchial cryobiopsies

The recent introduction of bronchoscopically recovered cryobiopsy of lung tissue has opened up new possibilities in the diagnosis of neoplastic and non-neoplastic lung diseases in various aspects. Most notably the morphological diagnosis of peripheral lung biopsies promises to achieve a better yield with a high quality of specimens. To better understand this phenomenon, its diagnostic options and perspectives, this study morphometrically compares 15 cryobiopsies and 18 transbronchial forceps biopsies of peripheral lung tissue a priori without considering clinical hit ratio or integration of results in the clinical diagnostic processing. Cryotechnically harvested specimens were significantly larger (mean: 17.1 ± 10.7 mm2 versus 3.8 ± 4.0 mm2) and contained alveolar tissue more often. If present, the alveolar part in cryobiopsies exceeded the one of forceps biopsies. The alveolar tissue of crybiopsy specimens did not show any artefacts. Based on these results cryotechnique seems to open up new perspectives in bronchoscopic diagnosis of lung disease

DNA index determination with Automated Cellular Imaging System (ACIS) in Barrett's esophagus: Comparison with CAS 200

BACKGROUND: For solid tumors, image cytometry has been shown to be more sensitive for diagnosing DNA content abnormalities (aneuploidy) than flow cytometry. Image cytometry has often been performed using the semi-automated CAS 200 system. Recently, an Automated Cellular Imaging System (ACIS) was introduced to determine DNA content (DNA index), but it has not been validated. METHODS: Using the CAS 200 system and ACIS, we compared the DNA index (DI) obtained from the same archived formalin-fixed and paraffin embedded tissue samples from Barrett's esophagus related lesions, including samples with specialized intestinal metaplasia without dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. RESULTS: Although there was a very good correlation between the DI values determined by ACIS and CAS 200, the former was 25% more sensitive in detecting aneuploidy. ACIS yielded a mean DI value 18% higher than that obtained by CAS 200 (p < 0.001; paired t test). In addition, the average time required to perform a DNA ploidy analysis was shorter with the ACIS (30–40 min) than with the CAS 200 (40–70 min). Results obtained by ACIS gave excellent inter-and intra-observer variability (coefficient of correlation >0.9 for both, p < 0.0001). CONCLUSION: Compared with the CAS 200, the ACIS is a more sensitive and less time consuming technique for determining DNA ploidy. Results obtained by ACIS are also highly reproducible

Utility of multispectral imaging for nuclear classification of routine clinical histopathology imagery

<p>Abstract</p> <p>Background</p> <p>We present an analysis of the utility of multispectral versus standard RGB imagery for routine H&E stained histopathology images, in particular for pixel-level classification of nuclei. Our multispectral imagery has 29 spectral bands, spaced 10 nm within the visual range of 420–700 nm. It has been hypothesized that the additional spectral bands contain further information useful for classification as compared to the 3 standard bands of RGB imagery. We present analyses of our data designed to test this hypothesis.</p> <p>Results</p> <p>For classification using all available image bands, we find the best performance (equal tradeoff between detection rate and false alarm rate) is obtained from either the multispectral or our "ccd" RGB imagery, with an overall increase in performance of 0.79% compared to the next best performing image type. For classification using single image bands, the single best multispectral band (in the red portion of the spectrum) gave a performance increase of 0.57%, compared to performance of the single best RGB band (red). Additionally, red bands had the highest coefficients/preference in our classifiers. Principal components analysis of the multispectral imagery indicates only two significant image bands, which is not surprising given the presence of two stains.</p> <p>Conclusion</p> <p>Our results indicate that multispectral imagery for routine H&E stained histopathology provides minimal additional spectral information for a pixel-level nuclear classification task than would standard RGB imagery.</p

Comparison of DNA histograms by standard flow cytometry and image cytometry on sections in Barrett's adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to compare DNA histograms obtained by standard flow cytometry (FC) and high fidelity image cytometry on sections (ICS) in normal gastrointestinal mucosa and Barrett's adenocarcinoma (BAC).</p> <p>Methods</p> <p>Archival formalin-fixed paraffin-embedded tissue blocks of 10 normal controls from 10 subjects and 42 BAC tissues from 17 patients were examined. DNA FC was performed using standard techniques and ICS was carried out by Automated Cellular Imaging System (ACIS). DNA ploidy histograms were classified into diploid with peak DNA index (DI) at 0.9–1.1, and aneuploid with peak DI > 1.1. DI values of aneuploid peaks were determined. Additionally, for DNA ICS, heterogeneity index (HI) representing DNA content heterogeneity, and histograms containing cells with DI > G2 were also identified.</p> <p>Results</p> <p>All control samples were diploid by both FC and ICS analyses. In BAC, FC showed diploid peaks in 29%, diploid peaks with additional aneuploid or tetraploid peaks in 57%, and 14% of the samples, respectively. In contrast, ICS showed aneuploid peaks in all the cases with peak DI > 1.25; 37 cases had peak DI between 1.25 and 2.25; and 5 cases had peak DI > 2.25. HI values (mean ± SD) were 11.3 ± 1.1 in controls and 32.4 ± 8.5 in BAC (p < 0.05). Controls had no G2 exceeding cells. However, 19/37 (51%) of the cases with primary peak DI < 2.25 had cells exceeding 9N.</p> <p>Conclusion</p> <p>ICS detects DNA aneuploidy in all BAC samples while FC missed the diagnosis of aneuploidy in 29%. In addition, ICS provides more information on HI and G2 exceeding rates.</p

Development of Fluorine-18 Labeled Metabolically Activated Tracers for Imaging of Drug Efflux Transporters with Positron Emission Tomography

Increased activity of efflux transporters, e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier is a pathological hallmark of many neurological diseases, and the resulting multiple drug resistance represents a major clinical challenge. Noninvasive imaging of transporter activity can help to clarify the underlying mechanisms of drug resistance and facilitate diagnosis, patient stratification, and treatment monitoring. We have developed a metabolically activated radiotracer for functional imaging of P-gp/BCRP activity with positron emission tomography (PET). In preclinical studies, the tracer showed excellent initial brain uptake and clean conversion to the desired metabolite, although at a sluggish rate. Blocking with P-gp/BCRP modulators led to increased levels of brain radioactivity; however, dynamic PET did not show differential clearance rates between treatment and control groups. Our results provide proof-of-concept for development of prodrug tracers for imaging of P-gp/BCRP function in vivo but also highlight some challenges associated with this strategy