Andrew Lesniak

Digital transplantation pathology: combining whole slide imaging, multiplex staining and automated image analysis.

Conventional histopathology is the gold standard for allograft monitoring, but its value proposition is increasingly questioned. “‐Omics” analysis of tissues, peripheral blood and fluids and targeted serologic studies provide mechanistic insights into allograft injury not currently provided by conventional histology. Microscopic biopsy analysis, however, provides valuable and unique information: (a) spatial‐temporal relationships; (b) rare events/cells; (c) complex structural context; and (d) integration into a “systems” model. Nevertheless, except for immunostaining, no transformative advancements have “modernized” routine microscopy in over 100 years. Pathologists now team with hardware and software engineers to exploit remarkable developments in digital imaging, nanoparticle multiplex staining, and computational image analysis software to bridge the traditional histology—global “‐omic” analyses gap. Included are side‐by‐side comparisons, objective biopsy finding quantification, multiplexing, automated image analysis, and electronic data and resource sharing. Current utilization for teaching, quality assurance, conferencing, consultations, research and clinical trials is evolving toward implementation for low‐volume, high‐complexity clinical services like transplantation pathology. Cost, complexities of implementation, fluid/evolving standards, and unsettled medical/legal and regulatory issues remain as challenges. Regardless, challenges will be overcome and these technologies will enable transplant pathologists to increase information extraction from tissue specimens and contribute to cross‐platform biomarker discovery for improved outcomes.

Experience with multimodality telepathology at the University of Pittsburgh Medical Center

Several modes of telepathology exist including static (store-and-forward), dynamic (live video streaming or robotic microscopy), and hybrid technology involving whole slide imaging (WSI). Telepathology has been employed at the University of Pittsburgh Medical Center (UPMC) for over a decade at local, national, and international sites. All modes of telepathology have been successfully utilized to exploit our institutions subspecialty expertise and to compete for pathology services. This article discusses the experience garnered at UPMC with each of these teleconsultation methods. Static and WSI telepathology systems have been utilized for many years in transplant pathology using a private network and client-server architecture. Only minor clinically significant differences of opinion were documented. In hematopathology, the CellaVision® system is used to transmit, via email, static images of blood cells in peripheral blood smears for remote interpretation. While live video streaming has remained the mode of choice for providing immediate adequacy assessment of cytology specimens by telecytology, other methods such as robotic microscopy have been validated and shown to be effective. Robotic telepathology has been extensively used to remotely interpret intra-operative neuropathology consultations (frozen sections). Adoption of newer technology and increased pathologist experience has improved accuracy and deferral rates in teleneuropathology. A digital pathology consultation portal (https://pathconsult.upmc.com/) was recently created at our institution to facilitate digital pathology second opinion consults, especially for WSI. The success of this web-based tool is the ability to handle vendor agnostic, large image files of digitized slides, and ongoing user-friendly customization for clients and teleconsultants. It is evident that the practice of telepathology at our institution has evolved in concert with advances in technology and user experience. Early and continued adoption of telepathology has promoted additional digital pathology resources that are now being leveraged for other clinical, educational, and research purposes.

Preexisting epithelial diversity in normal human livers: A tissue‐tethered cytometric analysis in portal/periportal epithelial cells

Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte‐BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the canals of Hering and/or metaplasia of preexisting mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high‐resolution whole‐slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes preexist in normal adult human livers, which might provide an alternative explanation for disease‐induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Conclusion: Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here. (HEPATOLOGY 2013)

Digital pathology consultations-a new era in digital imaging, challenges and practical applications.

Digital pathology has grown dramatically in the last 10 years and has created opportunities to not only support the triaging of difficult cases among specialists within an organization, but also enable remote pathology consultations with external organizations across the world. This study investigated one organizations need for a vendor agnostic Digital Pathology Consultation workflow solution that overcomes the challenges associated with the transfer of large studies across a local area network or across the Internet. The organization investigated is a large multifacility healthcare organization that consists of 20 hospitals spread across a wide geographical area. The organization has one of the largest academic pathology departments in the USA, with more than 100 diagnostic anatomic pathologists. This organization developed a set of web-based tools to support the workflow of digital pathology consultations and allow the viewing of whole slide images. The challenges and practical implementations of two different use cases are addressed: the occasional end user (professional or patient) requesting a second opinion and the external laboratory or hospital looking for an established consultative relationship with a large volume of cases. The solution presented in this study addresses the challenges associated with the distribution of large images and the lack of established imaging standards, while providing for a convenient and secure portal for pathologist report entry and distribution.

Role for mammalian chitinase 3-like protein 1 in traumatic brain injury

Traumatic brain injury (TBI) is accompanied by inflammatory infiltrates and CNS tissue response. The astrocytosis associated with TBI has been proposed to have both beneficial and detrimental effects on surviving neural tissue. We recently observed prominent astrocytic expression of YKL‐40/chitinase 3‐like protein 1 (CHI3L1) associated with severity of brain injury. The physiological role of CHI3L1 in the CNS is unknown; however, its distribution at the perimeter of contusions and temporal course of expression suggested that in TBI it might be an important component of the astrocytic response to modulate CNS inflammation. To address this hypothesis, we used serially sectioned brains to quantitatively compare the neuropathological outcomes of TBI produced by controlled cortical impact in wild type (WT) and chi3l1 knockout (KO) mice where the murine YKL‐40 homologue, breast regression protein 39 (BRP‐39/CHI3l1), had been homozygously disrupted. At 21 days post‐injury, chi3l1 KO mice displayed greater astrocytosis (increased GFAP staining) in the hemispheres ipsilateral and contralateral to impact compared with WT mice. Similarly, Iba1 expression as a measure of microglial/macrophage response was significantly increased in chi3l1 KO compared with WT in the hemisphere contralateral to impact. We conclude that astrocytic expression of CHI3L1 limits the extent of both astrocytic and microglial/macrophage facets of neuroinflammation and suggests a novel potential therapeutic target for modulating neuroinflammation.

Elevated ST2 Distinguishes Incidences of Pediatric Heart and Small Bowel Transplant Rejection.

Elevated serum soluble (s) suppressor of tumorigenicity‐2 is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n = 41) and SBTx recipient (n = 18) sera. At times of biopsy‐diagnosed HTx rejection (cellular and/or antibody‐mediated), serum sST2 was elevated compared to rejection‐free time points (1714 ± 329 vs. 546.5 ± 141.6 pg/mL; p = 0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536 ± 1561 vs. 2662 ± 543.8 pg/mL; p = 0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2 > 600 pg/mL could discriminate time points of HTx rejection and nonrejection (area under the curve [AUC] = 0.724 ± 0.053; p = 0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC = 0.6921 ± 0.0820; p = 0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed that rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted tumor necrosis factor‐α and IL‐1β as upstream activators. In total, our data indicate that alloimmune‐associated pro‐inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the noninvasive diagnosis of rejection.

Inhibition of large T antigen ATPase activity as a potential strategy to develop anti-polyomavirus JC drugs.

INTRODUCTION This study evaluates polyomavirus JC (JCV) large T antigen (LTA) as a potential target for drug development. LTA is a hexameric protein with a helicase activity that is powered by ATP binding and hydrolysis. The helicase and ATPase function is critical for viral replication. METHODS Recombinant JCV LTA was produced in an Escherichia coli based expression plasmid. ATPase activity was measured using the malachite green assay. A high throughput screen was completed using a brain-biased library of 75,000 drug-like compounds selected for physicochemical properties consistent with blood-brain barrier permeability. RESULTS Five compounds showed non-competitive inhibition of ATPase activity with an EC50 ⩽ 15 μM. Modest antiviral activity was demonstrated in an immunofluorescence assay for JCV VP-1 expression in COS7 cells (EC50 15, 18, 20, 27, and 52 μM respectively). The compounds also inhibited viral replication in a real time PCR assay at comparable concentrations. LD50 in the MTS96 and Cell TiterGlo assays was >100 μM for all compounds in COS7 as well as HEK293 cells. However, two compounds inhibited cell proliferation in culture with IC50 values of 43 and 34 μM respectively. Despite substantial amino acid similarity between polyomavirus JC, BK and SV40 proteins, these compounds differ from those previously reported to inhibit SV40 LTA ATPase in chemical structure as well as a non-competitive mechanism of inhibition. CONCLUSION LTA ATPase is a valid target for discovery. Additional screening and chemical optimization is needed to develop clinically useful compounds with less toxicity, which should be measured by metabolic as well as cell proliferation assays.

Prospective Validation of Molecular Prognostic Markers in Cutaneous Melanoma: A Correlative Analysis of E1690.

Purpose: To validate the prognostic impact of combined expression levels of three markers (SPP1, RGS1, and NCOA3) in melanoma specimens from patients enrolled in the E1690 clinical trial of high-dose or low-dose IFNα-2b versus observation. Experimental Design: Tissue was available from 248 patients. Marker expression was determined by digital imaging of immunohistochemically stained slides. The prognostic impact of each marker was first assessed by recording its expression value relative to the median. A multimarker index was then developed to combine marker expression levels by counting for each patient the number of markers with high expression. The impact of the multimarker index on relapse-free survival (RFS) and overall survival (OS) was assessed using Kaplan–Meier analysis, and both univariate and multivariate Cox regression analyses. Results: By Kaplan–Meier analysis, high multimarker expression scores were significantly predictive of RFS (P < 0.001) and OS (P < 0.001). Stepwise multivariate Cox regression analysis with backward elimination that included routine clinical and histologic prognostic factors revealed high multimarker expression scores and tumor thickness as the only factors significantly and independently predicting RFS and OS. Stepwise multivariate Cox regression analyses that also included treatment type and number of positive lymph nodes generated identical results for both RFS and OS. In the molecularly defined low-risk subgroup, patients treated with high-dose IFN had a significantly improved RFS compared with patients in the other two subgroups (P < 0.05). Conclusions: These results validate the independent impact of combined expression levels of SPP1, RGS1, and NCOA3 on survival of melanoma in a prospectively collected cohort. Clin Cancer Res; 23(22); 6888–92. ©2017 AACR.

Comparison of the diagnostic utility of digital pathology systems for telemicrobiology.

Introduction: Telemicrobiology is a growing component of clinical microbiology informatics. However, few studies have been performed to assess the diagnostic utility of telemicroscopy systems in evaluating infectious agents. Objective: Evaluate multiple contemporary digital pathology platforms for use in diagnostic telemicrobiology. Materials and Methods: A mix of thirty cases that included viral, bacterial, fungal, and parasitological findings were evaluated by four experts using ×40 whole slide imaging (WSI) scans, ×83 oil-immersion WSI scans, ×100 oil-immersion WSI scans, digital photomicrographs, and glass slides. Results: The ×83 WSI, ×100 WSI, and photomicrograph interpretations were not significantly different in quality and accuracy when compared to glass slide interpretations. The ×40 WSI interpretations were of lower quality and were more likely to be incorrect when compared to glass slide interpretations. Conclusions: In this study, high magnification, oil-immersion digital pathology platforms are better suited to support telemicrobiology applications and yield interpretations on par with glass slide evaluations.