Mount Sections Research Articles

Abstract 5502: Multiplex data utilizing a single-step staining and imaging workflow for the investigation of multiple sample types

Abstract Background: Tissue consists of heterogenous cell types, each with diverse functions and functional states, where spatial organization can impact patient health status. Resolving this complexity at the subcellular level has historically been challenged by image resolution, the number of targets that can be simultaneously assessed, and throughput. Such barriers can be broken using high plex and whole slide biomarker imaging data, in one single cycle thus also mitigating issues that arise with multiple cyclic rounds. Methods: A range of whole slide healthy and diseased tissue types were stained with 15 to 18-plex validated biomarker panels. The tissues’ inherent autofluorescence was isolated into discrete fluorescence channels for enhanced information about each tissue’s morphology. Whole slide spatial staining and imaging was conducted on the Orion™ spatial biology platform, and H&E staining was performed after immunofluorescence (IF) imaging on the same section and imaged by brightfield microscopy. The full protocol is fairly quick and simple, using standard histology tools: - Mount sections on glass slides- De-paraffinize and perform antigen retrieval- Quench autofluorescence- Stain slides with a panel of ArgoFluor™ conjugated antibodies- Coverslip with ArgoFluor Mounting Medium and cure overnight- Image whole slides at 20X magnification using the Orion instrument- Process to ome.TIFF and analyze- De-coverslip in aqueous solution- Perform H&E staining and scanning on same section. Results: Imaging data within each tissue type presented here reveals important biological insights which can then be used for quantitative analysis. The little to no tissue degradation due to the one round staining and imaging process also enables H&E capabilities after IF, further enhancing biomarker quantitation. The combination of speed and resolution proved effective for comprehensive phenotypic profiling and characterization of tissue architecture, tumor heterogeneity, and the immune response. Conclusions: There is a need to show tissue biology in a high quality, high resolution, subcellular fashion as it provides a more in-depth understanding of each biological sample and thus can lead to improved spatial research of various human diseases. The single-stain and scan method described here improves the acquisition of subcellular spatial context and greatly reduces/removes errors in multiplex image data compared to cyclic methods. A single stain and image cycle across a 12-18 plex sample provides accurate downstream biomarker quantitation, offering better prediction of patient outcomes. Citation Format: Selena Larkin, Tad George, Eric Kaldjian. Multiplex data utilizing a single-step staining and imaging workflow for the investigation of multiple sample types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5502.

Abstract 6886: Orion™: 17-plex single-step stain and imaging of normal lung section adjacent to non-small cell lung carcinoma

Abstract Background: The ability to image tissue microenvironments (TME) at high-plex over a whole slide without requiring multiple staining steps allows for unprecedented ability to study tissue architecture and molecular mechanisms of immune and disease processes. Here we investigate a sample of normal lung section adjacent to non-small cell lung carcinoma using whole slide, single-step stain and imaging at single-cell resolution. Methods: These images profiled a whole slide tissue section of normal lung section adjacent to non-small cell lung carcinoma, stained with a 17-plex immuno-oncology biomarker panel. In this profile, we designed a high-plex panel of 17 biomarkers where the tissue autofluorescence was imaged and isolated as an additional fluorescence channel. All markers were stained in a single staining procedure and imaged in a single scan. Whole slide spatial staining and imaging was conducted on the Orion spatial biology platform. The full protocol is quick and simple, using standard histology tools: •Mount sections on glass slides•De-paraffinize and perform antigen retrieval•Quench autofluorescence•Stain slides with a panel of ArgoFluor™ conjugated antibodies•Coverslip with ArgoFluor Mounting Medium and cure overnight•Image whole slides at 20X magnification using the Orion instrument•Process to ome.TIFF and analyze. Results: Data revealed intact normal lung alveolar architecture, with scattered immune cell infiltrates. Multiplexed imaging revealed epithelial cells, which make up alveoli, and endothelial vasculature as well as immune cells infiltrates in the alveolar space. Various immune cell populations were observed to be present (T cells, B cells, macrophages) and immune cell activation could be distinguished using PCNA and/or Ki-67 markers. Various T cell subsets could also be identified among the infiltrate using markers for CD4, CD8 and T-regulatory marker FOXP3. Through the use of multiplex staining, immune cell activity could also be characterized with respect to bronchiolar, artery, and capillary tissue architecture. Conclusions: High-quality subcellular imaging of TME can lead to greater spatial investigation and more in-depth understanding of immune function in normal and diseased tissue. Here, multiplexed imaging identified distinct immune cell collections among cancer-adjacent normal lung tissue, providing validation of instrumentation and methods to obtain benchmarks for spatial quantitation of immune cells in control regions adjacent to cancer tissue. Citation Format: Selena Larkin, Tad George, Eric Kaldjian. Orion™: 17-plex single-step stain and imaging of normal lung section adjacent to non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6886.

Highly accurate and effective deep neural networks in pathological diagnosis of prostate cancer.

To established an AI system to make the pathological diagnosis of prostate cancer. Prostate histopathological whole mount (WM) sections from patients underwent robot-assisted laparoscopic prostatectomy were prepared. All the prostate WM pathological sections were converted to digital image data and marked with different colors on the basis of the ISUP Gleason grade group. The image was then fed into a segmentation algorithm. We chose modified U-Net as our fundamental network architecture. 172 patients were involved in this study. 896 pieces of prostate WM pathological sections from 160 patients, in which 826 pieces of WM sections from 148 patients were assigned to the training set randomly. After image segmentation there were totally 2,138,895 patches, of which 1,646,535 patches were valid for training. The other WM section was arranged for testing. Based on the whole image testing, AI and pathologists presented the same answers among 21 of 22 pieces of sections. To evaluate the diagnostic results at the pixel level, we anticipated correct cancer or non-cancer diagnose from this AI system. The area under the ROC curve as 96.8%. The value of pixel accuracy of three methods (binary analysis, clinically oriented analysis and analysis for different ISUP Gleason grade) were 96.93%, 95.43% and 93.88%, respectively. The value of frequency weighted IoU were 94.32%, 92.13% and 90.21%, respectively. This AI system is able to assist pathologists to make a final diagnosis, indicating the great potential and a wide-range of applications of AI in the medical field.

A comprehensive comparison between mpMRI of the prostate, MR-US fusion biopsy and whole mount histopathology.

To compare multiparametric magnetic resonance imaging (mpMRI) findings, US-MR fusion prostate biopsy results and whole-mount thin-section histopathology after radical prostatectomy. Overall 259 patients,who had undergone mpMRI with lesions reported as PI-RADS 3-5, underwent a MR-US fusion biopsy between 2018 and 2020.Overall 186 biopsies yielded prostate cancer and 104 patients subsequently underwent endoscopic extraperitoneal radical prostatectomy. Histopathology of biopsies was compared to the final histopathology in whole mount thin sections after radical prostatectomy by means of descriptive statistics, and further, the lesions from mpMRT were compared to whole mount histology. Prostate cancer was diagnosed in 186 (71.8%) of 259 patients (median age 69.2 y, range 42-82 y, median PSA 7.8ng/ml, range 2.1-31.3ng/ml). Of those, 95 (51,1%) underwent radical endoscopic prostatectomy, and 80 (43%) chose radiotherapy or active surveillance. In 52/95 (54,7%) with RPE additional lesions were found in the final histological whole mount sections not described at mpMRI. 22/95 (23,2%) of RPE patients had ≥ 1 additional Gleason score ≥ 7 lesions, 23 /259 (8,4%) of biopsies, respectively. The Gleason score after surgery was upgraded in 37/95 (38,9%) and downgraded in 18/95 (18,9%) patients. If we compare all 259 performed biopsies with the final histological whole mount sections which showed additional lesions with Gleason ≥ 7 (23,2%), it can be assumed that up to 10% ofclinical significant carcinomas are missed during primary assessment via mpMRI. The majority of additional findings after RP were intermediate/high risk tumors. Upgrades from low-risk to intermediate or high-risk occurred.

Value of magnetic resonance imaging/ultrasound fusion prostate biopsy to select patients for focal therapy.

To investigate the role of transrectal MRI fusion biopsy to select patients for prostate cancer focal therapy. Patients with suspected prostate cancer underwent transrectal MRI fusion biopsy with the Koelis trinity device. Two focal therapy eligibility criteria were subsequently defined: Group 1: PSA ≤ 15ng/ml, unilateral csPCa, ISUP grade ≤ 2, no contralateral PIRADS 3-5 lesion; Group 2: same criteria but ISUP grade 3. These subgroups were correlated with histopathological post-prostatectomy parameters for stage pT2, unilateral csPCa, no ISUP upgrading. In addition, parameters of csPCa detection were analyzed for patients undergoing primary and re-biopsy. Four hundred fourteen consecutive patients were analyzed (314 for primary biopsy, 100 for re-biopsy). Post-prostatectomy whole mount section analysis was available from 155 patients. 39 and 62 of these patients met focal therapy inclusion criteria for group 1 and group 2, respectively. A correlation with final pathology parameters following radical prostatectomy (stage pT2, unilateral csPCa, no ISUP upgrading) revealed a positive predictive value of only 53.8% and 64.5% for Group 1 and 2, respectively. The overall csPCa detection rate was 73.7%. In the re-biopsy group 20% additional patients with csPCa were detected by targeted biopsy. Despite high csPCa detection rates following MRI fusion biopsy our study demonstrated that, using final pathology to confirm locally advanced tumor stage, presence of bilateral csPCa and ISUP upgrading, between 35.5 and 46.2% of patients would have been incorrectly selected for focal therapy.

Comparison Between Micro-Ultrasound and Multiparametric MRI Regarding the Correct Identification of Prostate Cancer Lesions.

Multiparametric MRI (mpMRI) has become the standard imaging technique for the diagnosis of prostate cancer. However, mpMRI pathways are depending on experience, expertise, and information transfer from radiology to urology. Micro-ultrasound (Micro-US) is a new system, using high frequency (up to 29 MHz) and high resolution (down to 75 µm) ultrasound images. We evaluated the diagnostic performance of Micro-US in the detection of the prostate cancer index lesion and compared its performance to mpMRI using pathological whole mount sections as the reference. We retrospectively reviewed the data of 32 patients with diagnosis of prostate cancer and scheduled for radical prostatectomy and who underwent Micro-US before surgery. Still images and cineloops of Micro-US were recorded. Sixteen patients had also mpMRI images with acceptable quality and complete sequences available. For validation purposes each prostate was partitioned into 12 sectors for a total of 192 sectors evaluated. Micro-US and mpMRI images were both scored according to a validated system (PRI-MUS and Pi-RADS) where a score ≥3 was suspicious for both scores. Preoperative and postoperative results regarding the identification of the index lesion, the biggest lesion visible, were then compared and sensitivity, specificity, negative and positive predictive values, and accuracy were calculated. Median age was 67 years, median PSA was 6.2ng/ml, and median cancer volume of the index lesion was 3.1cc. The sensitivity of Micro-US in the index lesion detection was 76.5%, specificity 76.6%, negative predictive value 85.6%, positive predictive value 64.1% and 76.6% of accuracy. The sensitivity of mpMRI was 65.1%, specificity 93.4%, negative predictive value 83.2%, positive predictive value 84.3%, and 81.8% of accuracy (all p> .05). Micro-US showed good reliability in identifying prostate cancer index lesions. Its performance is comparable to that of mpMRI.

Well-differentiated liver cancers reveal the potential link between ACE2 dysfunction and metabolic breakdown

Angiotensin-converting enzyme 2 (ACE2) is the receptor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus disease 2019 (COVID-19). Transmembrane serine protease 2 (TMPRSS2) is a coreceptor. Abnormal hepatic function in COVID-19 suggests specific or bystander liver disease. Because liver cancer cells express the ACE2 viral receptor, they are widely used as models of SARS-CoV-2 infection in vitro. Therefore, the purpose of this study was to analyze ACE2 and TMPRSS2 expression and localization in human liver cancers and in non-tumor livers. We studied ACE2 and TMPRSS2 in transcriptomic datasets totaling 1503 liver cancers, followed by high-resolution confocal multiplex immunohistochemistry and quantitative image analysis of a 41-HCC tissue microarray. In cancers, we detected ACE2 and TMPRSS2 at the biliary pole of tumor hepatocytes. In whole mount sections of five normal liver samples, we identified ACE2 in hepatocyte’s bile canaliculi, biliary epithelium, sinusoidal and capillary endothelial cells. Tumors carrying mutated β-catenin showed ACE2 DNA hypomethylation and higher mRNA and protein expression, consistently with predicted β-catenin response sites in the ACE2 promoter. Finally, ACE2 and TMPRSS2 co-expression networks highlighted hepatocyte-specific functions, oxidative stress and inflammation, suggesting a link between inflammation, ACE2 dysfunction and metabolic breakdown.

Endothelial Tyrosine Kinase Tie1 Is Required for Normal Schlemm's Canal Development-Brief Report.

Schlemm's canal (SC) is a large vessel residing in the iridocorneal angle and is required to regulate aqueous humor outflow. Normal SC structure and function is indispensable for maintaining normal intraocular pressure, and elevated intraocular pressure is a risk factor for development of glaucoma. Recent reports have identified a key role of the angiopoietin-Tie2 pathway for SC development and function; however, the role of the orphan receptor Tie1 has not been clarified. We used Tie1 knock out mice to study the function of Tie1 in SC development and function. Real-time quantitative polymerase chain reaction and Western blot analyses were used to verify Tie1 deletion. High-resolution microscopy of mouse SC whole mount and cross sections were used to study SC morphology. Measurement of intraocular pressure in live mice was used to study the impact of Tie1 on SC function. Tie1 is highly expressed in both human and mouse SC. Tie1 knock out mice display hypomorphic SC and elevated intraocular pressure as a result of attenuated SC development. Tie1 is indispensable for SC development and function, supporting it as a novel target for future SC-targeted glaucoma therapies and a candidate gene for glaucoma in humans.

V14-03 TRANSURETHRAL WATER VAPOR ABLATION (STEAM) TO TREAT LOCALIZED PROSTATE CANCER (INVESTIGATIONAL USE ONLY)

You have accessJournal of UrologyProstate Oncology II & Testis Oncology & Misc. Oncology (V14)1 Sep 2021V14-03 TRANSURETHRAL WATER VAPOR ABLATION (STEAM) TO TREAT LOCALIZED PROSTATE CANCER (INVESTIGATIONAL USE ONLY) Christopher Dixon, and Christopher Warlick Christopher DixonChristopher Dixon More articles by this author , and Christopher WarlickChristopher Warlick More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002111.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Convective water vapor is a unique technology that uses the thermal energy of water vapor (steam) to ablate tissue. The objective was in vivo validation of Transurethral Vapor Ablation (TUVA) in men with clinically significant localized prostate cancer by; Group 1 - TUVA/RRP assessed by immediate whole mount sectioning, Group 2 - TUVA assessed by pre/post treatment MRI in an early feasibility trial and Both Groups by intraoperative real-time ultrasound and AE assessment. METHODS: A tabletop generator and a transurethral endoscopic delivery device, along with real-time TRUS, were used to direct water vapor into any anatomic zone of the prostate to ablate malignant prostate tissue. The procedure technique currently used, including the endoscopic and real-time ultrasound monitoring of needle deployment, advancement and vapor delivery is demonstrated. To evaluate the convective principles of TUVA, real-time ultrasound patterns of vapor delivery and 7-day post treatment gadolinium MRI images were studied. Ablation effectiveness was assessed in two independent groups. In Group 1 by whole mount sectioning immediately following TUVA / radical prostatectomy. In Group 2 by assessing pre- and 7-day post TUVA MRI nonenhancement patterns as well as PIRADSv2 resolution. Immediate safety was assessed by intraoperative inspection, gross examination of the prostate (Group 1) and immediate post op adverse events along with 7-day MRI’s (Group 2). RESULTS: Real-time ultrasound vapor patterns confirmed convective spread within the zonal anatomy and boundaries of the prostate. Real-time ultrasound during treatment demonstrated the convective principles and the ability to “see” the treatment. Collectively, whole mount sectioning along with the non-enhancing areas seen on MRI’s at 7-days demonstrated ablation to ALL anatomic zones and boundaries consistent with the unique ablation capabilities of convection. In Group 2, after one treatment session, 14/17 PIRADSv2 lesions ≥3 were no longer visible within the ablation zone on 7-day MRI. There were no serious adverse events intraoperatively or within one week of the treatment. CONCLUSIONS: Transurethral delivery of water vapor thermal energy can effectively ablate any zone of the prostate from apex to base, anterior to posterior, and laterally to the prostatic “capsule”. Early feasibility data using this investigational device are favorable. Source of Funding: Francis Medical, Maple Grove, MN © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1165-e1166 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christopher Dixon More articles by this author Christopher Warlick More articles by this author Expand All Advertisement Loading ...

Digital whole mount sections of the prostate: heading towards new ways of communicating with clinicians and patients without microscope.

article: Digital whole mount sections of the prostate: heading towards new ways of communicating with clinicians and patients without microscope - Minerva Urology and Nephrology 2022 February;74(1):127-9 - Minerva Medica - Journals

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view ofunperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

A STUDY ON CHROMOBLASTOMYCOSIS IN A TERTIARY CARE HOSPITAL (DMCH), LAHERIASARAI, BIHAR

Background: Chromoblastomycosis, a chronic subcutaneous mycosis, is caused by several dematiaceous Fungi, the most common being Fonsecaea pedrosoi. A majority of cases from India have been reported from the sub-Himalayan belt and South India. Aim: The aim was to study chromoblastomycosis in around Darbhanga reason of Bihar including demographic and clinicomycological prole. Materials and Methods: This report is a retrospective hospital record-based analysis of all cases of chromoblastomycosis who presented to the dermatology outpatient department of our tertiary care hospital during the past 3 years. Results : A total of 11 cases of chromoblastomycosis were diagnosed during the above period. The disease was seen predominantly in middle-aged male. The lower extremity (72.7%) was more commonly affected. Verrucous and nodular lesions are the common clinical presentation. Sclerotic bodies are demonstrated in potassium hydroxide mount and histopathological section in 81.8% and 90.9% cases, respectively. The causative fungus was isolated in 90.9% and cass with F. pedrosoi, as the most common species

The male external urethral sphincter is autonomically innervated.

The aim of the present study was to describe autonomic urethral sphincter (US) innervation using specific muscular and neuronal antibody markers and 3D reconstruction. We performed en-bloc removal of the entire pelvis of three male human fetuses between 18 and 40 weeks. Serial whole mount sections (5 μm intervals) were stained and investigated. The sections were stained with Masson's trichrome and Eosin Hematoxylin, and immunostained with: anti-SMA antibody for smooth muscle; anti-S100 antibody for all nerves; and anti-PMP22 antibody, anti-TH antibody, anti-CGRP antibody, anti-NOS antibody for somatic, adrenergic, sensory and nitrergic nerve fibers, respectively. The slides were digitized for 3D reconstruction to improve topographical understanding. An animated reconstruction of the autonomic innervation of the US was generated. The external and internal US are innervated by autonomic nerves of the inferior hypogastric plexus (IHP). These nerves are sympathetic (positive anti-TH antibody), sensory (positive anti-CGRP antibody), and nitrergic (positive anti-NOS antibody). Some autonomic fibers run within the neurovascular bundles, posterolaterally. Others run from the IHP to the posteromedial aspect of the prostate apex, above an through the rectourethral muscle. The external US is also innervated by somatic nerves (positive anti-PMP22 antibody) arising from the pudendal nerve, joining the midline but remaining below the rectourethral. This study provides anatomical evidence of an autonomic component in the innervation of the external US that travels in the neurovascular bundle. During radical prostatectomy, the rectourethral muscle and the neurovascular bundles are to be preserved, particularly during apical dissection.

Abstract 152: Adipose-derived Stromal Cells Within Transplanted Fat Hone To Blood Vessels And Assume A Pericyte Structure

Purpose: Fat grafting can restore volume defects and improve the texture, quality, and appearance of overlying skin. The adipose-derived stromal cells (ASCs) within transplanted fat are thought to drive these regenerative effects, either by direct differentiation or by paracrine signaling. The survival and differentiation fate of transplanted ASCs post grafting, however, remains incompletely understood. We therefore performed lineage tracing of transplanted human ASCs in vivo using a mouse model of fat grafting. Methods: ASCs were isolated from human lipoaspirate and transduced to express green fluorescent protein (GFP). Successfully labeled cells were isolated using fluorescence-activated cells sorting (FACS). Next, CD1 nude mice were grafted with fresh human lipoaspirate enriched with GFP+ ASCs (10,000 cells/200ul lipoaspirate/graft). Mice were harvested at 2, 4, 6, and 8 weeks (n=5 mice/group). Grafted fat was explanted and assessed by whole tissue mounting, confocal microscopy, and image reconstruction software to characterize the three-dimensional structure of fat with reference to the GFP+ ASCs. Results: Transplanted ASCs were evident in whole mount sections through 6-weeks post transplantation. GFP+ ASCs grouped together and honed to blood vessels, and adipocyte size was correlated with proximity to GFP+ ASCs. ASCs co-stained for the known pericytes markers Tie2, smooth muscle actin (SMA), and nerve growth factor 2 (NGF2). Although ASCs became fewer in number with time post grafting, fat enriched with ASCs was more vascular throughout the graft compared to fat not enriched with ASCs. Conclusions: In this mouse model of fat grafting we show that the transplanted human ASCs may survive and hone to blood vessels, assuming a pericyte-like position. These data suggest a role for ASCs in directing and supporting angiogenesis and neovascularization by direct differentiation. Future work may further profile the exact cytokine profile of transplanted ASCs throughout grafting to determine the mechanism by which they drive their regenerative effects.

Abstract WMP80: Live Animal Imaging Reveals Delayed But Sustained Neutrophil Recruitment to Both Ischemic and Non-Ischemic Cortical Cerebrovasculature in a Mouse Transient Stroke Model

Background: Treatment for acute ischemic stroke (AIS) relies on early restoration of blood flow; however, injury persists despite reperfusion. The innate immune response plays a pivotal role in reperfusion injury, but the mechanisms and timing of neutrophil recruitment to the cerebrovasculature (CBV) during AIS remain unclear. We sought to characterize the neutrophil response in the CBV following ischemia/reperfusion using a transient middle cerebral artery occlusion (tMCAO) AIS model. Methods: Fluorescent reporter mice underwent 90-minute tMCAO confirmed with LSCI or surgery without occlusion (sham). Real-time confocal intravital microscopy of the CBV was performed at 24 and 72 hrs post-reperfusion through cranial windows in ischemic and non-ischemic hemispheres. Neutrophil recruitment (adhesion, rolling) and extravasation were quantified in vivo over both hemispheres. Neutrophil extravasation was further quantified using whole mount sections. Infarct volume was measured by 2,3,5-triphenyltetrazolium chloride staining. Results: An increase in neutrophil rolling, adhesion and extravasation was observed within the ischemic hemisphere compared to sham (p < 0.01) at 24 and markedly 72 hrs. Non-ischemic hemispheres showed increased neutrophil rolling and adhesion, but not extravasation, compared to sham surgery (p < 0.05). Whole mount sections demonstrated increased cortical and subcortical neutrophil extravasation in the ischemic hemisphere only (p < 0.01). Conclusions: Early neutrophil recruitment to the ischemic hemisphere following tMCAO was present at both the CBV and within the infarcted parenchyma at 72 hours. Acute neutrophil recruitment at the CBV in the non-ischemic hemisphere, but not the cortex, suggests that AIS triggers a global inflammatory response within the CBV. Targeted neutrophil-specific therapies during reperfusion may reduce global inflammation at the CBV, and potentially mitigate secondary neuronal injury.

Visualization of Mouse Choroidal and Retinal Vasculature Using Fluorescent Tomato Lectin Perfusion.

PurposeTo develop a reliable and simplified method to assess choroid and retinal vasculature on whole mount and cross sections in mice using tomato lectin (TL; Lycopersicon esculentum).MethodsAlbino mice (n = 27) received 1 mg/mL of TL (conjugated to Dylight-594) intravascularly through the tail vein, jugular vein, or cardiac left ventricle. Whole mounts of the retina and choroid were evaluated using fluorescence microscopy. Perfusion with GSL-IB4 conjugated to Dylight-594 and fluorescein isothiocyanate was performed to compare against labeling with TL. Co-labeling of choroidal endothelial cells with perfused TL on cross-sections with antibodies directed against the choriocapillaris-restricted endothelial cell marker CA4 was performed. The percentage of perfused choroidal and retinal vessels was assessed semiquantitatively. One mouse was subjected to thermal laser damage before perfusion to cause retinal and choroidal vasculature ablation.ResultsIntravascular injection of TL led to consistent, robust labeling of retinal and choroidal vascular walls. On cross-sections, choriocapillaris was co-labeled with CA4 and TL. On flat mount, TL perfusion resulted in better labeling of choroidal vessels using tail/jugular vein injection compared with cardiac perfusion (P < .01). More consistent labeling of the choroidal and retinal vascular trees was observed with TL than with GSL-IB4. Vascular damage caused by laser ablation was detected readily using this method.ConclusionsTL injection intravascularly can reliably label normal and ablated choroid and retinal vasculature in mouse in a quick, simple manner.Translational RelevanceThese data will help to facilitate modeling in rodents for diseases such as age-related macular degeneration, diabetes, and other ischemic/angiogenic processes that can also be used for treatment evaluation.

Application of modified transperineal template-guided prostate biopsy in the diagnosis of prostate cancer

Objective To investigate the clinical value of modified transperineal template-guided prostate biopsy (mTTPB) in the detection of prostate cancer. Methods A total of 217 patients were enrolled in this study. All the patients were randomly divided into 2 groups. The control group (n=112) underwent transperineal template-guided prostate biopsy (TTPB) which was traditional transperineal template-guided 11-region biopsy. On the basis of the control group, the apex of prostate was divided into four areas for biopsy in the observation group (mTTPB). The positive rate of apex and the incidence of complications were analyzed. The prostatic specimens from the radical prostatectomy underwent whole mount sections examination. The prostate biopsy results were compared with the postoperative pathological results. Results The average age of the control group and the observation group were (68.5±7.9) years and (67.3±8.5) years, PSA were (31.2±18.9) ng/ml and (29.7±19.5) ng/ml, prostate volume were (44.6±15.2) ml and (41.3±17.3) ml, respectively. In the control group, the positive rates of prostate cancer in 1-10 region were 24.1% (27/112), 27.7%(31/112), 23.2% (26/112), 28.6% (32/112), 26.8% (30/112), 25.0% (28/112), 26.8% (30/112), 19.6% (22/112), 25.9% (29/112), 25.0% (28/112), respectively, with an average of 25.3%. In the observation group, the positive rates in 1-10 region were 27.6% (29/105), 28.6% (30/105), 22.9% (24/105), 26.7% (28/105), 25.7% (27/105), 24.8% (26/105), 27.6% (29/105), 21.9% (23/105), 27.6% (29/105), 26.7% (28/105), respectively, with an average of 26.0%. There was no statistical difference between the two groups (P=0.904). The positive rate of apical prostate cancer in the control group and observation group was 37.5% (42/112) and 44.8% (47/105), respectively, and there was no statistical difference between the two groups (P=0.277). Patients were grouped according to PSA>20 ng/ml and PSA≤20 ng/ml. When PSA>20 ng/ml, the positive rate of apex was 58.6% (34/58) and 56.6% (30/53) respectively in the control group and the observation group, and there was no statistical difference between the two groups (P=0.830). When PSA≤20 ng/ml, the positive rate of apex was 14.8% (8/54) in the control group and 32.7% (17/52) in the observation group, with statistically significant differences (P=0.030). Before radical prostatectomy, 12 cases (57.1%) in the control group and 19 cases (73.1%) in the observation group showed apical invasion by biopsy. Results of whole mount sections examination in the control group showed that there were 19 cases (90.5%) with apical invasion, which was statistically different from that before surgery (P=0.035). The results of whole mount sections examination in the observation group showed that there were 23 cases (88.5%) with apex invasion, which had no statistical difference compared with that before surgery (P=0.291). There were no significant differences in the incidence of hematuria, fever, urinary retention and perineal discomfort between the observation group and the control group (all P>0.05). Conclusions mTTPB can significantly improve the detection rate of apical prostate cancer without increasing the incidence of complications, especially for patients with PSA≤20 ng/ml. Hence is safe and efficacious. Key words: Prostatic neoplasms; Biopsy; Diagnosis

High Accuracy and Effectiveness with Deep Neural Networks in Pathological Diagnosis of Prostate Cancer

PURPOSE: Due to the multifocality and heterogeneity, the pathological diagnosis of prostate cancer still faces some challenge. To establish an artificial intelligence (AI) system based on prostate histopathological whole mount (WM) sections to make the pathological diagnosis of prostate cancer. EXPERIMENTAL DESIGN: All WM pathological digital images was marked on the basis of the ISUP Gleason grade group by two pathologists. After image segmentation there were totally 2,138,895 patches, of which 1,646,535 patches were valid for training using modified U-Net as fundamental network architecture. Another 22 pieces of WM images from 12 patients were selected for the result testing. Comparisons of Gleason score distributions between training and testing sets were performed by the chi square test. ROC curves for the binary analysis plotted the true positive rate (sensitivity) versus the false positive rate (1 - specificity). The AUC was calculated as proposed by Obuchowski. RESULTS: 826 pieces of WM sections from 148 patients were assigned to the training set randomly. The value of pixel accuracy of three methods (binary analysis, clinically-oriented analysis and analysis for different ISUP Gleason grade) mentioned above were 96.93%, 95.43% and 93.88%, respectively. The value of frequency weighted IoU were 94.32%, 92.13% and 90.21%, respectively. CONCLUSIONS: The AI system not only identified cancer from non-cancer, but also evaluated the malignant degree by distinguishing the ISUP Gleason grade group with high accuracy at pixel level, which is comparable as a professional pathologist. FUNDING STATEMENT: This study was funded by the National Natural Science Foundation of China (81572519, 81772710 and 81802535), Invigorating Health Care through Science (ZDXKB2016014), Key Project Supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (YKK 18064 and YKK18056). DECLARATION OF INTERESTS: The authors declare no competing interests. ETHICS APPROVAL STATEMENT: All the patients involved in the study were provided signed informed consent, which was approved by the ethics committee of Nanjing Drum Tower Hospital.

Does tumor location affect prostate cancer prognosis?

45 Background: Does tumor location affect prostate cancer prognosis? To clarify this question we conducted a retrospective study to characterize the incidence and prognostic significance of tumor location of prostate cancer. Methods: From 2000 to 2017, radical prostatectomy with no neoadjuvant therapy was performed in 916 cases in our hospital. Serial whole mount sections were reviewed to determine the incidence, clinicopathological features and prognostic significance of tumor location in the prostate gland. For the tumor location, we defined the subzones, which were made from subdivision of the McNeal’s zonal anatomy, are shown in Table 1 in detail. The peripheral zone (PZ) is composed of 8 subzones including A1, A2, A3 , M1, M2, M3, M4, M5. The transition zone (TZ) is composed of 5 subzones including T1, T2, T3, T4 and B1. The central zone (CZ) is composed of 3 subzones including M6, B2 and B3. Results: The median age was 67 and PSA was 8.6 ng/ml. The subzonal tumor incidence divided by all cases was the highest in A2, followed by M4, A1, M3, T2 in that order, while the lowest in B3, followed by M6, T4, T3, B1 in that order. The median follow-up time was 67 months. A 5-year PSA failure rate (5Y-PSAFR) was 23%. Among the subzones, the highest 5Y-PSAFR was seen in B3, followed by M6, B2, T4, A3, and the lowest was seen in M4, A2, T2, A1 in that order. A multivariate analysis for PSAF risk among subzones showed that B3 (HR 8.6, p &lt;0.0001) and M6 (HR 3.3, p = 0.03) were the independent high risk subzones. Conclusions: We demonstrated that the cancer incidence and prognosis varies according to the location within the prostate gland. The B3 and M6 around the ejaculatory duct showed the lowest incidence, while these locations also had the highest recurrence risk. [Table: see text]

Tracking Macrophage Infiltration in a Mouse Model of Pancreatic Cancer with the Positron Emission Tomography Tracer [11C]PBR28

BackgroundThe tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) contains abundant immunosuppressive tumor-associated macrophages. High level of infiltration is associated with poor outcome and is thought to represent a major roadblock to lymphocyte-based immunotherapy. Efforts to block macrophage infiltration have been met with some success, but noninvasive means to track tumor-associated macrophagess in PDAC are lacking. Translocator protein (TSPO) is a mitochondrial membrane receptor which is upregulated in activated macrophages. We sought to identify if a radiotracer-labeled cognate ligand could track macrophages in PDAC. Materials and MethodsA murine PDAC cell line was established from a transgenic mouse with pancreas-specific mutations in KRAS and p53. After confirming lack of endogenous TSPO expression, tumors were established in syngeneic mice. A radiolabeled TSPO-specific ligand ([11C] peripheral benzodiazepine receptor [PBR]28) was delivered intravenously, and tumor uptake was assessed by autoradiography, ex vivo, or micro-positron emission tomography imaging. ResultsResected tumors contained abundant macrophages as determined by immunohistochemistry and flow cytometry. Immunoblotting revealed murine macrophages expressed TSPO with increasing concentration on activation and polarization. Autoradiography of resected tumors confirmed [11C]PBR28 uptake, and whole mount sections demonstrated the ability to localize tumors. To confirm the findings were macrophage specific, experiments were repeated in CD11b-deficient mice, and the radiotracer uptake was diminished. Micro-positron emission tomography imaging validated radiotracer uptake and tumor localization in a clinically applicable manner. ConclusionsAs new immunotherapeutics reshape the PDAC microenvironment, tools are needed to better measure and track immune cell subsets. We have demonstrated the potential to measure changes in macrophage infiltration in PDAC using [11C]PBR28.