Color Standardization and Stain Intensity Calibration for Whole Slide Image-Based Immunohistochemistry Assessment.

Automated quantification of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) using whole slide imaging (WSI) is expected to eliminate subjectivity in visual assessment. However, the color intensity in WSI varies depending on the staining process and scanner device. Such variations affect the image analysis results. This paper presents methods to diminish the influence of color variation produced in the staining process using a calibrator slide consisting of peptide-coated microbeads. The calibrator slide is stained along with tissue sample slides, and the 3,3'-diaminobenzidine (DAB) color intensities of the microbeads are used for calibrating the color variation of the sample slides. An off-the-shelf image analysis tool is employed for the automated assessment, in which cells are classified by the thresholds for the membrane staining. We have adopted two methods for calibrating the color variation based on the DAB color intensities obtained from the calibrator slide: (1) thresholds for classifying the DAB membranous intensity are adjusted, and (2) the color intensity of WSI is corrected. In the experiment, the calibrator slides and tissue of breast cancer slides were stained together on different days and used to test our protocol. With the proposed protocol, the discordance in the HER2 evaluation was reduced to one slide out of 120 slides.

Alanine racemase a promising Helicobacter pylori drug target inhibited by propanoic acid.

Eradication of Helicobacter pylori, the class 1 carcinogen, faces several obstacles, which demand alternative options to conventional drug development methods. Alanine racemase (Alr) was proposed as H. pylori drug target, inhibited by propanoic acid (PA), in a previous in silico study. We investigated the possible treatment of H. pylori infection through Alr inhibition. A new model of H. pylori Alr was built, validated, and the binding of PA to the active site was modelled via molecular docking with a good docking score. PA minimum inhibitory concentration (MIC) against H. pylori ATCC 43504 and six H. pylori clinical isolates ranged from 312.5 to 416.7 ± 180 µg/ml and remained unchanged after 14 serial passages in increasing PA concentrations. The minimum bactericidal concentration of PA was 625 µg/ml. Selective Alr inhibition was confirmed by a significant PA MIC increase with increasing d-alanine concentrations. Similar PA MIC in other tested pathogens was recorded (312.5-625 µg/ml). PA lacked cytotoxicity in tested cell lines and efficiently eradicated H. pylori in a rat infection model. In conclusion, Alr is a promising broad-spectrum drug target, inhibited by PA without resistance development by repeated exposure for 14 serial passages.

Aspartate α-decarboxylase a new therapeutic target in the fight against Helicobacter pylori infection.

Effective eradication therapy for Helicobacter pylori is a worldwide demand. Aspartate α-decarboxylase (ADC) was reported as a drug target in H. pylori, in an in silico study, with malonic acid (MA) as its inhibitor. We evaluated eradicating H. pylori infection through ADC inhibition and the possibility of resistance development. MA binding to ADC was modeled via molecular docking. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MA were determined against H. pylori ATCC 43504, and a clinical H. pylori isolate. To confirm selective ADC inhibition, we redetermined the MIC in the presence of products of the inhibited enzymatic pathway: ß-alanine and pantothenate. HPLC was used to assay the enzymatic activity of H. pylori 6x-his tagged ADC in the presence of different MA concentrations. H. pylori strains were serially exposed to MA for 14 passages, and the MICs were determined. Cytotoxicity in different cell lines was tested. The efficiency of ADC inhibition in treating H. pylori infections was evaluated using a Sprague-Dawley (SD) rat infection model. MA spectrum of activity was determined in different pathogens. MA binds to H. pylori ADC active site with a good docking score. The MIC of MA against H. pylori ranged from 0.5 to 0.75 mg/mL with MBC of 1.5 mg/mL. Increasing ß-alanine and pantothenate concentrations proportionally increased MA MIC. The 6x-his tagged ADC activity decreased by increasing MA concentration. No resistance to ADC inhibition was recorded after 14 passages; MA lacked cytotoxicity in all tested cell lines. ADC inhibition effectively eradicated H. pylori infection in SD rats. MA had MIC between 0.625 to 1.25 mg/mL against the tested bacterial pathogens. In conclusion, ADC is a promising target for effectively eradicating H. pylori infection that is not affected by resistance development, besides being of broad-spectrum presence in different pathogens. MA provides a lead molecule for the development of an anti-helicobacter ADC inhibitor. This provides hope for saving the lives of those at high risk of infection with the carcinogenic H. pylori.

Pathological Evaluation of Rectal Cancer Specimens Using Micro-Computed Tomography.

Whole-block imaging (WBI) using micro-computed tomography (micro-CT) allows the nondestructive reconstruction of a three-dimensional view of tissues, implying that WBI may be used for accurate pathological evaluation of patients with rectal cancer. HOWEVER, the clinical impact of this approach is unclear. We aimed to clarify the efficacy of WBI in the whole-mount specimens of locally advanced rectal cancer. A total of 237 whole-mount formalin-fixed paraffin-embedded blocks from 13 patients with rectal cancer who underwent surgical treatment were enrolled and scanned with micro-CT to generate three-dimensional images. WBI was evaluated following the conventional pathological review of the corresponding whole-slide imaging (WSI). WBI identified all tumor sites detected using WSI. Furthermore, WBI revealed one additional tumor site, which was not detected using WSI. Tumor resection margin was significantly closer to the soft-tissue edge when measured using WBI (7.7 mm vs. 6.6 mm, p < 0.01). Seventy-six percent of tumor deposits on WSI were changed according to the evidence of tumor interaction with the surrounding tissues confirmed using WBI. Furthermore, WBI revealed 25 additional lymph nodes, six of which were metastatic. The combination of conventional hematoxylin and eosin-stained imaging and WBI may contribute to an accurate pathological assessment.

An evaluation of the effectiveness of perampanel in people with epilepsy who have previously undergone resective surgery and/or implantation of a vagal nerve stimulator.

About 30% of people with epilepsy (PWE) are drug-resistant. Those with focal seizures may be suitable for epilepsy surgery. Those not amenable to resective surgery can be considered for vagus nerve stimulation (VNS). However, after operative procedures, around 50% of patients continue to experience seizures. A multi-center retrospective study assessing perampanel effectiveness and tolerability for PWE who have undergone surgical resection and/or VNS implantation was performed. The primary outcome was ≥50% reduction in seizure frequency while secondary outcomes included side effects (SEs), dose-related effectiveness, and toxicity. The median perampanel dose was 6 mg. Only one PWE became seizure free. A ≥50% decrease in seizure frequency was observed in 52.8% of the post-resection group and 16.9% of the VNS group (p < 0.001), while SEs were seen in 44.8% and 41.1%, respectively. Perampanel doses greater than 8 mg led to better response in both groups, especially in the post-VNS cohort. SEs were not dose-related and the safety profile was similar to previous observational studies. Perampanel can be beneficial in these two super-refractory epilepsy groups, particularly in PWE with seizures after surgical resection. Doses of more than 8 mg appear to be well tolerated and may be more effective than lower doses in PWE after surgical interventions.

Three-Dimensional Vessel Segmentation in Whole-Tissue and Whole-Block Imaging Using a Deep Neural Network: Proof-of-Concept Study.

In the field of pathology, micro-computed tomography (micro-CT) has become an attractive imaging modality because it enables full analysis of the three-dimensional characteristics of a tissue sample or organ in a noninvasive manner. However, because of the complexity of the three-dimensional information, understanding would be improved by development of analytical methods and software such as those implemented for clinical CT. As the accurate identification of tissue components is critical for this purpose, we have developed a deep neural network (DNN) to analyze whole-tissue images (WTIs) and whole-block images (WBIs) of neoplastic cancer tissue using micro-CT. The aim of this study was to segment vessels from WTIs and WBIs in a volumetric segmentation method using DNN. To accelerate the segmentation process while retaining accuracy, a convolutional block in DNN was improved by introducing a residual inception block. Three colorectal tissue samples were collected and one WTI and 70 WBIs were acquired by a micro-CT scanner. The implemented segmentation method was then tested on the WTI and WBIs. As a proof-of-concept study, our method successfully segmented the vessels on all WTI and WBIs of the colorectal tissue sample. In addition, despite the large size of the images for analysis, all segmentation processes were completed in 10 minutes.

Identification of Potential Drug Targets in Helicobacter pylori Using In Silico Subtractive Proteomics Approaches and Their Possible Inhibition through Drug Repurposing.

The class 1 carcinogen, Helicobacter pylori, is one of the World Health Organization's high priority pathogens for antimicrobial development. We used three subtractive proteomics approaches using protein pools retrieved from: chokepoint reactions in the BIOCYC database, the Kyoto Encyclopedia of Genes and Genomes, and the database of essential genes (DEG), to find putative drug targets and their inhibition by drug repurposing. The subtractive channels included non-homology to human proteome, essentiality analysis, sub-cellular localization prediction, conservation, lack of similarity to gut flora, druggability, and broad-spectrum activity. The minimum inhibitory concentration (MIC) of three selected ligands was determined to confirm anti-helicobacter activity. Seventeen protein targets were retrieved. They are involved in motility, cell wall biosynthesis, processing of environmental and genetic information, and synthesis and metabolism of secondary metabolites, amino acids, vitamins, and cofactors. The DEG protein pool approach was superior, as it retrieved all drug targets identified by the other two approaches. Binding ligands (n = 42) were mostly small non-antibiotic compounds. Citric, dipicolinic, and pyrophosphoric acid inhibited H. pylori at an MIC of 1.5-2.5 mg/mL. In conclusion, we identified potential drug targets in H. pylori, and repurposed their binding ligands as possible anti-helicobacter agents, saving time and effort required for the development of new antimicrobial compounds.

Detection and assessment of capsular invasion, vascular invasion and lymph node metastasis volume in thyroid carcinoma using microCT scanning of paraffin tissue blocks (3D whole block imaging): a proof of concept.

In the modern era, detailed pathologic characteristics of a thyroid tumor are crucial to achieve accurate diagnosis and guide treatment. The presence of capsular invasion (CI) is diagnostic for carcinoma, whereas vascular invasion (VI) and nodal metastasis (NM) are included in risk stratification. However, the very definition of CI and VI is surrounded by controversies and an accurate assessment of NM is lacking. Whole Block Imaging (WBI) by microCT is a new imaging modality to create 3D reconstruction of whole tissue block with microscopic level resolution without the need for tissue sectioning. In this study, we aimed to define CI, VI, and NM volume using WBI by microCT. Twenty-eight paraffin blocks (PBs) from 26 thyroid tumors were scanned. Ten PBs contained CI, whereas 7 had VI. 3D microCT images were compared with whole slide images (WSI) of corresponding H&E slides. In 2 cases with VI and/or CI, WSI of serial H&E slides were obtained and underwent 3D-reconstruction to be compared with the WBI. Satellite tumor nodules beyond tumor capsule were shown to be CI by demonstrating the point of penetration using microCT and 3D reconstruction. Additional foci of CI were detected using microCT. VI was seen using microCT. Fibrin associated with tumor thrombus was not always present on serially sectioned H&E slides. WBI by microCT scanner was able to assess the volume of NM. In conclusion, WBI is able to detect CI, VI, and assess the volume of NM in thyroid carcinoma without tissue sectioning. It is the ultimate method for the complete sampling of the tumor capsule. It has the potential to increase the detection rate of CI, better define criteria for CI and VI, and provide an accurate assessment of the volume of nodal disease. This technology may impact the future practice of surgical pathology.

Is one benign fine needle aspiration enough?

BACKGROUND: Fine needle aspiration (FNA) is used to diagnose thyroid nodules, but the follow-up of benign FNA is unclear. We sought to determine whether routine repeat FNAs after initial benign FNA reduces false negatives. METHODS: We identified 265 patients who had at least one benign FNA that either progressed to surgery or had at least one repeat FNA. We reviewed their ultrasonography, FNA cytology, and surgical pathology. RESULTS: Of 127 patients with initial benign FNA that had surgery, 13 had a malignancy, yielding a 10.2% false-negative rate. Of 22 patients who had surgery after at least two benign FNAs, one had a malignancy, yielding a 4.5% false-negative rate. Initially benign cytology (Bethesda II) was upgraded to a cytology requiring surgical intervention (Bethesda IV-VI) in 7 of 129 (5.4%) patients after two FNAs. Suspicious features on ultrasound, including size >4 cm, calcifications, or increased vascularity were found in 90% of patients with a false-negative FNA. CONCLUSIONS: The overall false-negative rate of thyroid FNAs is 10.2%, which is reduced to 4.5% with a second benign FNA. Ninety percent of patients with a false-negative FNA had suspicious sonographic features. Reaspiration should be considered in patients with sonographically suspicious nodules.