Slide Staining Research Articles

Routine Elastic Staining Helps Detection of Vascular Invasion in Colorectal Cancer: A Comprehensive Analysis of T3 or Higher Tumors Without Lymph Node Metastasis.

Vascular invasion (VI) in colorectal carcinoma (CRC) is an independent prognostic feature and a high-risk indicator for adjuvant chemotherapy in stage II CRC. This study evaluated the effect of elastic staining on VI detection. The VI was assessed using elastic staining in 154 patients with CRC. Based on hematoxylin and eosin (H&E) staining, cases were classified into three groups: absent (n=80), equivocal (n=23), and suspected (n=51). Two sections per case were evaluated for VI using elastic staining, and the presence of VI on one or both slides was confirmed. Finally, the correlation between the VI and other clinicopathological factors was analyzed. The overall detection rate of VI using elastic staining was 67/154 (51.4%). VI was detected in 17/80 (21.3%), 3/23 (13.0 %), and 47/51 (92.2%) patients in the absent, equivocal, and suspected groups, respectively. VI was detected in both sections of the elastic staining slides in 28 cases, and in only one section in 38 cases. The VI was significantly associated with perineural invasion, M stage, and synchronous metastasis. VI detection using H&E staining alone is not reliable, emphasizing the importance of elastic staining in improving VI detection. Therefore, we recommend the incorporation of elastic staining into routine pathological practice for all pT3 and pT4 CRC cases.

Comparison of inferior vena cava puncture under continuous cardiac perfusion with cardiac puncture in blood acquisition of the laboratory mouse.

Obtaining sufficient blood volume from mice significantly facilitates experimental research. This study explored the inferior vena cava puncture under continuous cardiac perfusion (IVCP-UCCP) technique and evaluated its efficiency in comparison with conventional cardiac puncture (CP). In an initial dose-finding study, 50 mice were randomly assigned to one of 10 groups with escalating perfusion volume from 0.5 to 4.5 ml in 0.5-ml increments. The minimum perfusion volume was determined to be 2 ml in collecting whole circulating blood. In the next comparison using the conventional method, 40 mice were randomly assigned to one of two groups denoting different blood collection methods: Group 1: CP, Group 2: IVCP-UCCP. The results showed 1) that the cells and undiluted blood volume collected via IVCP-UCCP was over twofold higher than that by CP (p < 0.001), confirmed by the cell counts and hematoxylin-eosin staining of different tissues slides (p < 0.001); 2) the new technique did not alter the cellular composition or viability, which was verified by routine blood tests and flow cytometry (p > 0.05); 3) the blood collected via the novel technique was diluted 2.1 times: the hemato-biochemical indicator results multiplied by 2.1 were identical with the test results of blood from CP (p > 0.05). Together, the refined blood collection method of IVCP-UCCP completely extracted the limited blood resources in mice, significantly enhanced the utilization of each mouse, and thus offered scientific and ethical benefits. This technique may be also applicable for other small animal models.

Melatonin Attenuates Diabetic Retinopathy by Regulating EndMT of Retinal Vascular Endothelial Cells via Inhibiting the HDAC7/FOXO1/ZEB1 Axis

ABSTRACTDiabetic retinopathy (DR) is characterized as a microvascular disease. Nonproliferative diabetic retinopathy (NPDR) presents with alterations in retinal blood flow and vascular permeability, thickening of the basement membrane, loss of pericytes, and formation of acellular capillaries. Endothelial–mesenchymal transition (EndMT) of retinal microvessels may play a critical role in advancing NPDR. Melatonin, a hormone primarily secreted by the pineal gland, is a promising therapeutic for DR. This study explored the EndMT in retinal microvessels of NPDR and its related mechanisms. The effect of melatonin on the retina of diabetic rats was evaluated by electroretinogram (ERG) and histopathologic slide staining. Furthermore, the effect of melatonin on human retinal microvascular endothelial cells (HRMECs) was detected by EdU incorporation assay, scratch assay, transwell assay, and tube formation test. Techniques such as RNA‐sequencing, overexpression or knockdown of target genes, extraction of cytoplasmic and nuclear protein, co‐immunoprecipitation (co‐IP), and multiplex immunofluorescence facilitated the exploration of the mechanisms involved. Our findings reveal, for the first time, that melatonin attenuates diabetic retinopathy by regulating EndMT of retinal vascular endothelial cells via inhibiting the HDAC7/FOXO1/ZEB1 axis. Collectively, these results suggest that melatonin holds potential as a therapeutic strategy to reduce retinal vascular damage and protect vision in NPDR.

LncRNA OIP5-AS1 regulates ferroptosis and mitochondrial dysfunction-mediated apoptosis in spinal cord injury by targeting the miR-128-3p/Nrf2 axis

BackgroundFerroptosis is an important way of neuronal cell death in acute phase and participates in the inflammatory cascade after spinal cord injury (SCI). It is reported that microRNA (miRNA) and long non-coding RNA (lncRNA) are key mediators in the regulation of ferroptosis. This study will explore the inhibitory effect of LncRNA OIP5-AS1 on ferroptosis and mitochondrial dysfunction-mediated apoptosis in SCI. MethodsThe ferric ammonium citrate (FAC)-induced cell model and the SCI rat model were established. The expression of LncRNA OIP5-AS1, miR-128-3p and Nrf2 were transfected to evaluated the effect on the viability and apoptosis of FAC-induced cell. The interaction between LncRNA OIP5-AS1 and miR-128-3p or miR-128-3p and Nrf2 were analyzed. In addition, expressions of markers related to ferroptosis and mitochondrial dysfunction were analyzed in vitro and in vivo. Histopathologic slide staining was used to analyze spinal cord injury in vivo. ResultsLncRNA OIP5-AS1 expression was abnormally down-regulated in FAC-induced SCI cell model and SCI rats. The LncRNA OIP5-AS1 deficiency induced decreased Nrf2 level by less sponging miR-128-3p, thus, aggravating spinal cord injury and inducing more apoptosis, ferroptosis and mitochondrial dysfunction in neural stem cells with SCI. However, overexpression of LncRNA OIP5-AS1 inhibited apoptosis, ferroptosis and mitochondrial dysfunction, thus effectively ameliorating spinal cord injury. ConclusionThis finding demonstrates that LncRNA OIP5-AS1 overexpression could enhance the recovery of spinal cord injury by regulating the miR-128-3p/Nrf2 axis.

Robust, credible, and interpretable AI-based histopathological prostate cancer grading.

Prostate cancer (PCa) is among the most common cancers in men and its diagnosis requires the histopathological evaluation of biopsies by human experts. While several recent artificial intelligence-based (AI) approaches have reached human expert-level PCa grading, they often display significantly reduced performance on external datasets. This reduced performance can be caused by variations in sample preparation, for instance the staining protocol, section thickness, or scanner used. Another limiting factor of contemporary AI-based PCa grading is the prediction of ISUP grades, which leads to the perpetuation of human annotation errors. We developed the prostate cancer aggressiveness index (PCAI), an AI-based PCa detection and grading framework that is trained on objective patient outcome, rather than subjective ISUP grades. We designed PCAI as a clinical application, containing algorithmic modules that offer robustness to data variation, medical interpretability, and a measure of prediction confidence. To train and evaluate PCAI, we generated a multicentric, retrospective, observational trial consisting of six cohorts with 25,591 patients, 83,864 images, and 5 years of median follow-up from 5 different centers and 3 countries. This includes a high-variance dataset of 8,157 patients and 28,236 images with variations in sample thickness, staining protocol, and scanner, allowing for the systematic evaluation and optimization of model robustness to data variation. The performance of PCAI was assessed on three external test cohorts from two countries, comprising 2,255 patients and 9,437 images. Using our high-variance datasets, we show how differences in sample processing, particularly slide thickness and staining time, significantly reduce the performance of AI-based PCa grading by up to 6.2 percentage points in the concordance index (C-index). We show how a select set of algorithmic improvements, including domain adversarial training, conferred robustness to data variation, interpretability, and a measure of credibility to PCAI. These changes lead to significant prediction improvement across two biopsy cohorts and one TMA cohort, systematically exceeding expert ISUP grading in C-index and AUROC by up to 22 percentage points. Data variation poses serious risks for AI-based histopathological PCa grading, even when models are trained on large datasets. Algorithmic improvements for model robustness, interpretability, credibility, and training on high-variance data as well as outcome-based severity prediction gives rise to robust models with above ISUP-level PCa grading performance.

Assessing venous invasion in stage II colon cancer: optimal elastin stains and survival analysis

AimsVenous invasion (VI) in colorectal carcinoma influences treatment strategies, especially in early stages. Despite elastin staining effectiveness in detecting VI, guidelines for its routine application, including the optimal number of...

CD163+ macrophages in the triple-negative breast tumor microenvironment are associated with improved survival in the Women’s Circle of Health Study and the Women’s Circle of Health Follow-Up Study

BackgroundTumor-associated macrophages (TAMs) are a prominent immune subpopulation in the tumor microenvironment that could potentially serve as therapeutic targets for breast cancer. Thus, it is important to characterize this cell population across different tumor subtypes including patterns of association with demographic and prognostic factors, and breast cancer outcomes.MethodsWe investigated CD163+ macrophages in relation to clinicopathologic variables and breast cancer outcomes in the Women’s Circle of Health Study and Women’s Circle of Health Follow-up Study populations of predominantly Black women with breast cancer. We evaluated 611 invasive breast tumor samples (507 from Black women, 104 from White women) with immunohistochemical staining of tissue microarray slides followed by digital image analysis. Multivariable Cox proportional hazards models were used to estimate hazard ratios for overall survival (OS) and breast cancer-specific survival (BCSS) for 546 cases with available survival data (median follow-up time 9.68 years (IQR: 7.43–12.33).ResultsWomen with triple-negative breast cancer showed significantly improved OS in relation to increased levels of tumor-infiltrating CD163+ macrophages in age-adjusted (Q3 vs. Q1: HR = 0.36; 95% CI 0.16–0.83) and fully adjusted models (Q3 vs. Q1: HR = 0.30; 95% CI 0.12–0.73). A similar, but non-statistically significant, association was observed for BCSS. Macrophage infiltration in luminal and HER2+ tumors was not associated with OS or BCSS. In a multivariate regression model that adjusted for age, subtype, grade, and tumor size, there was no significant difference in CD163+ macrophage density between Black and White women (RR = 0.88; 95% CI 0.71–1.10).ConclusionsIn contrast to previous studies, we observed that higher densities of CD163+ macrophages are independently associated with improved OS and BCSS in women with invasive triple-negative breast cancer.Trial registrationNot applicable.

Abstract PO3-13-11: Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies

Abstract Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² &amp;gt; 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-11.

The Effectiveness of Ylang Ylang Oil (Cananga Odorata) as a Deparaffinizing Agent in Hematoxylin-Eosin Staining

Histological staining usually requires deparaffinization before starting the staining process. Deparaffinization is a paraffin removal step that often uses a xylol solution. Deparaffinization with xylol has disadvantages, including being toxic, harmful to the human body, and hazardous to the environment. A safer alternative to xylol is required. Ylang-ylang oil, which contains β-caryophyllene, is non-polar and can be used as a substitute for xylol. This study aimed to compare the staining quality of guinea pig liver tissue slides with and without heating as a deparaffinization agent in hematoxylin-eosin (HE) staining. The research set up a quasi-experimental approach using samples of guinea pig liver tissue slides. The sample size was calculated using the Federer formula (n-1) (t-1) ≥ 15, resulting in a total sample of 27 slides divided into three treatment groups: xylol, ylang-ylang oil with and without heating. The Kruskal-Wallis test was used to examine the data. The quality staining of guinea pig liver slides deparaffinized with xylol was 100% good, and deparaffinization of ylang-ylang oil with heating was 100% good, whereas deparaffinization with ylang-ylang oil without heating was 11.1% less good, and the quality was good with an 88.9%. The normality test indicated that the data were not normally distributed (p0.05). The Kruskal-Wallis test was then used.

Noninvasive virtual biopsy using micro-registered optical coherence tomography (OCT) in human subjects.

Histological hematoxylin and eosin-stained (H&E) tissue sections are used as the gold standard for pathologic detection of cancer, tumor margin detection, and disease diagnosis. Producing H&E sections, however, is invasive and time-consuming. While deep learning has shown promise in virtual staining of unstained tissue slides, true virtual biopsy requires staining of images taken from intact tissue. In this work, we developed a micron-accuracy coregistration method [micro-registered optical coherence tomography (OCT)] that can take a two-dimensional (2D) H&E slide and find the exact corresponding section in a 3D OCT image taken from the original fresh tissue. We trained a conditional generative adversarial network using the paired dataset and showed high-fidelity conversion of noninvasive OCT images to virtually stained H&E slices in both 2D and 3D. Applying these trained neural networks to in vivo OCT images should enable physicians to readily incorporate OCT imaging into their clinical practice, reducing the number of unnecessary biopsy procedures.

Conducting practical classes of phase II MBBS students (CBME batch) during COVID 19 pandemic: Experience of a paraclinical resident

ABSTRACT The Medical Council of India, now the National Medical council changed the teaching pattern of the Under Graduate Medical Course (MBBS) after 22 years. The new revised curriculum and teaching pattern is known as Competency Based Medical Education (CBME). CBME pattern started from the 2019 MBBS batch. In the second year of MBBS, they have to learn para clinical subjects, Pathology, Microbiology, and Pharmacology. Teaching Practical classes to MBBS students for the first year PG residents is a new experience for them, as they just start their paraclinical specialty journey, and it is even more difficult for them when they are teaching the CBME batch. To take one practical class MBBS, PG residents must start the preparation before at least one week. In that one week, we read the topic thoroughly with concepts. They must prepare the PowerPoint presentation followed by a microteaching presentation to the departmental faculties and have to do the correction to the presentation. PG residents make many arrangements like smear slides for staining, culture plates, and biochemical reactions to demonstrate to MBBS students but when only 4–5 students were attending the physical demonstration classes, all the preparation look useless.

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&amp;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&amp;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&amp;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.

Abstract 1027: Immunohistochemistry (IHC) to assess tumor cell-associated OX40 expression in lymphoma PDXs

Abstract Background. Tumor necrosis factor receptor superfamily member 4, or OX40, is an immunostimulatory receptor usually expressed in activated T-cells. The OX40-OX40L (ligand of OX40) interaction plays a crucial role in the enhancement of T-cell proliferation, survival, and cytokine production, including those in tumor microenvironment (TME) leading to increased anti-tumor immunity1. OX40 agonist antibodies have been widely tested for immunotherapy for solid tumors2,3. Interestingly, in the treatment of lymphoma, the situation may become more complicate, where OX40 could also be found to be expressed on lymphoma cells, in addition to TME. This phenomenon might or might not impact OX40 agonist therapy. This report aims at developing an immunohistochemistry (IHC) assay to assess tumor-associated OX40 by utilizing morphology approach to understand OX40 expression in lymphoma cells, as opposed on tumor-infiltrate T-lymphocytes (TILs) present in TME. FFPE samples from patient-derived xenografts (PDXs), recapitulating the main pathological features of the original tumors, but exclusive of human TILs, were used as the first stage of the assay development, avoiding TIL interference prior to human sample testing. Methods. Ninety leukemia and lymphoma PDXs are comprehensively annotated, including RNAseq and histopathology (HuBaseTM: https://hubase.crownbio.com). IHC staining of PDX FFPE slides or TMA (tissue microarray) of various cancers was performed using a commercial OX40 antibody (CST, #61637) and Bond RX Automated IHC/ISH Staining System (Leica Biosystems), followed by whole slide imaging by NanoZoomer NDP2.0-HT Digital Slide System (Hamamatsu) and quantitative analysis by HALOTM image analysis software (Indica labs). OX40 IHC scores were also compared to the RNA expression determined by RNAseq. Results. A total of 90 leukemia and lymphoma PDXs were comprehensively evaluated. RNASeq data (human mRNA) indeed revealed OX40 gene expression (cutoff as Log2 (FPKM)&amp;gt;3) in 8.9% of models (8/90 models); on the other hand, IHC data also confirmed these OX40 protein expression; and the IHC scores largely correlated with the RNA data. Together, our data firmly confirmed the expression of OX40 in certain hematological cancers, beyond just in TME. The ongoing studies is investigating the characteristics of the OX40 positive lymphoma/leukemia, and the preliminary data will be presented in the annual conference. Furthermore, we are also using the same assay to explore other cancer types beyond hematological cancers. Conclusion. A tumor associated OX40 expression IHC workflow was preliminarily established and validated to facility the OX40 immunotherapy development. Citation Format: Jie Lin, Xiaolong Tu, Likun Zhang, Hongjiang Yu, Cheng JIang, Tao Yang, Henry Qixiang Li. Immunohistochemistry (IHC) to assess tumor cell-associated OX40 expression in lymphoma PDXs [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 1027.

Abstract 4939: Computational pathology approach for prognostic advancements and therapeutic benefits in gastrointestinal cancer: A multi-centric retrospective study

Abstract Background: The current cancer staging methods cannot accurately predict survival and therapeutic benefits in cancer patients. Digital pathomics is an emerging field with potential to revolutionize disease evaluation. The present study aims to develop and validate a deep learning pathomics signature from digital hematoxylin-eosin (H&amp;E) staining slides to predict the survival of pan-gastrointestinal cancer, and further investigated its associations with chemotherapy and immunotherapy response. P atients and Methods: This international multicenter study included 2,463 patients with pan-gastrointestinal cancer from twelve cohorts of seven cancer centers, among which 1653 patients were diagnosed with GC. We proposed a deep learning pathomics signature (DLPS) by integrating information on three scales from the whole slide images (WSIs) of H&amp;E, including pathomics nucleus, pathomics microenvironment, and single-cell spatial distribution features. Next, we evaluated the predictive accuracy of DLPS for prognosis, chemotherapy response, and immunotherapy response. To interpret the predictive ability of the DLPS, we analyzed multi-omics data and employed the Shapley value strategy to provide its biological insights and importance ranking. Results: The DLPS was significantly associated with overall survival in patients with GC (hazard ratio range: 1.203-2.717, all p &amp;lt; 0.001), which was further validated in other gastrointestinal cancers (all p &amp;lt; 0.02). The DLPS remained an independent predictor of prognosis in multivariable analysis (all p &amp;lt; 0.001). Furthermore, a nomogram incorporating the DLPS and TNM stage shows significantly improved accuracy in predicting cancer prognosis compared to that with TNM stage alone (all p &amp;lt; 0.05). Shapley value analysis highlighted DLPS as the strongest predictor for prognosis. Importantly, GC patients with a low-DLPS (but not those with a high-DLPS) exhibited substantial benefits from adjuvant chemotherapy (all p &amp;lt; 0.05). Furthermore, we found the objective responses of anti-PD-1 immunotherapy is significantly higher in the low-DLPS group (29.6%) than in the high-DLPS group (8.3%, p &amp;lt; 0.05). Upon analyzing multi-omics data, we found that a higher DLPS was positively correlated with tumor promoting, chemotherapy resistance, immune tumor microenvironment and metabolic signaling. Conclusion: The DLPS enabled improved assessment on prognosis, and has the potential to identify patients who will benefit from adjuvant chemotherapy and immunotherapy, which can further be extended to many gastrointestinal cancers or other solid tumors. Citation Format: Taojun Zhang, Zepang Sun, Zhe Li, M. Usman Ahmad, Md Tauhidul Islam, Fan Yang, Zhenhui Li, Yuming Jiang. Computational pathology approach for prognostic advancements and therapeutic benefits in gastrointestinal cancer: A multi-centric retrospective study [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 4939.

Discontinuity third harmonic generation microscopy for label-free imaging and quantification of intraepidermal nerve fibers

Label-free imaging methodologies for nerve fibers rely on spatial signal continuity to identify fibers and fail to image free intraepidermal nerve endings (FINEs). Here, we present an imaging methodology-called discontinuity third harmonic generation (THG) microscopy (dTHGM)-that detects three-dimensional discontinuities in THG signals as the contrast. We describe the mechanism and design of dTHGM and apply it to reveal the bead-string characteristics of unmyelinated FINEs. We confirmed the label-free capability of dTHGM through a comparison study with the PGP9.5 immunohistochemical staining slides and a longitudinal spared nerve injury study. An intraepidermal nerve fiber (IENF) index based on a discontinuous-dot-connecting algorithm was developed to facilitate clinical applications of dTHGM. A preliminary clinical study confirmed that the IENF index was highly correlated with skin-biopsy-based IENF density (Pearson's correlation coefficient R= 0.98) and could achieve differential identification of small-fiber neuropathy (p= 0.0102) in patients with diabetic peripheral neuropathy.

Quantitative assessment of H&E staining for pathology: development and clinical evaluation of a novel system

BackgroundStaining tissue samples to visualise cellular detail and tissue structure is at the core of pathology diagnosis, but variations in staining can result in significantly different appearances of the tissue sample. While the human visual system is adept at compensating for stain variation, with the growth of digital imaging in pathology, the impact of this variation can be more profound. Despite the ubiquity of haematoxylin and eosin staining in clinical practice worldwide, objective quantification is not yet available. We propose a method for quantitative haematoxylin and eosin stain assessment to facilitate quality assurance of histopathology staining, enabling truly quantitative quality control and improved standardisation.MethodsThe stain quantification method comprises conventional microscope slides with a stain-responsive biopolymer film affixed to one side, called stain assessment slides. The stain assessment slides were characterised with haematoxylin and eosin, and implemented in one clinical laboratory to quantify variation levels.ResultsStain assessment slide stain uptake increased linearly with duration of haematoxylin and eosin staining (r = 0.99), and demonstrated linearly comparable staining to samples of human liver tissue (r values 0.98–0.99). Laboratory implementation of this technique quantified intra- and inter-instrument variation of staining instruments at one point in time and across a five-day period.ConclusionThe proposed method has been shown to reliably quantify stain uptake, providing an effective laboratory quality control method for stain variation. This is especially important for whole slide imaging and the future development of artificial intelligence in digital pathology.

Algorithm-assisted diagnosis of Hirschsprung’s disease – evaluation of robustness and comparative image analysis on data from various labs and slide scanners

BackgroundDifferences in the preparation, staining and scanning of digital pathology slides create significant pre-analytic variability. Algorithm-assisted tools must be able to contend with this variability in order to be applicable in clinical practice. In a previous study, a decision support algorithm was developed to assist in the diagnosis of Hirschsprung's disease. In the current study, we tested the robustness of this algorithm while assessing for pre-analytic factors which may affect its performance.MethodsThe decision support algorithm was used on digital pathology slides obtained from four different medical centers (A-D) and scanned by three different scanner models (by Philips, Hamamatsu and 3DHISTECH). A total of 192 cases and 1782 slides were used in this study. RGB histograms were constructed to compare images from the various medical centers and scanner models and highlight the differences in color and contrast.ResultsThe algorithm was able to correctly identify ganglion cells in 99.2% of cases, from all medical centers (All scanned by the Philips slide scanner) as well as 95.5% and 100% of the slides scanned by the 3DHISTECH and Hamamatsu brand slide scanners, respectively. The total error rate for center D was lower than the other medical centers (3.9% vs 7.1%, 10.8% and 6% for centers A-C, respectively), the vast majority of errors being false positives (3.45% vs 0.45% false negatives). The other medical centers showed a higher rate of false negatives in relation to false positives (6.81% vs 0.29%, 9.8% vs 1.2% and 5.37% vs 0.63% for centers A-C, respectively). The total error rates for the Philips, Hamamatsu and 3DHISTECH brand scanners were 3.9%, 3.2% and 9.8%, respectively. RGB histograms demonstrated significant differences in pixel value distribution between the four medical centers, as well as between the 3DHISTECH brand scanner when compared to the Philips and Hamamatsu brand scanners.ConclusionsThe results reported in this paper suggest that the algorithm-based decision support system has sufficient robustness to be applicable for clinical practice. In addition, the novel method used in its development – Hierarchial-Contexual Analysis (HCA) may be applicable to the development of algorithm-assisted tools in other diseases, for which available datasets are limited. Validation of any given algorithm-assisted support system should nonetheless include data from as many medical centers and scanner models as possible.

Histopathologic spectrum of morphea: a single-center retrospective study.

Morphea is a rare autoimmune disease that often affects skin and subcutaneous tissues. The aim of this study was to determine the association between patient demographic parameters, lesion site, clinical subtype of morphea, and histological findings. Between 2016 and 2022, we investigated 78 patients with morphea at the Department of Pathology, Prof. Dr. Cemil Taşcıoğlu City Hospital in Turkey. Case-specific hematoxylin and eosin stain slides were obtained from the pathology archive and assessed blindly by two pathologists. Flattening of rete ridges, location of inflammatory infiltrate, grade of inflammatory infiltrate, presence of plasma cells, presence of eosinophils, homogenization of dermal collagen, decrease of skin appendages, basal pigmentation and melanin incontinence were evaluated. Statistical analyses were performed using SPSS Statistics v.20 (IBM, Armonk, NY, USA). The most common clinical presentation was plaque type (87.5%), while histopathological findings included homogenization of dermal collagen (100%) and decrease of skin appendages (98.7%). Flattening of the rete ridges was observed in 46.2% of patients. Severity of the inflammatory infiltrate was found to be higher in these patients (p=0.028). Basal pigmentation was observed in 59% of patients. Line sign was more common in lower extremity lesions among all localizations (p=0.015). The histopathologic features of morphea are variable and confusing. Particularly, in cases with collagen homogenization, morphea should be considered in differential diagnosis with clinical correlation. In addition, the line sign could be helpful for identifying lesions located in the lower extremities.

Covid-19-Associated Mucormycosis: Histopathology of the Deadly Fungal Infection.

Introduction Many patients suffered from rhino-orbital-cerebral mucormycosis during the coronavirus disease 2019 (COVID-19) pandemic in India. Diabetes is a known risk factor of COVID-19 infection and mucormycosis. Objective The present study was done to describe the clinical spectrum and histopathological findings of mucormycosis in COVID-19 patients and their outcomes. Methods A cross-sectional study was done over a period of two and half months. The biopsy samples or scrapings from sinonasal or periorbital tissue of 38 patients were analyzed. Hematoxylin & Eosin (H&E stain) slides were evaluated along with Grocott-Gomori methenamine-silver and Periodic acid-Schiff stains to highlight the fungal elements. Results The male to female ratio was 2.5:1, and the mean age of the subjects was 53 years old. A total of 68.4% ( n = 26/38) of the patients had diabetes as a comorbidity, 84.2% ( n = 32/38) had a history of steroid intake, and 55.3% ( n = 21/38) were given supplemental oxygen during their treatment. The common presentations were nasal blockage, discharge, eye pain, headache, and altered mentation. The sites of biopsy were: nasal cavity 76.3% ( n = 29/38), periorbital fat/orbit 21.1% ( n = 8/38), maxillary sinus 15.8% ( n = 6/38) and ethmoid sinus 13.2% ( n = 5/38). In 76.3% ( n = 29/38) cases, broad, irregular, nonseptate, and right-angle branching hyphae were seen on H&E-stained tissue sections. Conclusion COVID-19 led to various complications in individuals affected by it. Mucormycosis was one such lethal complication. An early diagnosis and prompt treatment is crucial to control the progression of the disease and improve outcomes.