Laboratory Investigation最新文献

{"title":"Quantifying Cardiac Tissue Composition Using QuPath and Cellpose: An Accessible Approach to Postmortem Diagnosis","authors":"Pernille Heimdal Holm ,&nbsp;Kristine Boisen Olsen ,&nbsp;Richard Denis Maxime De Mets ,&nbsp;Jytte Banner","doi":"10.1016/j.labinv.2024.102181","DOIUrl":"10.1016/j.labinv.2024.102181","url":null,"abstract":"<div><div>Sudden death can be the first symptom of cardiac disease, and establishing a precise postmortem diagnosis is crucial for genetic testing and follow-up of relatives. Arrhythmogenic cardiomyopathy is a structural cardiomyopathy that can be challenging to diagnose postmortem because of differences in structural findings and propagation of the disease at the time of death. Cases can have minimal or no structural findings and later be diagnosed according to genotype, known as concealed cardiomyopathy. Postmortem diagnosis often lacks clinical information, whereas antemortem diagnosis is based on paraclinical investigations that cannot be performed after death. However, the entire substrate is available, which is unique to postmortem diagnosis and research and can provide valuable insights when adding new methods. Reactive changes in the heart, such as myocardial fibrosis and fat, are significant findings. The patterns of these changes in various diseases are not yet fully understood and may be limited by sampling material and conventional microscopic diagnostics. We demonstrate an automated pipeline in QuPath for quantifying postmortem picrosirius red cardiac tissue for collagen, residual myocardium, and adipocytes by integrating Cellpose into a versatile pipeline. This method was developed and tested using cardiac tissues from autopsied individuals. Cases diagnosed with arrhythmogenic cardiomyopathy and age-matched controls were used for validation and testing. This approach is free and easy to implement by other research groups using this paper as a template. This can potentially lead to the development of quantitative diagnostic criteria for postmortem cardiac diseases, eliminating the need to rely on diagnostic criteria from endomyocardial biopsies that are not applicable to postmortem specimens. We propose that this approach serves as a template for creating a more efficient process for evaluating postmortem cardiac measurements in an unbiased manner, particularly for rare cardiac diseases.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 1","pages":"Article 102181"},"PeriodicalIF":5.1,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623193","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Selecting Preclinical Animal Models in Hepatology Research: A Call for Uniform Guidelines","authors":"Devaraj Ezhilarasan ,&nbsp;Mustapha Najimi","doi":"10.1016/j.labinv.2024.102179","DOIUrl":"10.1016/j.labinv.2024.102179","url":null,"abstract":"","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 1","pages":"Article 102179"},"PeriodicalIF":5.1,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cefadroxil Targeting of SLC15A2/PEPT2 Protects From Colistin Nephrotoxicity","authors":"Raul Fernandez-Prado ,&nbsp;Lara Valiño ,&nbsp;Aranzazu Pintor-Chocano ,&nbsp;Ana B. Sanz ,&nbsp;Alberto Ortiz ,&nbsp;Maria Dolores Sanchez-Niño","doi":"10.1016/j.labinv.2024.102182","DOIUrl":"10.1016/j.labinv.2024.102182","url":null,"abstract":"<div><div>Acute kidney injury (AKI) and chronic kidney disease (CKD) are considered interconnected syndromes, as AKI episodes may accelerate CKD progression, and CKD increases the risk of AKI. Genome-wide association studies (GWAS) may identify novel actionable therapeutic targets. Human GWAS for AKI or CKD were combined with murine AKI transcriptomics data sets to identify 13 (<em>ACACB</em>, <em>ACSM5</em>, <em>CNDP1</em>, <em>DPEP1</em>, <em>GATM</em>, <em>SLC6A12</em>, <em>AGXT2L1</em>, <em>SLC15A2</em>, <em>CTSS</em>, <em>ICAM1</em>, <em>ITGAX</em>, <em>ITGAM</em>, and <em>PPM1J</em>) potentially actionable therapeutic targets to modulate kidney disease severity across species and the AKI-CKD spectrum. Among them, <em>SLC15A2</em>, encoding the cell membrane proton-coupled peptide transporter 2, was prioritized for data mining and functional intervention studies in vitro and in vivo because of its known function to transport nephrotoxic drugs such as colistin and the possibility for targeting with small molecules already in clinical use, such as cefadroxil. Data mining disclosed that <em>SLC15A2</em> was upregulated in the tubulointerstitium of human CKD, including diabetic nephropathy, and the upregulation was localized to proximal tubular cells. Colistin elicited cytotoxicity and proinflammatory response in cultured human and murine proximal tubular cells that was decreased by concomitant exposure to cefadroxil. In proof-of-concept in vivo studies, cefadroxil protected from colistin nephrotoxicity in mice. The GWAS association of <em>SLC15A2</em> with human kidney disease may be actionable and related to the modifiable transport of nephrotoxins causing repeated subclinical episodes of AKI and/or chronic nephrotoxicity.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 1","pages":"Article 102182"},"PeriodicalIF":5.1,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lymphocyte Activation Gene 3 Expression, γδ T-Cell/Major Histocompatibility Complex Class I Interactions, and Prognosis in Merkel Cell Carcinoma","authors":"Jonathan Lai ,&nbsp;Vrinda Madan ,&nbsp;Aasheen Qadri ,&nbsp;Ludmila Danilova ,&nbsp;Long Yuan ,&nbsp;Victoria Jacobs ,&nbsp;Aleksandra Ogurtsova ,&nbsp;Logan L. Engle ,&nbsp;Joel C. Sunshine","doi":"10.1016/j.labinv.2024.102178","DOIUrl":"10.1016/j.labinv.2024.102178","url":null,"abstract":"<div><div>Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy with a poor prognosis. One of the major mechanisms of immune evasion in MCC involves downregulation of major histocompatibility complex class I (MHC-I). Anti-PD-1/programmed death ligand 1 checkpoint inhibitors have revolutionized treatment for MCC, producing objective responses in approximately 50% of patients, and are now the standard of care; however, a substantial proportion of patients either fail to respond or develop resistance to checkpoint inhibitors. Given these recent successes, identification of other targetable immune checkpoints in the MCC tumor microenvironment is of great interest. Additionally, γδ T cells may play critical roles in response to MHC-I–deficient cancers; therefore, evaluating γδ T cells as a prognostic biomarker is warranted. We characterized the expression of programmed death ligand 1, PD-1, CD3, CD8, lymphocyte activation gene 3 (LAG-3), MHC-I, and γδ T cells by immunohistochemistry in a preimmunotherapy retrospective cohort of 54 cases of MCC and quantified expression levels and marker density using HALO software. The increased density of LAG-3 and γδ T cells correlated with other markers of an inflamed tumor microenvironment, with significant positive associations across all 6 markers (<em>P</em> &lt; .002). Reflective of their putative role in the response to MHC-I–suppressed cancers, cases with low human leukocyte antigen I density showed a trend toward a higher ratio of γδ T cells:CD3+ T cells (Spearman <em>r</em> = −0.1582; <em>P</em> = .21). Importantly, high CD3 density (hazard ratio [HR], 0.23; <em>P</em> = .002), LAG-3 density (HR, 0.47; <em>P</em> = .037), γδ T-cell density (HR, 0.26; <em>P</em> = .02), and CD8 density (HR, 0.27; <em>P</em> = .03) showed associations with improved progression-free survival. Conditional tree analysis demonstrated that high CD8 and TCRδ expression were nonsignificant predictors of improved progression-free survival and overall survival. Overall, LAG-3 is expressed in MCC infiltrates and is prognostic in preimmunotherapy MCC, suggesting a potential role for LAG-3 inhibition in MCC. Additionally, CD8 and γδ T cells may play a critical role in the response to MCC, and γδ T-cell density may represent a novel biomarker in MCC.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 1","pages":"Article 102178"},"PeriodicalIF":5.1,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142604508","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Machine Vision–Detected Peritumoral Lymphocytic Aggregates Are Associated With Disease-Free Survival in Patients With Papillary Thyroid Carcinoma","authors":"Shayan Monabbati ,&nbsp;Pingfu Fu ,&nbsp;Sylvia L. Asa ,&nbsp;Tilak Pathak ,&nbsp;Joseph E. Willis ,&nbsp;Qiuying Shi ,&nbsp;Anant Madabhushi","doi":"10.1016/j.labinv.2024.102168","DOIUrl":"10.1016/j.labinv.2024.102168","url":null,"abstract":"<div><div>Papillary thyroid carcinoma (PTC) is the most prevalent form of thyroid cancer, with a disease recurrence rate of around 20%. Lymphoid formations, which occur in nonlymphoid tissues during chronic inflammatory, infectious, and immune responses, have been linked with tumor suppression. Lymphoid aggregates potentially enhance the body’s antitumor response, offering an avenue for attracting tumor-infiltrating lymphocytes and fostering their coordination. Increasing evidence highlights the role of lymphoid aggregate density in managing tumor invasion and metastasis, with a favorable impact noted on overall and disease-free survival (DFS) across various cancer types. In this study, we present a machine vision model to predict recurrence in different histologic subtypes of PTC using measurements related to peritumoral lymphoid aggregate density. We demonstrated that quantifying peritumoral lymphocytic presence not only is associated with better prognosis but also, along with tumor-infiltrating lymphocytes within the tumor, adds additional prognostic value in the absence of well-known second mutations including <em>TERT</em>. Annotations of peritumoral lymphoid aggregates on 171 well-differentiated PTCs in the Cancer Genome Atlas Thyroid Carcinoma (TCGA-THCA) data set were used to train a deep-learning model to predict regions of lymphoid aggregates across the entire tissue. The fractional area of the tissue regions covered by these lymphocytes was dichotomized to determine the following 2 risk groups: a significant and low density of peritumoral lymphocytes. DFS prognosticated using these risk groups via the Kaplan-Meier analysis revealed a hazard ratio (HR) of 2.51 (95% CI: 2.36, 2.66), tested on 170 new patients also from the TCGA-THCA data set. The prognostic performance of peritumoral lymphocyte aggregate density was compared against the univariate Kaplan-Meier analysis of DFS using the fractional area of intratumoral lymphocytes within the primary tumor with an HR of 2.04 (95% CI: 1.89, 2.19). Combining the lymphocyte features in and around the tumor yielded a statistically significant improvement in prognostic performance (HR, 3.17 [95% CI: 3.02, 3.32]) on training and were independently evaluated against 62 patients outside TCGA-THCA with an HR of 2.44 (95% CI: 2.19, 2.69). Multivariable Cox regression analysis on the validation set revealed that the density of peritumoral and intratumoral lymphocytes was prognostic independent of histologic subtype with a concordance index of 0.815.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"104 12","pages":"Article 102168"},"PeriodicalIF":5.1,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142590999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deciphering the Intricate Relationship Between Macrophages, Pigmentation, and Prognosis in Uveal Melanoma","authors":"Jayanti Jha ,&nbsp;Mithalesh Kumar Singh ,&nbsp;Lata Singh ,&nbsp;Neelam Pushker ,&nbsp;Aanchal Kakkar ,&nbsp;Rachna Meel ,&nbsp;Neiwete Lomi ,&nbsp;Sameer Bakhshi ,&nbsp;Tapas Chandra Nag ,&nbsp;Chanda Panwar ,&nbsp;Seema Sen ,&nbsp;Seema Kashyap","doi":"10.1016/j.labinv.2024.102167","DOIUrl":"10.1016/j.labinv.2024.102167","url":null,"abstract":"<div><div>High pigmentation and the abundance of M2 macrophages have been identified as negative predictors in uveal melanoma (UM). Risk factors associated with UM that are prevalent in high-risk White populations are still present, although less common, in relatively low-risk Asian populations. Research indicates that proangiogenic M2 macrophages and monosomy 3 play significant roles in UM progression. Our aim was to investigate the impact of tumor-associated macrophages in UM and examine their correlation with monosomy 3 and pigmentation. Transmission electron microscopy was used to analyze the morphology of macrophages in UM. Forty UM samples underwent fluorescent in situ hybridization for monosomy 3 identification. Immunohistochemistry was done to assess M2/M1 macrophages on 82 UM tissue samples. IL-10 and IL-12 expressions were quantified in UM serum samples by enzyme-linked immunosorbent assay. The expression of all markers was correlated with pigmentation markers (tyrosinase-related protein 1, tyrosinase-related protein 2, silver protein, and microphthalmia-associated transcription factor). Prognostic outcomes were determined using the Cox proportional hazard model and log-rank tests. Increased expression of M2/M1 macrophages was observed in 31 UM cases, which correlated with the high expression of pigmentation markers. IL-10 concentration was high in UM cases. Monosomy 3 was evident in 50% of UM cases and significantly associated with increased immunoexpression of M2/M1 macrophages and pigmentation markers. Reduced metastasis-free survival was observed in patients with UM with high M2/M1 macrophage expression (<em>P</em> = .001). High pigmentation and increased M2 macrophage density could impact the tumor microenvironment in UM. This could contribute to ineffective antitumor immune responses in patients with UM. Our findings suggest avenues for developing novel therapeutic approaches to counteract these immunosuppressive effects in UM.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"104 12","pages":"Article 102167"},"PeriodicalIF":5.1,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142567313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nup210 Promotes Colorectal Cancer Progression by Regulating Nuclear Plasma Transport","authors":"Fangyi Han ,&nbsp;Xingdi Fan ,&nbsp;Minxuan Hu ,&nbsp;Jing Wen ,&nbsp;Junrao Wang ,&nbsp;Dan Zhang ,&nbsp;Shuyang Wang ,&nbsp;Yanqing Ding ,&nbsp;Yaping Ye ,&nbsp;Hongli Jiao","doi":"10.1016/j.labinv.2024.102149","DOIUrl":"10.1016/j.labinv.2024.102149","url":null,"abstract":"<div><div>The nuclear pore complex (NPC) regulates nucleoplasmic transport, transcription, and genomic integrity in eukaryotic cells. However, little is known about how NPC works in cancer. In this study, we investigated the role of the nuclear pore protein 210 (Nucleoporin 210, Nup210) in colorectal cancer (CRC). Bioinformatics analysis revealed that the expression of Nup210 was increased in CRC and was associated with poor patient prognosis, but it was not a statistically significant independent prognostic factor. Moreover, knockdown of Nup210 in CRC cells inhibited the proliferation, invasion, and metastasis of CRC cells in vivo and in vitro. Additionally, nuclear size and nuclear plasma material transport capacity decreased along with the number and density of NPCs on the surface of CRC cells when Nup210 expression was inhibited. Furthermore, Nup210 required nuclear localization sequences (NLS) to localize to the nuclear membrane surface and interact with importin-α/β, which in turn affected the transit of nuclear plasma material. Importazole, a small molecule inhibitor of importin, along with therapy that targets the Nup210 protein is anticipated to be a novel strategy for CRC treatment. Their combination may be able to more effectively lower CRC tumor load. In conclusion, Nup210 modulates cellular nucleoplasmic transport capability and cell surface NPC density via NLS, thus promoting CRC progression. This discovery validates the molecular function of NPC in the development of CRC and provides a theoretical foundation for NPC-regulated nuclear import targeting as a therapeutic strategy for CRC.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"104 11","pages":"Article 102149"},"PeriodicalIF":5.1,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Epigenomic and Transcriptomic Profiling of Solitary Fibrous Tumors Identifies Site-Specific Patterns and Candidate Genes Regulated by DNA Methylation","authors":"Hannah C. Beird ,&nbsp;Jeffrey M. Cloutier ,&nbsp;Nalan Gokgoz ,&nbsp;Christopher Eeles ,&nbsp;Anthony M. Griffin ,&nbsp;Davis R. Ingram ,&nbsp;Khalida M. Wani ,&nbsp;Rossana Lazcano Segura ,&nbsp;Luca Cohen ,&nbsp;Carl Ho ,&nbsp;Jay S. Wunder ,&nbsp;Irene L. Andrulis ,&nbsp;P. Andrew Futreal ,&nbsp;Benjamin Haibe-Kains ,&nbsp;Alexander J. Lazar ,&nbsp;Wei-Lien Wang ,&nbsp;Joanna Przybyl ,&nbsp;Elizabeth G. Demicco","doi":"10.1016/j.labinv.2024.102146","DOIUrl":"10.1016/j.labinv.2024.102146","url":null,"abstract":"<div><div>A solitary fibrous tumor (SFT) is a rare mesenchymal neoplasm that can arise at any anatomical site and is characterized by recurrent <em>NAB2::STAT6</em> fusions and metastatic progression in 10% to 30%. The cell of origin has not been identified. Despite some progress in understanding the contribution of heterogeneous fusion types and secondary mutations to SFT biology, epigenetic alterations in extrameningeal SFT remain largely unexplored, and most sarcoma research to date has focused on the use of methylation profiling for tumor classification. We interrogated genome-wide DNA methylation in 79 SFTs to identify informative epigenetic changes. RNA-seq data from targeted panels and data from the Cancer Genome Atlas (TCGA) were used for orthogonal validation of selected findings. In unsupervised clustering analysis, the top 500 most variable cytosine-guanine sites segregated SFTs by primary anatomical site. Differentially methylated genes associated with the primary SFT site included <em>EGFR</em>; <em>TBX15</em>; multiple <em>HOX</em> genes; and their cofactors <em>EBF1</em>, <em>EBF3</em>, and <em>PBX1</em>; as well as <em>RUNX1</em> and <em>MEIS1</em>. Of the 20 DMGs interrogated on the RNA-seq panel, 12 were significantly differentially expressed according to site. However, except <em>TBX15</em>, most of these also showed differential expression according to <em>NAB2::STAT6</em> fusion type, suggesting that the fusion oncogene contributes to the transcriptional regulation of these genes. Transcriptomic data confirmed an inverse correlation between gene methylation and the expression of <em>TBX15</em> in both SFT and TCGA sarcomas<em>. TBX15</em> also showed differential mRNA expression and 5′ UTR methylation between tumors in different anatomical sites in TCGA data. In all analyses, <em>TBX15</em> methylation and mRNA expression retained the strongest association with tissue of origin in SFT and other sarcomas, suggesting a possible marker to distinguish metastatic tumors from new primaries without genomic profiling. Epigenetic signatures may further help to identify SFT progenitor cells at different anatomical sites.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"104 11","pages":"Article 102146"},"PeriodicalIF":5.1,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365739","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Highly Multiplexed Immunofluorescence PhenoCycler Panel for Murine Formalin-Fixed Paraffin-Embedded Tissues Yields Insight Into Tumor Microenvironment Immunoengineering","authors":"Sachin S. Surwase ,&nbsp;Xin Ming M. Zhou ,&nbsp;Kathryn M. Luly ,&nbsp;Qingfeng Zhu ,&nbsp;Robert A. Anders ,&nbsp;Jordan J. Green ,&nbsp;Stephany Y. Tzeng ,&nbsp;Joel C. Sunshine","doi":"10.1016/j.labinv.2024.102165","DOIUrl":"10.1016/j.labinv.2024.102165","url":null,"abstract":"<div><div>Spatial proteomics profiling is an emerging set of technologies that has the potential to elucidate the cell types, interactions, and molecular signatures that make up complex tissue microenvironments, with applications in the study of cancer, immunity, and much more. An emerging technique in the field is CoDetection by indEXing, recently renamed as the PhenoCycler system. This is a highly multiplexed immunofluorescence imaging technology that relies on oligonucleotide-barcoded antibodies and cyclic immunofluorescence to visualize many antibody markers in a single specimen while preserving tissue architecture. Existing PhenoCycler panels are primarily designed for fresh frozen tissues. Formalin-fixed paraffin-embedded blocks offer several advantages in preclinical research, but few antibody clones have been identified in this setting for PhenoCycler imaging. Here, we present a novel PhenoCycler panel of 28 validated antibodies for murine formalin-fixed paraffin-embedded tissues. We describe our workflow for selecting and validating clones, barcoding antibodies, designing our panel, and performing multiplex imaging. We further detail our analysis pipeline for comparing marker expressions, clustering and phenotyping single-cell proteomics data, and quantifying spatial relationships. We then apply our panel and analysis protocol to profile the effects of 3 gene delivery nanoparticle formulations, in combination with systemic anti-PD1, on the murine melanoma tumor immune microenvironment. Intralesional delivery of genes expressing the costimulatory molecule 4-1BBL and the cytokine IL-12 led to a shift toward intratumoral M1 macrophage polarization and promoted closer associations between intratumoral CD8 T cells and macrophages. Delivery of interferon gamma, in addition to 4-1BBL and IL-12, not only further increased markers of antigen presentation on tumor cells and intratumoral antigen-presenting cells but also promoted greater expression of checkpoint marker PD-L1 and closer associations between intratumoral CD8 T cells and PD-L1–expressing tumor cells. These findings help explain the benefits of 4-1BBL and IL-12 delivery while offering additional mechanistic insights into the limitations of interferon gamma therapeutic efficacy.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 1","pages":"Article 102165"},"PeriodicalIF":5.1,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142567314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Concordance of Whole-Slide Imaging and Conventional Light Microscopy for Assessment of Pathologic Response Following Neoadjuvant Therapy for Lung Cancer","authors":"Julie S. Deutsch ,&nbsp;Daphne Wang ,&nbsp;Krista Y. Chen ,&nbsp;Ashley Cimino-Mathews ,&nbsp;Elizabeth D. Thompson ,&nbsp;Jaroslaw Jedrych ,&nbsp;Robert A. Anders ,&nbsp;Edward Gabrielson ,&nbsp;Peter B. Illei ,&nbsp;Sonali Uttam ,&nbsp;Alexa Fiorante ,&nbsp;Emily Cohen ,&nbsp;Michael Fotheringham ,&nbsp;Logan L. Engle ,&nbsp;Joel C. Sunshine ,&nbsp;Hao Wang ,&nbsp;Dimple Pandya ,&nbsp;Vipul Baxi ,&nbsp;Joseph Fiore ,&nbsp;Kurex Sidik ,&nbsp;Janis M. Taube","doi":"10.1016/j.labinv.2024.102166","DOIUrl":"10.1016/j.labinv.2024.102166","url":null,"abstract":"<div><div>Pathologic response is an endpoint in many ongoing clinical trials for neoadjuvant regimens, including immune checkpoint blockade and chemotherapy. Whole-slide scanning of glass slides generates high-resolution digital images and allows for remote review and potential measurement with image analysis tools, but concordance of pathologic response assessment on digital scans compared with that on glass slides has yet to be evaluated. Such a validation goes beyond previous concordance studies, which focused on establishing surgical pathology diagnoses, as it requires quantitative assessment of tumor, necrosis, and regression. Further, as pathologic response assessment is being used as an endpoint, such concordance studies have regulatory implications. The purpose of this study was 2-fold, which was as follows: first, to determine the concordance between pathologic response assessed on glass slides and that assessed on digital scans, and second, to determine if pathologists benefited from using measurement tools when determining pathologic response. To that end, hematoxylin and eosin–stained glass slides from 64 non–small cell lung carcinoma specimens were visually assessed for percent residual viable tumor (%RVT). The sensitivity and specificity for digital vs glass reads of pathologic complete response (0% RVT) and major pathologic response (≤10% RVT) were all &gt;95%. When %RVT was considered as a continuous variable, the intraclass correlation coefficient of digital vs glass reads was 0.94. The visual assessments of pathologic response were supported by pathologist annotations of residual tumor and tumor bed areas. In a separate subset of hematoxylin and eosin–stained glass slides, several measurement approaches to quantifying %RVT were performed. Pathologist estimates strongly reflected measured %RVT. This study demonstrates the high level of concordance between glass slides evaluated using light microscopy and digital whole-slide images for pathologic response assessments. Pathologists did not require measurement tools to generate robust %RVT values from slide annotations. These findings have broad implications for improving clinical workflows and multisite clinical trials.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 1","pages":"Article 102166"},"PeriodicalIF":5.1,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}