QuPath Software Research Articles

The microglial translocator protein (TSPO) in Alzheimer's disease reflects a phagocytic phenotype.

Translocator protein (TSPO) is a mitochondrial protein expressed by microglia, ligands for which are used as a marker of neuroinflammation in PET studies of Alzheimer's disease (AD). We previously showed increasing TSPO load in the cerebral cortex with AD progression, consistent with TSPO PET scan findings. Here, we aim to characterise the microglial phenotype associated with TSPO expression to aid interpretation of the signal generated by TSPO ligands in patients. Human post-mortem sections of temporal lobe (TL) and cerebellum (Cb) from cases classified by Braak group (0-II, III-IV, V-VI; each n = 10) were fluorescently double labelled for TSPO and microglial markers: Iba1, HLA-DR, CD68, MSR-A and CD64. Quantification was performed on scanned images using QuPath software to assess the microglial phenotype of TSPO. Qualitative analysis was also performed for TSPO with GFAP (astrocytes), CD31 (endothelial cells) and CD163 (perivascular macrophages) to characterise the cellular profile of TSPO. The percentage of CD68+TSPO+ double-labelled cells was significantly higher thanfor other microglial markers in both brain regions and in all Braak stages, followed by MSR-A+TSPO+ microglia. Iba1+TSPO+ cells were more numerous in the cerebellum than the temporal lobe, while CD64+TSPO+ cells were more numerous in the temporal lobe. No differences were observed for the other microglial markers. TSPO expression was also detected in endothelial cells, but not detected in astrocytes nor in perivascular macrophages. Our data suggest that TSPO is mainly related to a phagocytic profile of microglia (CD68+) in human AD, potentially highlighting the ongoing neurodegeneration.

Clinical values of nuclear morphometric analysis in fibroepithelial lesions.

Fibroepithelial lesions (FELs) of the breast encompass a broad spectrum of lesions, ranging from commonly encountered fibroadenomas (FAs) to rare phyllodes tumors (PTs). Accurately diagnosing and grading these lesions is crucial for making management decisions, but it can be challenging due to their overlapping features and the subjective nature of histological assessment. Here, we evaluated the role of digital nuclear morphometric analysis in FEL diagnosis and prognosis. A digital nuclear morphometric analysis was conducted on 241 PTs and 59 FAs. Immunohistochemical staining for cytokeratin and Leukocyte common antigen (LCA) was used to exclude non-stromal components, and nuclear area, perimeters, calipers, circularity, and eccentricity in the stromal cells were quantified with QuPath software. The correlations of these features with FEL diagnosis and prognosis was assessed. All nuclear features, including area, perimeter, circularity, maximum caliper, minimum caliper and eccentricity, showed significant differences between FAs and benign PTs (p ≤ 0.002). Only nuclear area, perimeter, minimum caliper and eccentricity correlated significantly with PT grading (p ≤ 0.022). For differentiation of FAs from benign PTs, the model integrating all differential nuclear features demonstrated a specificity of 90% and sensitivity of 70%. For PT grading, the nuclear morphometric score showed a specificity of 78% and sensitivity of 96% for distinguishing benign/borderline from malignant PTs. In addition, a relationship of nuclear circularity was found with PT recurrence. The Kaplan-meier analysis, using the best cutoff determined by ROC curve, showed shorter event free survival in benign PTs with high circularity (chi-square = 4.650, p = 0.031). Our data suggested the digital nuclear morphometric analysis could have potentials to objectively differentiate different FELs and predict PT outcome. These findings could provide the evidence-based data to support the development of deep-learning based algorithm on nuclear morphometrics in FEL diagnosis.

NCMP-13. EXPLORING THE PATHOPHYSIOLOGY OF HYDROCEPHALUS IN CHOROID PLEXUS PAPILLOMA

Abstract BACKGROUND Choroid plexus papilloma (CPP) is a benign intraventricular lesion associated with hydrocephalus and ventricular outflow obstruction. However, the molecular mechanism by which cerebrospinal fluid (CSF) is produced by choroid plexus (CP), or by which CSF production is altered in the setting of CPP to cause hydrocephalus, is unknown. METHODS A PRISMA systematic literature review was conducted to identify gene targets associated with CSF production. Primary tissue samples of CP and CPP from the same patient were obtained, and expression of target genes was assessed using immunohistochemistry and quantified using QuPath software H-scoring. Single-nucleus sequencing of both CP and CPP was also completed, and differential expression of identified target genes was evaluated by calculating log fold change. RESULTS Systematic literature review yielded 25 studies in animal models and 18 unique gene targets. 16 targets were expressed in primary CP and CPP tissue samples upon immunohistochemistry staining. 11 of 18 targets revealed significant differential expression between CP and CPP on single-nucleus sequencing. Genes with notable expression increases in CPP included ion transporters NKCC1 (Na-K-Cl cotransporter) and TRPV4 (non-selective cation channel), as well as carbonic anhydrase II. CONCLUSIONS We present evidence that choroid plexus papilloma has altered expression of genes previously implicated in CSF production. Upregulation of membrane transporters and cytoplasmic enzymes involved in Na+, K+, Cl-, and HCO3- transport may provide a mechanistic explanation for the development of hydrocephalus in CPP. Pharmacologic inhibition of CSF production may offer a route for non-surgical management of CPP, as well as other pathologies causing hydrocephalus.

Prognostic assessment of T-cells in primary colorectal cancer and paired synchronous or metachronous liver metastasis.

Prognostic value of T-cells between primary colorectal cancer (pCRC) and its paired synchronous and metachronous liver metastasis (LM) is underinvestigated and is the subject of the present study. We enrolled into this retrospective cohort study patients, who underwent resection of both pCRC and synchronous LM (N = 55) or metachronous LM (N = 44). After immunohistochemical staining for CD3+, CD8+, and CD45R0+ whole slides were scanned and T-cell densities were quantified using QuPath software in tumor center (TC), inner margin (IM), outer margin (OM), and peritumor zone (PT) of pCRC and LM. High densities of CD8+ T-cells in TC, OM and PT of synchronous LM were associated with longer disease-free survival (DFS). Greater densities of CD3+ T-cells in IM and PT and CD8+ T-cells in IM, OM and PT in synchronous LM over pCRC were associated with longer DFS. Greater densities of CD8+ T-cells in the TC and IM and CD3+ T-cells in the IM of pCRC were found in the metachronous over synchronous group. The first novel finding demonstrated that high density of CD8+ T cells in synchronous LM were associated with favorable outcome. The second finding of high CD8+ cell density in pCRC in metachronous over synchronous CRC may provide a mechanistic basis for the delay of metastatic spread. Both findings could be applied clinically with own reference values.

Neuroglia markers expression in the cingulate and retrosplenial cortex of mice after nonseptic dose of lipopolysaccharide

Aim. To study the expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1) in the cerebral cingulate and retrosplenial cortex of mice on Day 5 after intraperitoneal (i.p.) administration of bacterial lipopolysaccharide (LPS) at the dose with no nervous tissue inflammation provoked.Materials and methods. The work was performed on 10 female C57BL/6 mice aged 90 ± 3 days. At the same time for 4 days, animals of group 1 were intraperitoneally injected with saline (0.9% NaCl), and animals of group 2 were injected with E. coli LPS endotoxin at a dose of 1 mg/kg/day. On the fifth day, the mice were withdrawn from the experiment by decapitation with xylazine/tiletamine-zoletil premedication, after which histological preparations of the cingulate and retrosplenial cortex were made, stained with antibodies to GFAP and Iba1, and the number of: (1) GFAP-positive cells of cytoplasmic areas, (2) cells with a positive reaction of antibodies to Iba1 were counted using QuPath software. Groups were compared using Mann-Whitney U-test.Results. The number of GFAP-positive cells after LPS administration in Group 2 was significantly higher than in Group 1, exactly 22.5 (8.0; 32.0) vs 9.0 (4.3; 17.0), respectively, p = 0.0038. The number of Iba1-positive portions of cytoplasm also was significantly higher in Group 2, namely 207,5 (154,8; 295,8) vs 128 (89,3; 165,5), respectively, p = 0,014. Both groups showed neither signs of inflammation, excessive blood supply nor hemorrhages, as well as no perivascular edema or leukocytic migration.Conclusion. LPS, administered i.p. to mice at a dose of 1 mg/kg/day for 4 days, allows assessment of changes in glia of CNS in damage with no signs of inflammation there. In cerebral cingulate and retrosplenial cortex, the number of astrocytes with a positive reaction of antibodies to GFAP increases, as well as number macrophages with the expression of the Iba1 protein.

8500 Fvb/Ant Mice Carrying the Thr92Ala-Dio2 Polymorphism Have a Goiter

Abstract Disclosure: A. Batistuzzo: None. B. Bocco: None. E. McAninch: None. M.O. Ribeiro: None. A.C. Bianco: Consulting Fee; Self; Abbvie. Fvb/Ant Mice Carrying the Thr92Ala-Dio2 Polymorphism Have a Goiter Introduction: The Thr92Ala-DIO2 polymorphism is prevalent worldwide, with about 50% of the population carrying at least one polymorphic allele. Ala-D2 is ectopically located in the Golgi apparatus and causes endoplasmic reticulum (ER) stress. It is also about 40 % less active than Thr92-D2, thus T3 production could be compromised in carriers of the polymorphism. Objective: C57BL/6J (B6) mice carrying the Ala92-DIO2 polymorphism exhibit no significant thyroid phenotype as compared to control Thr92-Dio2 mice. Here we wished to test whether changing the genetic background of mice can influence the thyroidal impact of the Thr92Ala-DIO2 polymorphism. Therefore, we backcrossed B6-Thr92Ala-Dio2 mice to the FVB/Ant background for 8 generations. Methods: Mice were killed by asphyxiation in a CO2 chamber and blood collected from 7-18 weeks old female and male FVB/Ant Ala-Dio2 mice to measure plasma levels of TSH, T4, T3, and rT3. The thyroid gland was dissected, weighed and processed for histological analysis. The weight of the thyroid gland was normalized by femoral length. The QuPath Software was used for measuring follicular area, cell height, and the number of internalized follicular cells. Thr92-Dio2 mice of the same age and sex were used as controls. Data were analyzed using PRISM software and expressed as mean±SE (TH plasma levels and thyroid weight) or mean±SD (thyroid histology). A Student t-test was used to compare 2 groups. Results: Male and female Ala mice had significantly higher levels of TSH (males: 0.36±0.04 vs 1.1±0.21 ng/ml; p<0.01 and females: 0.017±0.006 vs 0.12±0.06 ng/ml; p<0.01), lower levels of plasmatic T4 (males: 2.9±0.2 vs 1.3±0.25 µg/dl; p<0.01 and females: 2.1±0.16 vs 1.15±0.16 µg/dl; p<0.001) and lower levels of rT3 (males: 23.8±2 vs 14.2±1.3 ng/dl; p<0.001 and females: 16.45±1.4 vs 10.4±1.17 ng/dl; p<0.01). Additionally, the thyroid gland of female polymorphic mice was heavier (4.3±0.39 vs 1.8±0.17 mg/cm; p<0.001) and both sexes presented enlarged thyroid follicle area (males: 1464±1464 vs 2055±2443 µm2; p<0.0001 and females: 1519±1364 vs 2597±2624 µm2; p<0.0001) and a higher number of internalized follicular cells (females: 0.06±0.007 vs 0.17±0.008 internalized cells/follicle; p<0.0001). No differences in the follicular cell height were observed. Discussion: In contrast to B6, FVB mice carrying the Thr92Ala-D2 polymorphism exhibit goiter, increased follicular area, slightly reduced serum T4 levels, and elevated serum TSH levels. These findings suggest that the impact of the Thr92Ala polymorphism varies according to the genetic background. They may explain why clinical studies of patients carrying the Thr92Ala-DIO2 polymorphism have yielded inconsistent results when different populations are analyzed. Presentation: 6/3/2024

Correlation of FAPI PET Uptake with Immunohistochemistry in Explanted Lungs from Patients with Advanced Interstitial Lung Disease.

Recent studies have demonstrated promising results of fibroblast activation protein (FAP) inhibitor (FAPI) PET in prognosticating and monitoring interstitial lung diseases (ILDs). As a first step toward successful translation, our primary aim was to validate the FAPI PET uptake through immunohistochemistry in patients with advanced ILD who underwent lung transplantation after a FAPI PET scan. Methods: This is a preliminary analysis of a single-center, open-label, single-arm, prospective exploratory biodistribution study of 68Ga-FAPI-46 PET imaging in patients with ILD (NCT05365802). Patients with ILD confirmed by high-resolution CT and scheduled for lung transplant were included. Tissue samples of explanted lungs were obtained from both the central and peripheral lung parenchyma of each lobe. Additional samples were obtained from areas of the lung corresponding to regions of FAPI PET activity. Immunohistochemical staining was performed with an anti-FAP antibody. Percentages of FAP immunohistochemistry-positive area were measured semiautomatically using QuPath software. SUVs in the areas of pathologic samples were measured on FAPI PET/CT by referencing the gross photomap of the explanted lung. A Spearman correlation coefficient test was used to assess the relationship between FAPI PET uptake and FAP immunohistochemical expression in each specimen. Results: Four patients with advanced ILD who underwent FAPI PET/CT before lung transplantation were included. The types of ILD were idiopathic pulmonary fibrosis (n = 2), rheumatoid arthritis-associated ILD (n = 1), and nonspecific interstitial pneumonia (n = 1). FAPI uptake was visualized mainly in the fibrotic area on CT. Twenty-nine surgical pathology samples from 3 patients were analyzed. FAP staining was predominantly positive in fibroblastic foci. FAPI PET SUVmax and SUVmean showed a positive correlation with the immunohistochemical FAP expression score (SUVmax: r = 0.57, P = 0.001; SUVmean: r = 0.54, P = 0.002). Conclusion: In this analysis conducted in patients who underwent lung transplantation after a FAPI PET scan, FAPI PET uptake was positively correlated with FAP immunohistochemistry. These findings provide a rationale for further investigation of FAPI PET as a potential imaging biomarker for ILD.

Clinical prognosticators and targets in the immune microenvironment of intrahepatic cholangiocarcinoma

ABSTRACT Background Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation. Methods Liver tissue samples were collected during 2008–2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53). Results CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells (“hot” cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression. Conclusions These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with “hot” TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn “cold” into “hot” TIME in ICC.

Comprehensive Postnatal Anatomical, Histological and Geometric Morphometric Analysis of Thymus Development in Dromedary Camels (Camelus dromedarius).

The thymus, a primary lymphoid organ, plays a critical role in T lymphocyte development and adaptive immunity. This study focuses on the anatomical, histological and geometric morphometric characteristics of the thymus in dromedary camels (Camelus dromedarius) during postnatal development. Thymus samples were collected from camels aged approximately 4, 8, 12 and 16 months. Using photogrammetry and 3D modelling, the samples were analysed to generate landmarks and conduct geometric morphometry with the 3D Slicer and ALPACA algorithm. Principal component analysis (PCA) was then performed to evaluate shape variations. Histologically, the samples underwent Haematoxylin and Eosin and Masson's trichrome staining. Image analysis using QuPath software quantified trabeculae, adipose tissue and Hassall's corpuscles. The results revealed significant anatomical and histological changes in the thymus across the different age groups. Notable variations in tissue composition and structural integrity were observed, with the PCA highlighting distinct morphometric patterns associated with age-related development. These findings provide a deeper understanding of thymus maturation in dromedaries and offer valuable data for comparative anatomy and veterinary medicine. This comprehensive analysis enhances our knowledge of species-specific immune development, with important implications for the health and resilience of these animals in arid environments.

Evaluation of tumor budding with virtual panCK stains generated by novel multi-model CNN framework

As the global incidence of cancer continues to rise rapidly, the need for swift and precise diagnoses has become increasingly pressing. Pathologists commonly rely on H&E-panCK stain pairs for various aspects of cancer diagnosis, including the detection of occult tumor cells and the evaluation of tumor budding. Nevertheless, conventional chemical staining methods suffer from notable drawbacks, such as time-intensive processes and irreversible staining outcomes. The virtual stain technique, leveraging generative adversarial network (GAN), has emerged as a promising alternative to chemical stains. This approach aims to transform biopsy scans (often H&E) into other stain types. Despite achieving notable progress in recent years, current state-of-the-art virtual staining models confront challenges that hinder their efficacy, particularly in achieving accurate staining outcomes under specific conditions. These limitations have impeded the practical integration of virtual staining into diagnostic practices. To address the goal of producing virtual panCK stains capable of replacing chemical panCK, we propose an innovative multi-model framework. Our approach involves employing a combination of Mask-RCNN (for cell segmentation) and GAN models to extract cytokeratin distribution from chemical H&E images. Additionally, we introduce a tailored dynamic GAN model to convert H&E images into virtual panCK stains, integrating the derived cytokeratin distribution. Our framework is motivated by the fact that the unique pattern of the panCK is derived from cytokeratin distribution. As a proof of concept, we employ our virtual panCK stains to evaluate tumor budding in 45 H&E whole-slide images taken from breast cancer-invaded lymph nodes . Through thorough validation by both pathologists and the QuPath software, our virtual panCK stains demonstrate a remarkable level of accuracy. In stark contrast, the accuracy of state-of-the-art single cycleGAN virtual panCK stains is negligible. To our best knowledge, this is the first instance of a multi-model virtual panCK framework and the utilization of virtual panCK for tumor budding assessment. Our framework excels in generating dependable virtual panCK stains with significantly improved efficiency, thereby considerably reducing turnaround times in diagnosis. Furthermore, its outcomes are easily comprehensible even to pathologists who may not be well-versed in computer technology. We firmly believe that our framework has the potential to advance the field of virtual stain, thereby making significant strides towards improved cancer diagnosis.

Hyaluronidase improves the efficacy of nab-paclitaxel after prolonged angiogenesis inhibition in preclinical models for esophagogastric cancer

BackgroundLong-term anti-angiogenesis leads to pruned vasculature, densely deposited extracellular matrix (ECM), and consequently reduced chemotherapy delivery in esophagogastric cancer (EGC). To address this issue, we evaluated the efficacy of adding a hyaluronidase or a NO-donor to the regimen of chemotherapy and anti-angiogenic drugs. MethodsA patient-derived EGC xenograft model was developed. Grafted mice were randomly assigned to four experimental groups and one control group. The experimental groups received DC101, a murine angiogenesis inhibitor, and nab-paclitaxel (NPTX), with the addition of hyaluronidase (PEGPH20), or NO-donor (nitroglycerine, NTG), or their combination, respectively. We compared tumor growth during 17 days of treatment. We performed immunohistochemistry for ECM components hyaluronan (HA) and collagen, CD31 for endothelial cells, and γH2AX for DNA damage. The positively stained areas were quantified, and vessel diameters were measured using QuPath software. ResultsProlonged DC101 treatment induced deposition of HA (p<0.01) and collagen (p<0.01). HA was effectively degraded by PEGPH20 (p<0.001), but not by NTG as expected. Both PEGPH20 (p<0.05) and NTG (p<0.01) dilated vessels collapsed in response to long-term DC101 treatment. However, only PEGPH20 (rather than NTG) was found to significantly inhibit tumor growth (p<0.05) in combination with NPTX and DC101. ConclusionsThese findings suggest that the mechanical barrier of HA is the major reason responsible for the resistance developed during prolonged anti-angiogenesis in EGC. Incorporating PEGPH20 into the existing treatment regimen is promising to improve outcomes for patients with EGC.

QuPath for automated analysis of digital images of oral epithelial dysplasia

Abstract Objectives: Grading of oral epithelial dysplasia (OED) has been plagued with intra-observer and inter-observer variations. To overcome this subjectivity, a more objective digital image analysis is obligatory using computer-aided software. The use of open-source software like QuPath, which is a new bio-image evaluation software program, may fulfil this growing need in virtual pathology. This study used the QuPath software for automatic analysis of morphometric parameters in hematoxylin and eosin (H and E)-stained digital images of oral epithelial dysplasia. Methodology: 150 H and E digital images of varying grades of OED captured under 40x magnification were processed using QuPath software for automatic analysis of cellular and nuclear parameters. Results: The parameters that showed statistical significance included nuclear hematoxylin OD, nuclear eosin OD, cellular hematoxylin OD, cellular eosin OD, cytoplasm hematoxylin OD, and cytoplasmic eosin OD (P &lt; 0.05), while none of the other parameters showed statistically significant differences. A prediction accuracy of 76%, 74%, and 70% for mild, moderate, and severe dysplasia was obtained, respectively. Conclusion: The quantitative results outlined in this paper are encouraging to indicate that the use of this technique may improve the diagnostic reliability of OED. Morphometric analysis of OED using Qupath software can be fast and reproducible and can be quantitated automatically.

Prognostic relevance of high expression of kynurenine pathway markers in glioblastoma

Glioblastoma (GBM) continues to exhibit a discouraging survival rate despite extensive research into new treatments. One factor contributing to its poor prognosis is the tumor's immunosuppressive microenvironment, in which the kynurenine pathway (KP) plays a significant role. This study aimed to explore how KP impacts the survival of newly diagnosed GBM patients. We examined tissue samples from 108 GBM patients to assess the expression levels of key KP markers—tryptophan 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenase (IDO1/2), and the aryl hydrocarbon receptor (AhR). Using immunohistochemistry and QuPath software, three tumor cores were analyzed per patient to evaluate KP marker expression. Kaplan–Meier survival analysis and stepwise multivariate Cox regression were used to determine the effect of these markers on patient survival. Results showed that patients with high expression of TDO2, IDO1/2, and AhR had significantly shorter survival times. This finding held true even when controlling for other known prognostic variables, with a hazard ratio of 3.393 for IDO1, 2.775 for IDO2, 1.891 for TDO2, and 1.902 for AhR. We suggest that KP markers could serve as useful tools for patient stratification, potentially guiding future immunomodulating trials and personalized treatment approaches for GBM patients.

Dura mater and survival time determination in individuals who died after traumatic brain injury: a preliminary study.

Traumatic brain injury (TBI) is one of the major causes of morbidity and mortality among young people and is a matter of concern for forensic pathologists. Many authors have tried to estimate a person's survival time (ST) after TBI using different approaches. The present study aimed to present an innovative workflow to estimate the ST after TBI by observing the inflammatory reaction of the dura mater (DM). The authors collected DM samples from 36 cadavers (20 with TBI and 16 with no history or signs of TBI). Each sample was labelled via immunohistochemistry with three different primary antibodies, CD15, CD68, and CD3, yielding 108 slides in total. The slides were digitalized and analysed using QuPath software. The DM is involved in the inflammatory response after TBI. CD15 immunoreactivity allowed us to distinguish between subjects who died immediately after TBI and those with an ST of minutes or hours. CD3 immunoreactivity can be used to differentiate subjects with an ST of days from those with other STs. Moreover, the DM samples showed an acceptable diagnostic yield even in samples with signs of putrefaction.

Pathomics and single-cell analysis of papillary thyroid carcinoma reveal the pro-metastatic influence of cancer-associated fibroblasts

BackgroundPapillary thyroid carcinoma (PTC) is globally prevalent and associated with an increased risk of lymph node metastasis (LNM). The role of cancer-associated fibroblasts (CAFs) in PTC remains unclear.MethodsWe collected postoperative pathological hematoxylin–eosin (HE) slides from 984 included patients with PTC to analyze the density of CAF infiltration at the invasive front of the tumor using QuPath software. The relationship between CAF density and LNM was assessed. Single-cell RNA sequencing (scRNA-seq) data from GSE193581 and GSE184362 datasets were integrated to analyze CAF infiltration in PTC. A comprehensive suite of in vitro experiments, encompassing EdU labeling, wound scratch assays, Transwell assays, and flow cytometry, were conducted to elucidate the regulatory role of CD36+CAF in two PTC cell lines, TPC1 and K1.ResultsA significant correlation was observed between high fibrosis density at the invasive front of the tumor and LNM. Analysis of scRNA-seq data revealed metastasis-associated myoCAFs with robust intercellular interactions. A diagnostic model based on metastasis-associated myoCAF genes was established and refined through deep learning methods. CD36 positive expression in CAFs can significantly promote the proliferation, migration, and invasion abilities of PTC cells, while inhibiting the apoptosis of PTC cells.ConclusionThis study addresses the significant issue of LNM risk in PTC. Analysis of postoperative HE pathological slides from a substantial patient cohort reveals a notable association between high fibrosis density at the invasive front of the tumor and LNM. Integration of scRNA-seq data comprehensively analyzes CAF infiltration in PTC, identifying metastasis-associated myoCAFs with strong intercellular interactions. In vitro experimental results indicate that CD36 positive expression in CAFs plays a promoting role in the progression of PTC. Overall, these findings provide crucial insights into the function of CAF subset in PTC metastasis.

Distribution of prostatic markers in glands of the female urethra and anterior vaginal wall-a rapid autopsy study.

There are varying reports of immunohistochemically detected prostatic marker protein distribution in glands associated with the female urethra that may be related to tissue integrity at the time of fixation. In this study we used tissue derived from rapid autopsies of female patients to determine the distribution of glandular structures expressing prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) along the female urethra and in surrounding tissues, including the anterior vaginal wall (AVW). Tissue blocks from 7 donors that contained the entire urethra and adjacent AVW were analyzed. These tissue samples were fixed within 4-12hours of death and divided into 5-mm transverse slices that were paraffin embedded. Sections cut from each slice were immunolabeled for PSA or PSAP and a neighboring section was stained with hematoxylin and eosin. The sections were reviewed by light microscopy and analyzed using QuPath software. In tissue from all donors, glandular structures expressing PSA and/or PSAP were located within the wall of the urethra and were present along its whole length. In the proximal half of the urethra from all donors, small glands expressing PSAP, but not PSA, were observed adjacent to the and emptying into the lumen. In the distal half of the urethra from 5 of the 7 donors, tubuloacinar structures lined by a glandular epithelium expressed both PSA and PSAP. In addition, columnar cells at the surface of structures with a multilayered transitional epithelium in the distal half of the urethra from all donors expressed PSAP. No glands expressing PSA or PSAP were found in tissues surrounding the urethra, including the AVW. Greater understanding of the distribution of urethral glands expressing prostatic proteins in female patients is important because these glands are reported to contribute to the female sexual response and to urethral pathology, including urethral cysts, diverticula, and adenocarcinoma. Strengths of the present study include the use of rapid autopsy to minimize protein degradation and autolysis, and the preparation of large tissue sections to demonstrate precise anatomical relations within all the tissues surrounding the urethral lumen. Limitations include the sample size and that all donors had advanced malignancy and had undergone previous therapy which may have had unknown tissue effects. Proximal and distal glands expressing prostate-specific proteins were observed in tissue from all donors, and these glands were located only within the wall of the urethra.

#2615 Altered peroxidasin expression pattern in IgA nephropathy

Abstract Background and Aims PXDN is a multifunctional heme peroxidase responsible for the crosslinking of type IV collagen protomers at basement membranes, playing a crucial role in the extracellular matrix dynamics. In renal pathophysiology, increased PXDN expression has been associated with pro-fibrotic and pro-carcinogenic mechanisms and we have shown that PXDN is expressed throughout all kidney compartments since the early stages of nephrogenesis. However, the role of PXDN in kidney health and disease remains largely unknown. In this work we performed a comparative analysis of PXDN expression between renal biopsy samples from IgA nephropathy (IgAN) patients and healthy kidney biopsy samples. IgAN was the selected CKD model due to its straightforward diagnosis by standard protocols for histopathological evaluation of kidney biopsies, and to the fact that the renal outcomes are consistently associated with several key pathologic features, including tubular atrophy and interstitial fibrosis. Correlations between PXDN expression and IgAN disease and kidney function parameters as well as with ECM proteins were also explored. Method Fluorescence immunohistochemistry for PXDN was performed in biopsy samples from IgAN patients (n = 20) and in biopsies from healthy kidney donors (n = 7). Proximal tubules were assessed by anti-CD15 fluorescent staining. Collagen III (COLIII) deposition was investigated in IgAN samples (n = 7) by immunofluorescence. Brightfield images were manually segmented into glomeruli, proximal and distal tubules, and interstitium, using QuPath software, and the fluorescence intensity measurements were obtained with ImageJ/Fiji. Results Results showed that, in comparison with healthy kidney samples, PXDN is significantly decreased in proximal and distal tubules of IgAN patients (P &amp;lt; 0.001 and P &amp;lt; 0.05, respectively) whereas it is increased in IgAN interstitium (P &amp;lt; 0.001). When IgAN samples were stratified by the presence or absence of chronic lesions (glomerular sclerosis and interstitial fibrosis) we observed that in the proximal tubules, PXDN is decreased even in areas without lesions while in the distal tubules the expression is compromised only in the lesioned areas. In the interstitium, PXDN shows a tendency of increase from healthy tissue to IgAN in areas without and with tubulointerstitial fibrosis, presenting a significant increase in the lesioned areas (P &amp;lt; 0.001). Concerning ECM deposition, results showed a significant correlation between the expression of PXDN and COLIII in sclerotic glomeruli (R = 0.8214; P = 0.0341) and in the tubulointerstitial fibrotic areas (R = 0.93; P = 0.0067). Moreover, although no differences were observed in the glomerular expression of PXDN between healthy and IgAN samples, when considering eGFR, a significant increase of PXDN was observed in both non-lesioned and sclerotic glomeruli from IgAN patients with eGFR bellow 60 ml/min/1,73 m2. In the interstitium, the expression of PXDN was also increased in tubulointerstitial fibrotic areas of IgAN patients with eGFR bellow 60 ml/min/1,73 m2, in comparison with the same areas in patients with higher eGFR values. Conclusions Overall, our study shows a differential expression of PXDN in IgAN, with loss in the tubular region that relates with the state of interstitial fibrosis, and gain in the glomerular and interstitial region where it correlates with COLIII compartment-specific deposition and with kidney function as assessed by eGFR. These data support, for the first time, the enrollment of PXDN in the topographic mechanism of IgA-mediated kidney lesion that deserves further investigations. Given that sclerosis and fibrosis are a common pathogenic mechanism of CKD, irrespective of the underlying etiology, we postulate that PXDN may serve as a novel therapeutic target for this disease.

PSIV-A-7 Effects of late gestational nutrient restriction of beef heifers on small intestinal crypt depth in neonatal calves

Abstract We hypothesized that late gestational nutrient restriction would impair small intestinal development in neonatal calves, including crypt cell depth and proliferation. Fall-calving crossbred beef heifers [body weight (BW) = 466 ± 30 (SD) kg; BCS: 5.4 ± 0.5] bred to a single sire were individually-fed 100% (control; n = 10) or 70% (nutrient restricted; n = 12) of NASEM metabolizable energy and metabolizable protein requirements for maintenance, pregnancy, and growth from d 160 of gestation to calving. Calves were removed from their dams immediately post-calving and fed artificial colostrum and milk replacer relative to body weight until euthanasia at 48.7 ± 0.4 h of age. The gastrointestinal tract was removed, and proximal jejunum samples were dissected from each calf and immersion-fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin blocks and cut into 5-µm sections that were mounted on glass slides. Slides were incubated with Ki-67 (marker of nuclear proliferation) and counterstained with Hematoxylin using an autostainer. Images were taken at 5× magnification using an upright microscope. QuPath software was used to measure crypt depth, starting at the base of the crypt region and ending at the furthest cluster of Ki-67 positive cells, perpendicular to the base of the mucosa. For each calf, 10 crypt depth measurements were taken from 4 images, resulting in 40 crypt depth measurements that were averaged. Nutritional plane was a fixed effect in the model, and maternal sire, date of study initiation, and calf sex (when P &amp;lt; 0.25) were considered covariates. We previously reported that calves born to nutrient restricted dams weighed less (P ≤ 0.05) at birth and 48 h than calves born to control dams. Small intestinal mass tended to be less (P = 0.11) in offspring of nutrient restricted dams but was not affected (P = 0.58) when expressed relative to body weight. Calves born to nutrient restricted dams also tended to have less (P = 0.11) small intestinal mass relative to brain weight. Maternal nutrient restriction did not affect (P = 0.95) jejunal crypt depth. Additionally, crypt depth relative to body weight was not affected (P = 0.20) by nutrient restriction. Although crypt depth was not affected, further analysis of proliferation and morphology will help to understand the relationship between maternal nutrient restriction and small intestine development in neonatal calves.

Abstract PO1-15-03: Spatially resolved analysis of tumor microenvironment in invasive lobular carcinoma

Abstract Background: Invasive lobular carcinoma (ILC) is the second most common histological breast cancer subtype; however, little is known about its tumor microenvironment (TME). Here, we aimed to study ILC TME using spatial transcriptomics (ST). Methods: We performed ST (Visium 10x Genomics) on frozen tumor samples from 43 primary hormone receptor positive (HR+), HER2-negative (HER2-) ILCs. Of note, 9 samples were coming from patients who experienced disease relapse. Relative hematoxylin/eosin (H&amp;E) slides were morphologically annotated (QuPath software). xCell was used to perform cell type enrichment analysis on sample pseudo-bulks. After cross-samples integration, ST spots were clustered using hierarchical clustering (STutility R package). intNMF algorithm was used to perform clustering at the patient-level by combining: RNA sequencing information (relative percentage of spot-level clusters in each sample), morphology and level of colocalization between cancer cells and the other cell types of the tumor microenvironment. METABRIC (HR+, HER2- ILC samples, n = 122) was used as external validation cohort. Survival analyses (univariable and multivariable adjusting for clinicopathological features) were performed using Cox proportional hazard models. Results: The patient-level classification revealed four groups showing different biological characteristics. Differences in terms of morphology (annotation) and pathway enrichment analysis based on marker genes (GSEA) were observed between groups. This information allowed us to annotate our groups as: proliferative (P, n = 12, enriched in tumor cells and proliferation-related pathways), normal-stroma enriched (NSE, n = 10, enriched in fibroblasts and carcinoma in situ), metabolic (M, n = 9, enriched in metabolic-related pathways) and metabolic-immune enriched (MIE, n = 10, enriched in adipose tissue, metabolic and immune-related pathways). Interestingly, a significantly higher presence of macrophages M2 was found in MIE group. Using group-specific gene signatures, we were able to reproduce the same 4 groups in the METABRIC. Of note, we observed significant differences in relapse-free survival (RFS) in METABRIC (p = 0.03), with NSE presenting better outcome, while P, M and MIE presented worse prognosis. Analysing the area of contact between adipocytes and cancer cells typical of MIE group, we noticed an enrichment in metabolic and M2 macrophages-related pathways. In doing so, we derived a 28 gene signature relative to the tumor-adipocytes contact area. Importantly, our signature was highly expressed in M and MIE groups, and it was not correlated with proliferation-related signatures. Interestingly, the adipocytes-related signature was significantly associated with shorter RFS in ILC patients in METABRIC (univariable: HR 1.4, p = 0.023; multivariable: HR 1.6, p = 0.005). Since both proliferation and metabolism showed to be key processes in defining prognosis in ILC, we built a prognostic index by integrating our adipocytes-related signature with genomic grade index (GGI, a proliferation-related signature). Our index outperformed other existing prognostic signatures (e.g., Oncotype DX, MammaPrint, EndoPredict, LobSig) in assessing prognosis (RFS) in ILC in METABRIC (univariable: HR 1.7, p &amp;lt; 0.001; multivariable: HR 1.7, p = 0.002). Conclusions: We identified 4 biologically driven HR+, HER2- ILC groups describing tumor microenvironment heterogeneity. Of note, two of the three groups associated to worse disease outcome were related to metabolism, highlighting the importance of such process in ILC biology and in the future development of new treatment strategies. Moreover, the prognostic power of our index has the potential to refine the assessment of the risk of relapse in ILC. Further validation is warranted. Citation Format: Matteo Serra, Mattia Rediti, Laetitia Collet, Frédéric Lifrange, David Venet, Nicola Occelli, Xiaoxiao Wang, Delphine Vincent, Ghizlane Rouas, Ligia Craciun, Denis Larsimont, Laurence Buisseret, Miikka Vikkula, François Duhoux, Françoise Rothé, Christos Sotiriou. Spatially resolved analysis of tumor microenvironment in invasive lobular carcinoma [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 PO1-15-03.

Improved Preservation of Mouse Intestinal Tissue Using a Formalin/Acetic Acid Fixative and Quantitative Histological Analysis Using QuPath.

The architecture and morphology of the intestinal tissue from mice or other small animals are difficult to preserve for histological and molecular analysis due to the fragile nature of this tissue. The intestinal mucosa consists of villi and crypts lined with epithelial cells. In between the epithelial folds extends the lamina propria, a loose connective tissue that contains blood and lymph vessels, fibroblasts, and immune cells. Underneath the mucosa are two layers of contractile smooth muscle and nerves. The tissue experiencessignificant changes during fixation, which can impair the reliability of histologic analysis. Poor-quality histologic sections are not suitable for quantitative image-based tissue analysis. This article offers a new fixative composed of neutral buffered formalin (NBF) and acetic acid, called FA. This fixative significantly improved the histology of mouse intestinal tissue compared to traditional NBF and enabled precise, reproducible histologic molecular analyses using QuPath software. Algorithmic training of QuPath allows for automated segmentation of intestinal compartments, which can be further interrogated for cellular composition and disease-related changes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Improved preservation of mouse intestinal tissue using a formalin/acetic acid fixative Support Protocol: Quantitative tissue analysis using QuPath.