CD34 Antibodies Research Articles

Skin Whole-Mount Immunofluorescent Staining Protocol, 3D Visualization, and Spatial Image Analysis.

The use of polychromatic immunofluorescent staining on whole-mount skin enables cell type characterization and aids in the delineation of the physiological and immunological strategies used by the skin to combat pathogens. Using whole-mount skin for polychromatic immunofluorescent staining removes the need for histological sectioning and enables the visualization of anatomical structures and immune cell types in three dimensions. Here we present a detailed protocol for immunostaining with fluorescence-conjugated primary antibodies in whole-mount skin to reveal structural landmarks and specific immune cell types using confocal laser scanning microscopy (CLSM) (Basic Protocol 1). The optimized staining panel reveals structural features such as blood vessels (CD31 antibody) and the lymphatic network (LYVE-1 antibody), in combination with MHCII antibodies for antigen-presenting cells (APCs), CD64 for macrophages and monocytes, CD103 for dendritic epidermal T cells (DETC), and CD326 for Langerhans cells (LC). Basic Protocol 2 describes image visualization pipelines using open-source software (ImageJ/FIJI), enabling four visualization options (z-projections, orthogonal views, 3D visualization, and animation). Basic Protocol 3 describes a quantitative analysis pipeline using CellProfiler to characterize the spatial relationship between cell types using mathematical indices such as Spatial Distribution Index (SDI), Neighborhood Frequency (NF), and Normalized Median Evenness (NME). These protocols will enable researchers to stain, record, analyze, and interpret data from whole-mount skin using commercially available reagents in a CLSM-equipped laboratory and freely available analysis software. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescent staining and imaging for whole-mount mouse skin Basic Protocol 2: File rendering and visualization using FIJI Basic Protocol 3: Spatial image analysis using CellProfiler.

Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application

BackgroundThrombosis is a common cause of vascular prosthesis failure. Antibody coating of prostheses to capture circulating endothelial progenitor cells to aid endothelialization on the device surface appears a promising solution to prevent thrombus formation. Compared with random antibody immobilization, oriented antibody coating (OAC) increases antibody-antigen binding capacity and reduces antibody immunogenicity in vivo. Currently, few OAC methods have been documented, with none possessing clinical application potential.ResultsDopamine and the linker amino-PEG8-hydrazide-t-boc were successfully deposited on the surface of cobalt chromium (CC) discs, CC stents and expanded polytetrafluoroethylene (ePTFE) grafts under a slightly basic condition. CD34 antibodies were immobilized through the reaction between aldehydes in the Fc region created by oxidation and hydrazides in the linker after t-boc removal. CD34 antibody-coated surfaces were integral and smooth as shown by scanning electron microscopy (SEM), had significantly reduced or no substrate-specific signals as revealed by X-ray photoelectron spectroscopy, were hospitable for HUVEC growth as demonstrated by cell proliferation assay, and specifically bound CD34 + cells as shown by cell binding testing. CD34 antibody coating turned hydrophobic property of ePTFE grafts to hydrophilic. In a porcine carotid artery interposition model, a confluent monolayer of cobblestone-shaped CD31 + endothelial cells on the luminal surface of the CD34 antibody coated ePTFE graft were observed. In contrast, thrombi and fibrin fibers on the bare graft, and sporadic cells on the graft coated by chemicals without antibodies were seen.ConclusionA universal, OAC method was developed. Our in vitro and in vivo data suggest that the method can be potentially translated into clinical application, e.g., modifying ePTFE grafts to mitigate their thrombotic propensity and possibly provide for improved long-term patency for small-diameter grafts.

Tumor immune microenvironment in BRAF V600E mutant lung adenocarcinoma.

e21003 Background: The efficacy of immune checkpoint inhibitors (ICIs) is closely related to tumor immune microenvironment (TIME). However, little is known about the role of BRAF V600E mutation in the TIME of lung adenocarcinoma (LUAD). Methods: Here, we analyzed the TIME of LUAD with the BRAF V600E mutation using RNA sequencing and multiplex immunofluorescent (MIF) stained with CD8, CD68, CD163, CD57, PD-1, and PD-L1 antibodies. Patients were divided into the BRAF V600E (n = 31) and driver gene ( ALK, BRAF, EGFR, HER2, KRAS, MET, RET, and ROS1) wild-type groups (WT, n = 32). Results: There were no significant differences between the BRAF V600E and driver gene WT groups in terms of immunological signature scores, including T cell–inflamed gene expression profiles, adhesion molecules, chemokines, cytolytic activity, immunocostimulators, immunoinhibitors, MHC Class I, MHC Class II, MHC Non-Class, IFNg signature, tertiary lymphoid structure, and most of infiltrating immune cell subtypes. MIF showed that the proportions of PD-L1+ cells and CD68-associated tumor-associated macrophages (TAMs) were significantly higher in the BRAF V600E group in the tumor, stroma, and total regions. Notably, the majority of CD68-associated macrophages were CD68+CD163- TAMs. However, the proportions of CD8+, CD8+PD-L1+, and CD8+PD-1+ tumor-infiltrating lymphocytes (Tils) in the BRAF V600E and driver gene WT groups were all comparable in the tumor, stroma, and total region. Conclusions: The TIME was generally similar between the BRAF V600E mutant and driver gene wild-type groups, particularly in the proportion of CD8+ Tils. This may support that BRAF V600E mutation does not affect the efficacy of immune checkpoint inhibitors in LUAD.

KAPPA MYELOMA ANTIGEN (KMA) AND LAMBDA MYELOMA ANTIGEN (LMA) ARE EXPRESSED ON B CELL MALIGNANCIES AND ARE PROMISING NOVEL TARGETS ON MALIGNANT PLASMA CELLS

Introduction: The kappa myeloma antigen (KMA) and the lambda myeloma antigen (LMA) are found on the surface of malignant plasma cells (PCs) in multiple myeloma (MM), other plasma cell dyscrasia patient bone marrow (BM) samples, human myeloma cell lines and in patient BM samples from plasmacytomas, lymphoplasmacytoid and B cell lymphoproliferative disorders (BLPD). KMA and LMA are not present on normal B cells in BM but are found on occasional mononuclear cells in tonsillar tissue and secondary mucosal lymphoid tissue. KappaMab is a therapeutic antibody that specifically targets KMA and a phase IIb study in combination with lenalidomide and dexamethasone has recently been completed. In early clinical trials, KappaMab was shown to have an excellent safety profile, with low-grade infusion-related reactions and no haematological toxicities. Two human therapeutic LambdaMabs (10B3 and 7F11) bind specifically to LMA expressed only on malignant cells in lambda restricted B cell diseases. Here we compare the expression of KMA and LMA to that of BCMA in MGUS, newly diagnosed MM and relapsed, refractory (RRMM) myeloma BM samples. We describe the difference in phenotype between KMA and LMA expressing malignant PCs and their enrichment in RRMM compared to early disease stages. Methods: Patient BM samples (κ = 70 and λ = 40) were analysed using multiparametric FCM immunophenotyping with APC labelled LambdaMab (10B3) and KappaMab Fab’2 fragments, CD38, CD138, CD269 (BCMA), CD319 (SLAM F7), CD56 and CD45 monoclonal antibodies. PCs were identified by initial gating using CD38 and CD138. LMA and KMA expression was determined and compared with the other cell markers. Results: KMA was expressed on the majority of kappa restricted BM PCs from MGUS through to RRMM, on lymphoplasmacytoid cells in 2 out of 3 BM samples and in all 4 plasmacytoma BM samples. LMA was expressed on the surface of BM PCs in lambda restricted MGUS through to RRMM and in all 4 AL amyloidosis BM samples. KMA and LMA antigen densities were both higher than BCMA in the RRMM population: KMA mean 1899 (range 468–7943) versus BCMA mean 1426 (range 350-2630) and LMA mean 2439 (range 263–6664) versus BCMA mean 817 (range 537–1065) molecules of PE/cell. Conclusions: Although there are smaller numbers of samples from other B cell malignancies such as lymphoplasmacytoid, amyloidosis and BLPDs, we have demonstrated that KMA or LMA are expressed on malignant BM PCs. In the myeloma cases, the antigen density of both KMA and LMA was increased compared to BCMA on RRMM BM PCs, which suggests a clonal advantage of KMA and LMA expressing plasma cells that are resistant to standard of care treatments. These data suggest that KappaMab and LambdaMabs have therapeutic potential in RRMM where the combination of high antigen density and restriction of the target antigen to the malignant subpopulation of PCs may confer clinical benefit by retaining normal plasma cells following treatment. Keywords: Immunotherapy, Multiple Myeloma Conflicts of interests pertinent to the abstract D. Gottlieb Consultant or advisory role David sits on the Scientific Advisory Board for HaemaLogiX Ltd. R. Dunn Employment or leadership position: Employee at HaemaLogiX Ltd. Stock ownership: Yes

Association of peripheral memory B cell population maintenance and long term survival after perioperative chemoimmunotherapy in NSCLC (NADIM trial).

8551 Background: In the context of perioperative immunotherapy, it is crucial to personalize adjuvant treatment, identifying which patients should or should not receive post-surgical immunotherapy. Here we describe the changes in B cell peripheral blood immunophenotype during perioperative immunotherapy and its potential to predict disease progression in patients from NADIM I clinical trial (NCT03081689). Methods: Blood samples from 42 patients were obtained at pre-, post-neoadjuvant chemoimmunotherapy (pre-surgery) and at 6 and 12 months of adjuvant IO. Peripheral mononuclear cells were stained with CD19, CD20, CD38, CD24, CD27, CD25, CD10 antibody panel and analyzed by flow cytometry using FlowJo. Statistical analysis in paired samples was performed with Wilcoxon test and differences in parameters’ fold changes between disease status groups (Progression-free at 34.2 months) were determined by Mann-Whitney U tests. Variables cutoffs were stablished using ROC curve analysis. Progression-free survival (PFS) was evaluated using Kaplan-Meier curves and long-rank test. The median follow-up was 38.0 months. Results: Regarding the impact of NADIM complete treatment scheme in patient’s B cells, there was a reduction of memory B cells (CD19+CD20+CD38-CD24+CD27+; p < 0.001) and CD20 levels in total B cells (CD19+CD20+; p = 0.001) (n = 17). Importantly, this decrease occurs in all patients regardless their pathological response or disease progression. However, the magnitude of the memory B cell percentage and CD20 levels reduction was significantly more pronounced in patients whose disease progress thereafter (p = 0.023, p = 0.052 at 6 and 12 months for memory B cells; and p = 0.002, p = 0.009 at 6 and 12 months for CD20 expression). Patients with a relative drop of memory B cells greater than 90% at 6 or 12 months, showed a shorter PFS (p = 0.028, p = 0.007). Likewise, patients with a relative decrease of CD20 expression on B cells higher than 60% at 6 or 12 months had shorter PFS (p = 0.016, p < 0.001). The AUC ROCs to identify patients with disease progression, were 0.824 and 0.885 at 6 and 12 months for memory B cells; and 0.912 and 0.981 at 6 and 12 months for CD20 levels. Moreover, memory B cells percentage and CD20 levels decrease occur between surgery and the first 6 months of adjuvant therapy (n = 21, p < 0.001, p = 0.025). After that, these levels are maintained from 6 to 12 months of adjuvant treatment (n = 20, p = 0.290, p = 0.682), thus the overall reduction is also observed between surgery and 12 months samples (n = 17, p < 0.001, p = 0.001). Conclusions: Perioperative IO produces a significant reduction in memory B cells and CD20 levels. Importantly, stronger decreases might be helpful to predict progression since they are associated to shorter PFS, indicating that the maintenance of the memory B cell peripheral compartment is relevant for long-term survival of these patients.

Ureteropelvic junction obstruction in children by polar vessels: histological examination result.

In children, ureteropelvic junction obstruction (UPJO) is mostly caused by intrinsic factors (IUPJO) such as abnormal amounts of muscle and collagen deposition; extrinsic UPJO are rare and often due to crossing vessels (CVs). What is not clear is whether there is also intrinsic UPJ pathology in patients with CV. The aim of our study was to compare the histology of the two types of obstruction and to determine whether these histologic features are distinguishable enough to enable to identify the cause of obstruction based on histologic appearance alone. We retrospectively reviewed pathology reports of 38 children with UPJO that underwent surgery in our hospital from 2008 to 2022. The intrinsic and extrinsic groups consisted of 18 and 20 patients, respectively. After ematoxylin-eosin and Gomori's trichrome staining the specimens were scored for fibrosis and muscular hypertrophy in histhopatology, and CD117 antibody were used to detect interstitial Cajal-like cells. In our study, histological analyses revealed no differences between the CV and IUPJO specimens in terms of presence and degree of fibrosis and muscular hypertrophy; likewise, for presence of interstitial Cajal-like cells.

Novel discovery of a lymphatic bridge connecting Schlemm's canal to limbal and conjunctival lymphatic pathway

PurposeSchlemm's canal (SC) is a critical structure regulating aqueous humor (AH) drainage and intraocular pressure (IOP). It is known that in the conventional outflow pathway, AH flows from SC to episcleral veins. We recently reported a high-resolution three-dimensional (3D) imaging technology for intact eyeballs, SC and ocular surface. Using this advanced technology, we herein report the discovery of a new structure, termed lymphatic bridge, that directly connects SC to the limbal and conjunctival lymphatic pathway. Further investigation on this novel outflow pathway may provide new mechanisms and therapeutic approaches for glaucoma. MethodsAs reported previously, intact eyeballs were harvested from Prox-1-GFP (green fluorescent protein) mice and processed by a tissue clearing technique with CLARITY. Samples were immunolabeled with specific antibodies for CD31 (pan-endothelial marker) and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) and imaged by light-sheet fluorescent microscopy. The limbal areas were examined to locate connecting channels between SC and limbal and conjunctival lymphatic vessels. Moreover, in vivo anterior chamber dye injection was performed with Texas Red dextran for functional analysis on AH outflow. ResultsA novel lymphatic bridge structure that expressed both Prox-1 and LYVE-1 was discovered between the SC and limbal lymphatic vessels connected with conjunctival lymphatic pathway. Results from the anterior chamber dye injection also confirmed AH drainage into the conjunctival lymphatic outflow pathway. ConclusionsThis study provides the first evidence on the direct connection between SC and conjunctival lymphatic pathway. This new pathway is distinctive from the traditional episcleral vein pathway and merits further investigation.

Canine Mammary Neoplasia Induces Variations in the Peripheral Blood Levels of CD20, CD45RA, and CD99.

The idea of using tumour biomarkers as diagnostic tools is progressively increasing. Of these, serum biomarkers are of particular interest, as they can provide rapid results. In the present study, serum samples from 26 bitches diagnosed with mammary tumours, plus 4 healthy bitches, were obtained. The samples were analysed using CD antibody microarrays targeting 90 CD surface markers and 56 cytokines/chemokines. A total of five CD proteins, namely CD20, CD45RA, CD53, CD59, and CD99, were selected and further analysed, utilizing immunoblotting techniques to validate the microarray results. CD45RA showed a significantly lower abundance in the serum samples from the bitches carrying mammary neoplasia in comparison to the healthy animals. Regarding CD99, the serum samples from the neoplastic bitches showed it in a significantly higher abundance than those from the healthy patients. Finally, CD20 showed a significantly higher abundance in bitches carrying a malignant mammary tumour in comparison to healthy patients, but no differential expression between malignant and benign tumours was observed. According to these results, both CD99 and CD45RA are indicators of mammary tumour presence, but without distinguishing between malignant and benign.

Exosomes obtained from adipose mesenchymal stem cells prevent ischemia-reperfusion injury after torsion-detorsion in rat testes.

To investigate the effect of exosomes obtainedfrom adipose-derived mesenchymal stem cells (AD-MSCs) on testicular ischemia-reperfusion (I/R) injury. AD-MSCs from rat adipose tissue were cultured. Characterization of cells was evaluated with CD44, CD90, CD34 and CD45 antibodies. Exosomes from AD-MSCs were obtained with the miRCURYexosomeisolation kit. 21 rats were divided into 3 groups. The I/R model was created as 720° torsion for 4h and reperfusion for 4h. In the Sham group (SG), only scrotal incision was made. 100µl of medium in the torsion-control group (T-CG) and 100µl of exosome in the treatment group (TG) were injected into the testicular parenchyma after detorsion. Johnsen scores of testicles were determined. Apoptosis was evaluated by the TUNEL method. It was observed that the seminiferous tubule structures were partially disrupted in T-CG, but normal in SG and TG. Johnsen scores in SG, T-CG, and TG were 8.64 ± 0.39, 7.71 ± 0.37, and 8.57 ± 0.39, respectively. Apoptotic cell distribution was 11.28 ± 5.25%, 60.58% ± 1.68% and 17.71 ± 8.34% in SG, T-CG and TG, respectively. In both parameters, the difference between SG and TG was insignificant (p > 0.05), the difference between T-CG/TG and SG/T-CG was significant (p < 0.05). Exosomes obtainedfromAD-MSCs are effective in preventing testicular I/R injury. This effect appears to occur because of suppression of apoptotic activity.

Immunohistochemical analysis of the ligamentum mucosum is the key to understand its clinical usefulness

Ligamentum Mucosum(LM) is a ligamentous structure within the synovial layer of the knee joint capsule. For a long time LM was perceived as the vestigial remnant from the embryological development of the knee. Being treated as an irrelevant structure LM was often the first victim of shaver during arthroscopy. However, last years have shown increased interest in this structure due to its potentially significant clinical role. Our aim was to classify LM based on its morphological characteristics and examine its microanatomy usingimmunohistochemical analysis to reveal the potential clinical value for surgeons. We have examined sixteen fresh frozen lower limbs, 6 females (mean age 83.1 ± 3.4 years) and 10 males (mean age 84.2 ± 6.8 years). Classical histological H+E stain was routinely conducted. Subsequently, CD31 antibody (DAKO, Monoclonal Mouse Anti-Human, Clone JC70A) was used to mark vascular epithelium. Monoclonal Mouse Anti-Human Neurofilament Protein (NFP) antibody (DAKO, Clone 2F11) was used to expose the nerves. Moreover, we have conducted arthroscopic visualizing and suturing LM to the torn ACL during routinely performed arthroscopic suturing of the ACL. The dissection process has revealed that LM was present only in 75% of cases.Histological examination confirmed the presence of longitudinal collagen fiber bundles in all samples. Tiny nerves were confirmed by NFP, along the subsynovial layer in all samples. CD-31 immunostain revealed the presence of many vascular vessels along the entire ligament, especially well developed at its distal end. Our study has revealed that LM contains rich vascular network. Thus, it may be a donor for the revascularization process after ACL tear or reconstruction which may improve the recovery. Another great advantage of the LM is the presence of nerves along the subsynovial layer, hopefully they may serve as the source of reinnervation and hence better clinical outcome. Based on our results we believe that seemingly irrelevant LM may be very useful during surgical procedures in the knee region. Suturing LM to the ACL may not only prevent the infrapatellar fat pad from subluxation but also improve the blood flow and reinnervation of the injured ACL. Until now there are only a few studies examining microanatomy of the LM. This basic knowledge may serve as the foundation for surgical procedures. Hopefully our findings may be useful for surgeons while planning surgical procedures or clinicians while diagnosing patients who suffer anterior knee pain.

Gastrointestinal Stromal Tumor (GIST) in its Mesenteric Location: about One Case

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the digestive tract but very rare in its mesenteric location. We report the case of a young 43-year-old patient followed for iron deficiency anemia. Exploration by imaging showed a tumor mass 5 cm long, probably at the expense of the root of the mesentery of the distal jejunum. The midline laparotomy showed a mesenteric mass in contact with the jejunum located 70 cm from the duodenojejunal angle. We performed total tumor resection associated with segmental resection of the jejunal loop and end-to-end grelo-grelic anastomosis. The postoperative was simple. The pathological report showed a mesenteric gastrointestinal stromal tumor and the immunohistochemical study showed the positivity of the tumor cells for the anti CD117 antibody. Given the size (5 cm), weight (100 g) and location of the tumor with complete surgical resection, adjuvant treatment was not instituted in our patient.

IL-33-expressing microvascular endothelial cells in human esophageal squamous cell carcinoma: Implications for pathological features and prognosis.

Accumulating evidence suggests that interleukin (IL)-33 plays a critical role in regulating angiogenesis and cancer progression. In this study, we characterized the pathological importance of IL-33 deployed by tumor microvascular endothelial cells (ECs) in human esophageal squamous cell carcinoma (ESCC). The expression of IL-33 in microvascular ECs in 80 cases of ESCC was examined with immunohistochemistry (IHC) and double immunofluorescence. IHC results showed that strong IL-33-immunoreactivity (IR) in microvessels, which were confirmed to be ECs by double immunofluorescence staining with IL-33/CD31 antibodies. Moreover, high proliferative activity was shown in IL-33-positive ECs, and the IL-33 functional receptor ST2 was expressed in microvascular ECs. Clinicopathological analysis revealed that IL-33-positive microvessel density (MVD) was positively correlated with node involvement in patients with ESCC. A log rank test showed a highly significant inverse correlation between the densities of IL-33-positive MVDs and overall survival rate, and patients with higher IL-33-positive MVDs tended to have a lower survival rate (both p<0.05). Therefore, we concluded that IL-33 deployed by microvascular ECs correlates with advanced pathological features and the long-term survival rate, which provides new insights into the regulatory mechanisms of tumor angiogenesis in the tumor microenvironment and might serve as a promising target in patients with ESCC.

Abstract 703: Dioxin-induced Smooth Muscle Cell Phenotypic Modulation And Atherosclerosis Worsens In The Absence Of AHR

Background: Dioxin (TCDD, 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin) is an active constituent of tobacco smoke and air pollution that activates the aryl hydrocarbon receptor (AHR). Dioxin has been linked to an increased risk of coronary artery disease (CAD). However, the corresponded molecular mechanisms remain unclear. Smooth muscle cells (SMC) play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. In this study, we aimed to understand the role of AHR in modulating the effect of dioxin on vascular SMC and atherosclerosis phenotype. Methods: SMC lineage-tracing (SMC Lin ) mice ( Tg Myh11-CreERT2 , ROSA tdT/tdT , ApoE -/- ) without any TCDD injection were used as control. TCDD were injected to both SMC Lin mice, and SMC-specific Ahr knockout mice. High fat diet (HFD) and TCDD injection started at 8 weeks of age and aortic roots were harvested at 24 weeks of age for either histology or single-cell sequencing (10X Genomics, triplicates). Histology analysis was performed at the aortic sinus of control mice (n=9), TCDD injected SMC Lin mice (n=12) and TCDD injected SMC-specific Ahr knockout mice (n=9). Immunohistochemistry was performed with anti-SM22α antibody and CD68 antibody. Results: The histological analysis of the aortic sinus showed increased lesion area after TCDD treatment in the SMC Lin control mice. In the SMC-specific knockout of Ahr , the lesion area was significantly larger compared to the SMC Lin control mice when treated with TCDD along with significantly increased presence of Tagln + cells in the lesion. These results indicated TCDD leads to adverse remodeling of atherosclerosis and that the presence of Ahr gene is protective against the athero-promoting effects of dioxin. The scRNA-seq of SMC Lin control mice treated with TCDD exposure showed that the SMC phenotypic modulation was markedly inhibited, leading to the presence of fewer fibromyocytes (FMC). Increased FMC to chondromyocytes (CMC) modulation resulted in further reduction of FMC population in the Ahr -KO mice treated with TCDD. Conclusion: Dioxin adversely remodels atherosclerotic plaque, and the lack of AHR in SMC can exacerbate the atherosclerotic process, suggesting that dioxin may exhibit its harmful effect via non-AHR dependent pathway.

Degree of fibrosis and its association with angiogenesis in the myelofibrotic bone marrow

Background: Primary and secondary myelofibrosis has become a global burden due to its increased mortality and morbidity. Angiogenesis is a significant driving force in the development of fibrogenesis in the bone marrow, which leads to myelofibrosis. The microvascular density (MVD) with immunomarker CD34 can be used to assess the degree of angiogenesis. The objective of this study was to examine the association between degree of myelofibrosis and angiogenesis in hematological malignancies. Methods: Forty-six trephine biopsy specimens of various hematological malignancies with myelofibrosis were studied at the Department of Pathology of Bangabandhu Sheikh Mujib Medical University. Extent of myelofibrosis in each case was assessed by examining the reticulin and Masson’s trichrome stained sections using a semiquantitative grading system of bone marrow fibrosis (MF) within a scale of MF-0 to MF-3. Angiogenesis was measured by counting MVD in the ‘hotspots’ after immunostaining with CD34 antibody. Results: The trephine biopsy cases were grouped into early fibrotic (MF-1) and advanced fibrotic (MF-2,3) consisting of 16 (34.8%) and 30 (65.2%) patients, respectively. Angiogenesis was estimated as mean MVD count which revealed 16.7 ± 5.4 and 32.0 ± 11.5 in these groups, respectively. Significant difference of mean MVD values (P&lt;0.001) between the early and advanced fibrotic groups revealed the association of angiogenesis and degree of myelofibrosis. Conclusion: MVD may be used to measure angiogenesis in myelofibrotic marrow along with other clinical and laboratory indices as a marker of disease activity in hematological malignancies, thus aiding disease prognosis.

Further explorations into the role of mast cells in deaths associated with fatal asphyxia: an immunohistochemical study utilizing CD 117 marker

Asphyxia-related deaths have always been a challenging task in the speciality of forensic pathology. Apart from helpful macroscopical signs (e.g., strangulation mark, cyanosis, petechial haemorrhage, and lung oedema), recent literature indicates that prolonged asphyxia is sufficient to induce an increase in mast cells (MC). Inflammatory cells migrate from the bone marrow to the lungs, aiding in the diagnosis of fatal asphyxial deaths. HIF1-α, a key regulator protein, is released from lung tissue capillaries during catastrophic hypoxia circumstances, as previously demonstrated in immunohistochemistry (IHC) research. The present study analyzed lung samples from 164 medico-legal autopsy cases, including 57 asphyxia/hypoxia deaths and 107 controls (non-asphyxial deaths). Peribronchial, perivascular and perialveolar MCs were detected using CD117 antibody, and the average of MCs in each of these locations was noted in each case. The results indicated a statistically significant increase in peribronchial and perialveolar mast cells (MC) in fatal asphyxial deaths, including those caused by hanging, drowning, or postural asphyxia. Peri-bronchial MC in lung sections of asphyxial deaths were in the range of 0.2–5.4 and in non-asphyxial samples were in the range of 0.0–2.2. Peri-alveolar MCs in lung sections of asphyxial deaths were in the range of 0.0–0.6 and in non-asphyxial samples were in the range of 0.0–0.2. Our data suggest that mast cells (MC) play an important role in fatal hypoxia-related mortality and CD 117 may be a reliable marker for detection of mast cells in asphyxial deaths. It could be very beneficial to forensic pathologists tasked with differentiating fatal asphyxia fatalities from other causes of death.

Expression of GPNMB in renal eosinophilic tumors and its value in differential diagnosis

Objective: To investigate the expression of glycoprotein non metastatic melanoma protein B (GPNMB) in renal eosinophilic tumors and to compare the value of GPNMB with CK20, CK7 and CD117 in the differential diagnosis of renal eosinophilic tumors. Methods: Traditional renal tumor eosinophil subtypes, including 22 cases of renal clear cell carcinoma eosinophil subtype (e-ccRCC), 19 cases of renal papillary cell carcinoma eosinophil subtype (e-papRCC), 17 cases of renal chromophobe cell carcinoma eosinophil subtype (e-chRCC), 12 cases of renal oncocytoma (RO) and emerging renal tumor types with eosinophil characteristics [3 cases of eosinophilic solid cystic renal cell carcinoma (ESC RCC), 3 cases of renal low-grade eosinophil tumor (LOT), 4 cases of fumarate hydratase-deficient renal cell carcinoma (FH-dRCC) and 5 cases of renal epithelioid angiomyolipoma (E-AML)], were collected at the Affiliated Drum Tower Hospital of Nanjing University Medical School from January 2017 to March 2022. The expression of GPNMB, CK20, CK7 and CD117 was detected by immunohistochemistry and statistically analyzed. Results: GPNMB was expressed in all emerging renal tumor types with eosinophil characteristics (ESC RCC, LOT, FH-dRCC) and E-AML, while the expression rates in traditional renal eosinophil subtypes e-papRCC, e-chRCC, e-ccRCC and RO were very low or zero (1/19, 1/17, 0/22 and 0/12, respectively); the expression rate of CK7 in LOT (3/3), e-chRCC (15/17), e-ccRCC (4/22), e-papRCC (2/19), ESC RCC (0/3), RO (4/12), E-AML(1/5), and FH-dRCC (2/4) variedly; the expression of CK20 was different in ESC RCC (3/3), LOT(3/3), e-chRCC(1/17), RO(9/12), e-papRCC(4/19), FH-dRCC(1/4), e-ccRCC(0/22) and E-AML(0/5), and so did that of CD117 in e-ccRCC(2/22), e-papRCC(1/19), e-chRCC(16/17), RO(10/12), ESC RCC(0/3), LOT(1/3), E-AML(2/5) and FH-dRCC(1/4). GPNMB had 100% sensitivity and 97.1% specificity in distinguishing E-AML and emerging renal tumor types (such as ESC RCC, LOT, FH-dRCC) from traditional renal tumor types (such as e-ccRCC, e-papRCC, e-chRCC, RO),respectively. Compared with CK7, CK20 and CD117 antibodies, GPNMB was more effective in the differential diagnosis (P<0.05). Conclusion: As a new renal tumor marker, GPNMB can effectively distinguish E-AML and emerging renal tumor types with eosinophil characteristics such as ESC RCC, LOT, FH-dRCC from traditional renal tumor eosinophil subtypes such as e-ccRCC, e-papRCC, e-chRCC and RO, which is helpful for the differential diagnosis of renal eosinophilic tumors.

Morphological features of epithelial-mesenchymal transformation and its effect on tumor progression of breast cancer

Introduction. Breast cancer is in the first place in the structure of morbidity among all malignant neoplasms in women. The prognosis of the disease depends on the tumor degree, including the presence of epithelial-mesenchymal transformation (EMT), the degree of invasion, the proliferative index, the preservation or absence of estrogen, progesterone, and epidermal growth factor receptors.Aim: To study the immunohistochemical and morphological characteristics of the epithelial-mesenchymal transformation of breast cancer.Material and Methods. Immunohistochemical study with antibodies to AE1/AE3, HMW, CK18, Snail, HER2/neu, E-cadherin, Vimentin, α-SMA, CD34, Ki-67 and p63 was performed in 60 patients of different age with breast cancer. Native preparations were stained with picrofuchsin according to van Gieson Alcian blue. Inflammatory infiltrate cells were examined for antibodies CD45, CD3, CD4, CD8, CD20, CD68.Results. In ductal carcinoma, positive expression for estrogen and progesterone was found in 82.7% and 86.3%, respectively, the proliferation index ranged before 66,6%, and p-53 was positive in 97%. In lobular cancer, positive expression to estrogen and progesterone was observed in 83.4% and 66.6%, respectively, the index of proliferative activity at the level of 50%, and p-53 was positive in 66.6%. Positive moderate expression of HER-2/neu was determined in 47% of ductal and 50% of lobular cancers. Estrogen plays an important role in the development of invasive breast cancer, leads to tumor progression and contributes to EMT. EMT, in turn, leads to the expression of E-cadherin associated with a worse survival prognosis. EMT indirectly leads to the intensification of angiogenesis, and the presence of a large number of newly formed vessels increases the risk of metastasis. Histochemical methods were used to determine the growth of fibrous tissue around invasively growing cancer complexes. Cells located perifocally looked like fibrobla ts, immunohistochemically moderately expressed Vimentin and weakly expressed pancytokeratin, which proved the tumor nature of the cells and the acquisition of mesenchymal features by them. The inflammatory infiltrate along the periphery of the tumor growth consisted mainly of T- and B-lymphocytes, and around the cancer complexes - of B-lymphocytes and macrophages.Conclusion. The study of the immunohistochemical tumor phenotype will make it possible to prescribe adequate polychemotherapy and determine the prognosis of the course of the disease.

Abstract 44: An in vivo platform of pre-characterized renal cell carcinoma (RCC) patient-derived xenograft models allows the preclinical evaluation of patient-tailored intervention strategies

Abstract Renal cell carcinoma (RCC), is the most common kidney cancer of adults, originating in the lining of the proximal convoluted tubule. Prognosis is poor in patients with advanced or metastasized RCC. Drug resistance towards Standard of Care (SoC, incl. everolimus, sorafenib, or sunitinib) drugs develops frequently within months. Therefore, development of novel options to target acquired TKI resistance mechanisms in advanced and metastatic RCC is still an urgent medical need. Preclinical models with high translational relevance can promote the implementation of novel personalized therapies. To evaluate novel targeted therapies and their combinations in preclinical settings, patient-derived xenograft (PDX) models represent valuable tools. Responsible local ethics committees approved usage of patient tissue and all animal procedures. In this study, RCC tissue from 167 patients was collected and xenotransplanted in mice. Partially, a multi-region approach, xenografting tissue from different regions of one tumor, was used. PDX models were characterized by immunohistochemistry (Ki-67, CD31, Pax2 and Pax8 antibodies), gene expression, copy number variations and mutational analyses. To evaluate in vivo drug response of RCC PDX models, mice transplanted with PDX tumors were treated with bevacizumab (i.p.), with everolimus, sorafenib, or sunitinib (p.o.). Adopted clinical response criteria for solid tumors (RECIST) were applied to classify the anti-tumor activity of the tested compounds in RCC PDX models. Next generation sequencing (NGS, panel) and transcriptome data were used to compare primary tumors and metastases. A comprehensive panel of subcutaneous RCC PDX models with well-conserved molecular and pathological features over multiple passages was established. The overall take for the RCC PDX in this study was 21%. Tumor growth characteristics were heterogeneous throughout the different models but were stable during in vivo passaging. Drug screening towards four SoC drugs, targeting the VEGF and PI3K/mTOR pathway, revealed individual and heterogeneous response profiles in the PDX, resembling the clinical situation. Intra-tumor heterogeneity can be assessed via PDX models from multi-tumor regions from one patient in our platform. Development of corresponding in vitro cell culture models from the PDX enables advanced high throughput drug screening in a personalized context. Analyzing novel targeted molecules is possible due to the pre-established molecular characterization of the PDX at the genomic and expression level. In conclusion, we established a new and molecularly characterized panel of RCC PDX models with high relevance for translational preclinical research. Citation Format: Dennis Kobelt, Dennis Gürgen, Michael Becker, Mathias Dahlmann, Susanne Flechsig, Elke Schaeffeler, Florian A. Büttner, Christian Schmees, Regina Bohnert, Jens Bedke, Matthias Schwab, Johann J. Wendler, Martin Schostak, Burkhard Jandrig, Wolfgang Walther, Jens Hoffmann. An in vivo platform of pre-characterized renal cell carcinoma (RCC) patient-derived xenograft models allows the preclinical evaluation of patient-tailored intervention strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 44.

Abstract 5786: Spatial relationships in the tumor microenvironment predict response to immune checkpoint inhibitors in urothelial and head and neck cancer

Abstract Background: Immune checkpoint inhibitors (ICI) can achieve remarkable clinical responses in urothelial cancer (UC). However, it remains unclear which aspects of the tumor microenvironment (TME) determine a patient’s response. The TME is usually characterized by immune cell density, which ignores cells’ spatial relationships relative to each other. Methods: Using multiplex immunofluorescence data (PanCK, CD20, CD68, CD3, CD8, and FoxP3 antibody panel) of 24 pre-ICI UC transurethral resections retrieved from the NABUCCO trial (NCT03387761), we spatially profiled cancer cells, macrophages, B-cells, and distinct T-cell populations. We first quantified the TME immune cell densities at the tumor and stroma tissue compartments. We then quantified the TME spatial relationships with a novel approach based on fitting a Weibull function to the first nearest neighbor (1-NN) distance distribution, allowing us to uniquely summarize spatial relationships with two parameters (Weibull approach). We compared this approach to conventional techniques (G-functions) that rely upon a predefined distance threshold. We performed a simulation study to identify sources of variation in the spatial relationship parameters. Lastly, we associated the TME parameters with ICI (ipilimumab + nivolumab) response. Results: Unlike the Weibull approach, the G-function quantifications manifested a variable effect size and statistical power in association studies because of its distance threshold dependence. We found that variation in density affected the spatial relationship metrics of rare cell types (i.e., B-cells) but not of abundant cell types (i.e., cancer cells). The spatial relationship metrics from the Weibull approach outperformed immune cell density in ICI response prediction. Specifically, immune cell density did not discriminate between ICI response groups (FDR&amp;gt;10%). In contrast, the spatial relationship between either CD8+ T-cells or macrophages to their closest cancer cell did associate with response to ICI (FDR=1%). Furthermore, non-responding tumors were characterized by CD8+ T-cells close to B-cells (FDR=9%). Importantly, we validated the association between proximity and response from CD8+ T-cells to cancer cells (FDR=1%) and from macrophages to cancer cells (FDR=1%) using data from 25 pre-ICI head and neck squamous cell carcinoma tumors from the IMCISION trial (NCT03003637, arm B, ipilimumab + nivolumab). Conclusion: We created a framework to quantify, study, interpret and analyze spatial relationships in the TME and illustrated its superior clinical relevance compared to density metrics for predicting ICI treatment response. Our findings emphasize the importance of spatial relationships in the TME for response and suggest that proximity between either macrophages or CD8+ T-cells to cancer cells are candidate biomarkers for ICI response. Citation Format: Alberto Gil-Jimenez, Nick van Dijk, Yoni Lubeck, Maurits L. van Montfoort, Dennis Peters, Erik Hooijberg, Annegien Broeks, Joris L. Vos, Charlotte L. Zuur, Bas van Rhijn, Daniel J. Vis, Michiel S. van der Heijden, Lodewyk F. Wessels. Spatial relationships in the tumor microenvironment predict response to immune checkpoint inhibitors in urothelial and head and neck cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5786.

Combined analysis of bulk and single-cell RNA sequencing reveals novel natural killer cell-related prognostic biomarkers for predicting immunotherapeutic response in hepatocellular carcinoma.

Natural killer (NK) cells play an irreplaceable and important role as a subtype of innate immune cells in the contemporary setting of antitumor immunity. We chose a total of 1,196 samples for this analysis from the public dataset's six separate cohorts. To identify 42 NK cell marker genes, we first carried out a thorough study of single-cell RNA sequencing data from the GSE149614 cohort of hepatocellular carcinoma (HCC). Using the NK cell marker genes in the TCGA cohort, we next created a seven-gene prognostic signature, separating the patients into two categories with distinct survival patterns. This signature's prognostic prediction ability was well verified across several validation cohorts. Patients with high scores had higher TIDE scores but lower immune cell infiltration percentages. Importantly, low-scoring patients had superior immunotherapy response and prognosis than high-scoring patients in an independent immunotherapy cohort (IMvigor210). Finally, we used CD56 and TUBA1B antibodies for immunohistochemical labeling of HCC tissue sections, and we discovered a lower number of CD56+ cells in the HCC tissue sections with high TUBA1B expression. In summary, our research created a unique prognostic profile based on NK cell marker genes that may accurately predict how well immunotherapy would work for HCC patients.