Semiquantitative Immunohistochemistry Research Articles

Kir4.1 and Aqp4 Contribution to Schisis Cystic Water Accumulation and Clearance in the Rs1 Exon-1 Del XLRS Rat Model.

The Rs1 exon-1-del rat (Rs1KO) XLRS model shows normal retinal development until postnatal day 12 (P12) when small cystic spaces start to form in the inner nuclear layer. These spaces enlarge rapidly, peak at P15, and then collapse by P19. We explored the possible involvement of Kir4.1 and Aqp4, the principal retina channels for water movement and homeostasis, along with Muller glia cells (MGCs), using semi-quantitative fluorescent immunohistochemistry at P7, P9, P12, and P30, in Rs1KO and WT littermates. Kir4.1 expression was reduced in Rs1KO retinas at all the early time points-P7, P9, and P12-as the schisis cavities began to form; downregulation would reduce water egress from the retina. Aqp4 was upregulated at P30 in Rs1KO retinas during schisis cavity closure but not as cavities formed at P12. When examined by GFAP expression, MGCs were not activated at the preschisis P12 age but showed considerable GFAP expression at P30 following retinal cystic structural damage at P15, indicating that MGCs were activated during the period of retina water removal and cavity closure. The study results implicate the downregulation of Kir4.1 in schisis formation and a role for both Kir4.1 and Aqp4 upregulation in subsequent schisis closure.

Prognostic Effectiveness of PD-L1 Tumoral Expression in Oral Cavity Squamous Cell Carcinoma.

PD-L1 is a glycoprotein present on antigen-presenting cells and malignant cells and activates the PD-L1 receptor on cells surfaces, leading to T-cell anergy and death. The objective of this study was to assess PD-L1 tumoral expression in 50 patients with oral squamous cell carcinoma, define its prognostic value, and investigate its association with 2-year overall survival, locoregional recurrence, distant metastasis, and a number of other clinicopathological parameters. In a 24-month prospective observational study, involving 50 oral cavity squamous cell carcinoma patients, PD-L1 tumoral expression was evaluated using semiquantitative immunohistochemistry analysis by an experienced pathologist. PD-L1 expression of ≥ 6% of tumor cells was associated with poor survival outcome and determined to be a pertinent clinical cutoff value. PD-L1 expression of ≥ 6% was significantly associated with a number of clinicopathological parameters in our study of 50 patients with OSCC. Pathological nodal staging (P = 0.00), lympho-vascular invasion (P = 0.03), extra-nodal extension (P = 0.04), overall staging (P = 0.01), locoregional recurrence (P = 0.00), and distant metastasis (P = 0.00) all showed statistically significant associations. Our study concluded a significant correlation with decreased 2-year overall survival and 2-year disease free survival by using the Kaplan- Meier survival plot for overall survival (p = 0.02) and (p = 0.0002), respectively. The presence of ≥ 6% PD-L1 (CD274) tumoral expression was found to be significantly associated with 2-year overall survival (OS), locoregional recurrence (LRC), distant metastasis (DM), and various clinicopathological parameters. Tumoral PD-L1 was found as a discrete prognostic biomarker which showed significant association with tumor aggressiveness.

Impact of LKB1 status on radiation outcome in patients with stage III non-small-cell lung cancer

Preclinical studies suggest that loss of LKB1 expression renders cancer cells less responsive to radiation partly through NRF2-mediated upregulation of antioxidant enzymes protecting against radiation-induced DNA damage. Here we investigated the association of an alteration in this pathway with radio-resistance in lung cancer patients. Patients with locally advanced non-small cell lung cancer (LA-NSCLC) who were treated with chemoradiotherapy (CRT) and analyzed for LKB1 expression using semiquantitative immunohistochemistry. Clinical characteristics and expression of LKB1 were analyzed for association with radiotherapy outcomes. We analyzed 74 available tumor specimens from 178 patients. After a median follow-up of 40.7 months, 2-year cumulative incidence of locoregional recurrence (LRR) in patients who had LKB1Low expression was significantly higher than those with LKB1High expression (68.8% vs. 31.3%, P = 0.0001). LKB1Low expression was found significantly associated with a higher incidence of distant metastases (DM) (P = 0.0008), shorter disease-free survival (DFS) (P = 0.006), and worse overall survival (OS) (P = 0.02) compared to LKB1High expression. Moreover, patients with LKB1Low expression showed a significantly higher 2-year cumulative incidence of LRR (77.6% vs. 21%; P = 0.02), higher DM recurrence (P = 0.002), and shorter OS (P < 0.0001) compared with the EGFR-mutant group. For all patients with LKB1Low who had LRR, these recurrences occurred within the field of radiation, in contrast to those with LKB1High expression having both in-field, marginal, and out-of-field failures. LKB1 expression may serve as a potential biomarker for poor outcomes after receiving radiation in LA-NSCLC patients. Further studies to confirm the association and application are warranted.

Comparative quantitation of liver-type fatty acid-binding protein localizations in liver injury and non-pathological liver tissue in dogs

Background and Aim: Liver injury results in the production of free radicals that can lead to hepatocytic degeneration, cirrhosis, and hepatocellular carcinoma (HCC). Liver-fatty acid-binding protein (L-FABP) is highly expressed in hepatocytes and is a key regulator of hepatic lipid metabolism and antioxidant characteristics. Interestingly, the increase in L-FABP expression could be used as a novel marker of liver injury. Therefore, this study aimed to use immunohistochemical techniques to investigate the expression of L-FABP in dogs with liver injury compared with dogs with non-pathological liver. Materials and Methods: Liver tissue samples were collected from dog biopsy specimens at the Veterinary Diagnostic Laboratory at the Faculty of Veterinary Medicine, Chiang Mai University. The tissues were prepared for immunohistochemistry and the expression and localization of L-FABP were investigated using one-way analysis of variance. Results: Immunohistochemical analysis showed that L-FABP was strongly expressed in the hepatocytes of dogs with lipidosis and HCC when compared with that in normal liver. Semi-quantitative immunohistochemistry evaluation showed the percentage of protein expression of L-FABP 0.023 ± 0.027 in the non-pathological liver. The percentage of L-FABP protein expression in lipidosis and HCC was found to be 8.517 ± 1.059 and 17.371 ± 4.026, respectively. Conclusion: L-FABP expression in dogs with liver injuries was significantly higher than that in dogs with non-pathological liver injury (p = 0.05). These results suggest that L-FABP has the potential as a novel marker for specific diagnosis and prognosis of dogs with liver injury. Keywords: canine, hepatocyte, immunohistochemistry, lipidosis, liver injury, liver-fatty acid-binding protein, pathology.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Tumor cell cytoplasmic metallothionein expression associates with differential tumor immunogenicity and prognostic outcome in high-grade serous ovarian carcinoma.

The underlying mechanism of high T-cell presence as a favorable prognostic factor in high-grade serous ovarian carcinoma (HGSOC) is not yet understood. In addition to immune cells, various cofactors are essential for immune processes. One of those are metallothioneins (MTs), metal-binding proteins comprising various isoforms. MTs play a role in tumor development and drug resistance. Moreover, MTs influence inflammatory processes by regulating zinc homeostasis. In particular, T-cell function and polarization are particularly susceptible to changes in zinc status. The aim of the present study was to investigate a possible role of MT-mediated immune response and its association with prognostic outcome in ovarian cancer. A retrospective study was conducted on a clinically well-characterized cohort of 24 patients with HGSOC treated at the University Hospital of Essen. Gene expression patterns for anti-cancer immunogenicity-related targets were performed using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel, consisting of 770 targets and 30 reference genes. Tumor-associated immunohistochemical MT protein expression was evaluated using a semi-quantitative four-tier Immunohistochemistry (IHC) scoring. MT immunoexpression was detected in 43% (10/23) of all HGSOC samples. MT immunoexpression levels showed a significant association to survival, leading to prolonged progression-free and overall survival in positively stained tumors. Furthermore, T-cell receptor signaling gene signature showed a strong activation in MT-positive tumors. Activated downstream signaling cascades resulting in elevated interferon-gamma expression with a shift in the balance between T helper cells (TH1 and TH2) could be observed in the MT-positive subgroup. In addition, a higher expression pattern of perforin and several granzymes could be detected, overall suggestive of acute, targeted anti-cancer immune response in MT-positive samples. This is the first study combining broad, digital mRNA screening of anti-tumor immune response-associated genes and their relation to MT-I/II in ovarian cancer. MT overexpression is associated with molecular characteristics of an anti-cancer immune response and is a strong prognostic marker in ovarian HGSOC. The observed immune cell activation associated with tumor MT expression comprises but is not limited to T cells and natural killer cells.

Linking epileptic phenotypes and neural extracellular matrix remodeling signatures in mouse models of epilepsy

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g. caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brain's excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and to a lesser extent GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link proteins Hapln1 and Hapln4 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype.

Age-Related Gene and Protein Expression in Mouse Mandibular Condyle Analyzed by Cap Analysis of Gene Expression and Immunohistochemistry

Introduction: Aging, an inevitable physiological process, leads to morphological and histological degenerative changes in the mandibular condylar cartilage (MCC); however, the molecular mechanism has not yet been elucidated, and little information is available on age-related factors. Therefore, this study was designed to identify age-related factors by investigating the age-related differentially expressed genes (DEGs) and localization of their translated protein expression in the mandibular condyle. Methods: Mandibular condyles were collected from 10- and 50-week-old mice. Total RNA was extracted from the samples and then analyzed using cap analysis of gene expression (CAGE) to identify age-related DEGs. Gene ontology (GO) enrichment analysis was performed to determine which biological processes were most affected by aging in terms of gene expression using Metascape. The mandibular condyle samples were processed for histology to investigate morphological changes caused by aging and for immunohistochemistry to localize the protein expression encoded by age-related genes identified with CAGE. Semi-quantitative immunohistochemistry was performed to assess age-related extracellular matrix (ECM) protein levels in the MCC. The histological sections were also used for Alcian blue histochemistry to detect glycosaminoglycans (GAGs). Results: GO enrichment analysis revealed that the genes related to “extracellular matrix organization,” including Acan, Col1a1, Col1a2, Col2a1, Mmp3, Mmp9, and Mmp13, were most differentially expressed in the aged mandibular condyle. Among these seven genes, Mmp3 was upregulated, and the others were downregulated with aging. Histological examination showed the age-related morphological and histological changes in the MCC. Immunohistochemical investigation showed the localization of matrix metalloproteinases (MMPs)-3, -9, and -13 and their substrate proteins, aggrecan, type I collagen, and type II collagen, in the mandibular condyle at 10 and 50 weeks, indicating different localizations between the young and the aged. In the aged MCC, semi-quantitative immunohistochemistry showed a significant decrease in the aggrecan protein level, and Alcian blue histochemistry showed a decrease in GAGs. Conclusion: MMP-3, MMP-9, and MMP-13 contribute to the remodeling of the ECM of the MCC and subchondral bone during aging by degrading ECM proteins at specific times and sites under the regulation of their production and secretion.

Cancer Stem Cells' Biomarker ALDH1&2 Increased Expression in Erosive Oral Lichen Planus Compared to Oral Leukoplakia.

ALDH1&2 has been considered an oral cancer stem cell (CSC) marker. Oral carcinogenesis is a process that usually passes through oral potentially malignant disorders (OPMD). Oral lichen planus (OLP) consists of immune-related chronic disorders that have been included in the OPMDs due to their possible transformation into oral cancer. The aim of this study was to investigate the early presence of ALDH1&2 in OLP compared to early oral leukoplakias (OL), especially mildly and non-dysplastic OL. The study type is experimental, and the study design is characterized as semiquantitative research which belongs to the branch of experimental research. The study sample consisted of paraffin-embedded OLP biopsy samples from the archives of the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece, during the period 2009-2019. The study sample contained 24 cases of OLP (14 erosive and 10 reticular) and 30 cases of OL (16 cases of moderately and severely dysplastic OL and 14 cases of mildly and non-dysplastic OL). The CSC-related biomarker ALDH1&2 was examined using semiquantitative immunohistochemistry (monoclonal antibody sc-166362, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:100). ALDH1&2 expression was evaluated through a scale of 1 to 3 depending on the percentage of positive epithelial cells and was compared to normal epithelium as well as cases of OL (the most prominent OPMD). The statistical analysis was performed with the Pearson chi-square test and the significance level was set at p≤0.05. The cytoplasmic staining of ALDH1&2 was observed mostly in the epithelial cells of the basal layer of the epithelium of OLP. Overall, this expression was significantly increased compared to normal epithelium. In addition, statistically significantly higher expression of ALDH1&2 was observed in the erosive form of OLP. Interestingly, this OLP positivity was higher compared to mild and non-dysplastic leukoplakias (p<0.001). ALDH1&2 is a confirmed CSC marker that was found to be clearly increased in OLP and characteristically in erosive OLP epithelium for the first time. Noteworthy, it was more prominent in erosive OLP rather than in mildly and non-dysplastic OL. Whether this pattern of expression raises the red flag of an early epithelial "CSC" phenotype in OLP or that ALDH1&2 expression indicates a response to the OLP inflammatory process requires further investigation.

The Landscape of MYB/MYBL1- and Peri-MYB/MYBL1-Associated Rearrangements in Adenoid Cystic Carcinoma

Approximately 60% of adenoid cystic carcinoma (AdCC) cases are positive for MYB::NFIB or MYBL1::NFIB, whereas MYB/MYBL1 oncoprotein, a key driver of AdCC, is overexpressed in most cases. Juxtaposition of superenhancer regions in NFIB and other genes into the MYB/MYBL1 locus is an attractive oncogenic hypothesis for AdCC cases, either negative or positive for MYB/MYBL1::NFIB. However, evidence supporting this hypothesis is insufficient. We examined 160 salivary AdCC cases for rearrangements in MYB/MYBL1 loci and peri-MYB/MYBL1 areas (centromeric and telomeric areas of 10 Mb each) using formalin-fixed, paraffin-embedded tumor sections. For the detection of the rearrangements, we employed conventional fluorescence in situ hybridization split and fusion assays and a 5 Mb fluorescence in situ hybridization split assay. The latter is a novel assay that enabled us to detect any possible splits within a 5 Mb distance of a chromosome. We found MYB/MYBL1- and peri-MYB/MYBL1-associated rearrangements in 149/160 patients (93%). AdCC cases positive for rearrangements in MYB, MYBL1, the peri-MYB area, and the peri-MYBL1 area numbered 105 (66%), 20 (13%), 19 (12%), and 5 (3%), respectively. In 24 peri-MYB/MYBL1 rearrangement-positive cases, 14 (58%) were found to have a juxtaposition of the NFIB or RAD51B locus into the MYB/MYBL1 loci. On comparing with a tumor group positive for MYB::NFIB, a hallmark of AdCC, other genetically classified tumor groups had similar features of overexpression of the MYB transcript and MYB oncoprotein as detected by semiquantitative RT-qPCR and immunohistochemistry, respectively. In addition, clinicopathological and prognostic features were similar among these groups. Our study suggests that peri-MYB/MYBL1 rearrangements may be a frequent event in AdCC and may result in biological and clinicopathological consequences comparable to MYB/MYBL1 rearrangements. The landscape of MYB/MYBL1 and peri-MYB/MYBL1 rearrangements shown here strongly suggests that juxtaposition of superenhancers into MYB/MYBL1 or peri-MYB/MYBL1 loci is an alteration that acts as a key driver for AdCC oncogenesis and may unify MYB/MYBL1 rearrangement-positive and negative cases.

Genome-wide DNA methylation profile analysis identifies an individualized predictive signature for melanoma immune response

The current evaluation methods for tumor infiltrating lymphocytes (TILs), particularly CD8 + TILs, mainly rely on semiquantitative immunohistochemistry with high variability. We aimed to construct an individualized DNA methylation-based signature for CD8 + TILs (CD8 + MeTIL) that may characterize melanoma immune microenvironment and guide therapeutic selection. The transcriptome profiles and DNA methylation data of 457 melanoma patients from The Cancer Genome Atlas (TCGA) database were analyzed. Differential methylation analysis between groups with high and low CD8 + TILs was performed to select differentially methylated positions (DMPs) and define CD8 + MeTIL. The prognostic value of CD8 + MeTIL and its predictive value for immunotherapy response were investigated using multiple melanoma cohorts. We successfully constructed the CD8 + MeTIL signature based on four DMPs. The survival analyses showed that higher CD8 + MeTIL score was associated with worse survival outcomes in TCGA-SKCM and GSE144487 cohorts. The ROC curve for the predictive analysis revealed that the survival prediction of CD8 + MeTIL score was superior compared with CD8 + TILs (CIBERSORT) and CD8B mRNA expression. Furthermore, we founded that tumors with higher CD8 + MeTIL score were marked with immunosuppressive characteristics, including low immune score and downregulated immune-related pathways. More importantly, the CD8 + MeTIL score showed a potential predictive value for the benefit from immunotherapy in two published cohorts. When combined CD8 + MeTIL with PD-L1 expression, the patient classification showed significantly different immunotherapy response rates and long-term survival outcomes. The CD8 + MeTIL signature might be as a novel method to evaluate CD8 + TILs and guide immunotherapy approaches.

Tofacitinib Inhibits STAT Phosphorylation and Matrix Metalloproteinase-3, -9 and -13 Production by C28/I2 Human Juvenile Chondrocytes.

PurposeThis in vitro study was designed to determine the effect of the pan-Janus kinase inhibitor, Tofacitinib, on basal and interleukin-6 (IL-6)-induced signal transducers and activators of transcription (STAT) phosphorylation and matrix metalloproteinase (MMP) gene expression and MMP production by C28/I2 human chondrocytes.MethodsC28/I2 chondrocytes were grown to a confluent high-density and treated either with recombinant human IL-6 (rhIL-6; 10–20ng/mL) or maintained in the basal state for up to 60 min. MMP gene expression was determined using RT-PCR and MMP production by semi-quantitative immunohistochemistry. The effect of IL-6 with or without Tofacitinib on activation of STAT proteins was determined from quantitative Western blots.ResultsC28/I2 chondrocytes produced STAT1, STAT3 and STAT5AB which were phosphorylated (p) following treatment with rhIL-6 for 30 min. Tofacitinib (2.5nM–100nM) decreased rhIL-6-induced activation of STAT1, STAT3, and STAT5AB as well as decreasing the expression of MMP3 and MMP13 but not MMP9, MMP1 or MMP2. In addition, Tofacitinib (50nM) reduced the number of rhIL-6-induced MMP3-, and MMP13- antibody-positive C28/I2 chondrocytes. However, Tofacitinib did decrease the number of MMP9-antibody-positive C28/I2 chondrocytes.ConclusionTaken together, these data showed that Tofacitinib, a pan-JAK small molecule inhibitor employed for the medical therapy of rheumatoid arthritis was a potent inhibitor of rhIL-6-induced STAT phosphorylation that appeared to be coupled to the inhibition of MMP-3, -9 and -13 production by C28/I2 chondrocytes.

Erythropoietin prevented the decreased expression of aquaporin1-3 in ureteral obstructive kidneys in juvenile rats.

Urinary tract obstruction is associated with impaired renal urinary concentration; even after the release of the obstruction, patients still suffer from polyuria. It has been reported that the decreased expression of aquaporins (AQPs) is associated with postobstructive polyuria, and erythropoietin (EPO) can promote the recovery of decreased AQP2 expression induced by bilateral ureteral obstruction. However, whether EPO can promote the recovery of the expression of AQP1-3 after the release of unilateral ureteral obstruction (UUO) has not yet been reported. To investigate the effects of EPO treatment on the expression of renal AQP1-3 after the release of UUO. UUO was established in rats by 24-h temporary unilateral obstruction of renal ureters. Three days following EPO treatment, the kidneys were removed to determine the expression levels of AQP1-3, NLRP3, caspase-1, and IL-1β via semiquantitative immunoblotting and immunohistochemistry. EPO inhibited the expression of NLRP3, caspase-1, and IL-1β; reduced plasma creatinine and urea; and promoted the recovery of AQP1-3 expression in UUO rats. EPO treatment prevented the decreased expression of renal AQPs and the development of impaired urinary concentration capacity after the release of UUO, which may partially occur by way of anti-inflammasome effects. EPO treatment could prevent the decreased expression of renal water transporter proteins AQP1-3 and the development of impaired renal functions, which may be associated with its anti-inflammasome effects. EPO regulated the expression of renal water transporter proteins AQP1-3, which could provide the potential for the treatment of postobstructive polyuresis. EPO treatment could be one of the effective methods by participating in multiple dimensions for patients with obstructive nephropathy.

Assessing and improving the validity of COVID-19 autopsy studies - A multicentre approach to establish essential standards for immunohistochemical and ultrastructural analyses.

SummaryBackgroundAutopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain about whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, such as overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing.MethodsWe assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 proteins in cultured cell lines and COVID-19 autopsy tissues. In a multicentre study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 IHC. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2.FindingsPublications show high variability in detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. We show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (N= 35; r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and provide recommendations for optimized sampling and analysis. 135 of 144 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM sections as a reference and for training purposes.InterpretationSince detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2.FundingGerman Federal Ministry of Health, German Federal Ministry of Education and Research, Berlin University Alliance, German Research Foundation, German Center for Infectious Research.

Dimethylbisphenol A inhibits the differentiation of stem Leydig cells in adult male rats by androgen receptor (NR3C4) antagonism

Dimethylbisphenol A (DMBPA) is a novel alternative to bisphenol A. Whether short-term exposure to DMBPA affects Leydig cell regeneration remains unknown. The Leydig cell regeneration model was generated by intraperitoneal injection of 75 mg/kg ethane dimethane sulfonate (EDS) to adult male Sprague-Dawley rats. Leydig cell regeneration began on day 14 after EDS. Rats were gavaged with 0, 10, 50, or 200 mg/kg DMBPA from days 14–28 post-EDS, and Leydig cell regeneration was assessed on days 28 and 56 post-EDS. DMBPA significantly reduced serum testosterone levels on days 28 and 56 at 10 mg/kg and higher doses and sperm count in the caudal epididymis on day 56 at 200 mg/kg, without affecting estradiol, luteinizing hormone, and follicle-stimulating hormone. DMBPA had no effect on Leydig cell number but significantly down-regulated Scarb1 expression at ≥ 10 mg/kg on day 28, Cyp17a1 expression on day 28 at 200 mg/kg and on day 56 at ≥ 10 mg/kg. DMBPA markedly upregulated Srd5a1 expression at doses of 50 and 200 mg/kg on day 56 after EDS. DMBPA significantly down-regulated the expression of Sod1 and Nr3c4 at a dose of 200 mg/kg on day 28. Further semi-quantitative immunohistochemistry showed that DMBPA reduced NR3C4 levels in Leydig and Sertoli cells at 50 and 200 mg/kg. In vitro DMBPA treatment of immature Leydig cells for 24 h showed that it significantly reduced testosterone production at 10 and 50 μM, and further mechanistic studies showed that an NR3C4 agonist 7α-methyl-19-nortestosterone significantly reversed DMBPA-mediated suppression on testosterone output, but the estrogen receptor antagonist ICI 182,780 and G-coupled estrogen receptor 1 agonist G15 had no effect. In conclusion, DMBPA delays Leydig cell regeneration after short-term exposure during early Leydig cell regeneration via NR3C4 antagonism.

Using American Type Culture Collection Cell Lines to Evaluate Interlaboratory Variables for Estrogen Receptor and Human Epidermal Growth Factor Receptor 2 Immunostaining.

Most laboratories currently use patient tissues for validating immunohistochemical stains. To explore advantages of using cell lines with known antigenicity as a validation method. Five American Type Culture Collection (ATCC) cell lines with known negative, low positive, and moderate to strong estrogen receptor (ER) expression as well as negative, equivocal, and positive human epidermal growth factor receptor 2 (HER2) expression were cultured and made into cell blocks. One block from each cell line was fixed in formalin and another in ethanol before cell block preparation. Two sets of paired unstained slides from each block were sent to 10 different laboratories for HER2 and ER staining to be stained on runs from different days according to each laboratory's defined protocol. The 10 study participants evaluated 40 slides in a blinded fashion. For ER expression, all 80 interpretations for the ER strong and moderate positive cell lines had the target ER-positive result, and 74 of 80 ER-negative cell lines (92.5%) had agreement with the intended negative result. The ER low positive cell line showed varied but positive expression among all observers. The HER2 (3+)-positive cell lines yielded a target interpretation of 3+ in 65 of 80 interpretations (81.2%). For the HER2-negative cell line 69 of 78 interpretations (88.5%) were consistent with the target response (0 or 1+). No significant variation was observed between the ethanol- and non-ethanol-exposed cell lines, or between runs by the same laboratory. Variation from target results clustered within laboratories. This study indicates that variability between laboratories can be identified by using cell lines for quantitative or semiquantitative immunohistochemistry when using cultured cell lines of known antigenicity. These cell lines could potentially play a role in aiding anatomic pathology laboratories in validating immunohistochemistry tests for formalin- and ethanol-fixed tissues.

PD-L1 Protein Expression Is Associated With Good Clinical Outcomes and Nomogram for Prediction of Disease Free Survival and Overall Survival in Breast Cancer Patients Received Neoadjuvant Chemotherapy.

ObjectiveThis study aims to investigate the potential prognostic significance of programmed death ligand-1 (PD-L1) protein expression in tumor cells of breast cancer patients received neoadjuvant chemotherapy (NACT).MethodsUsing semiquantitative immunohistochemistry, the PD-L1 protein expression in breast cancer tissues was analyzed. The correlations between PD-L1 protein expression and clinicopathologic characteristics were analyzed using Chi-square test or Fisher’s exact test. The survival curve was stemmed from Kaplan-Meier assay, and the log-rank test was used to compare survival distributions against individual index levels. Univariate and multivariate Cox proportional hazards regression models were accessed to analyze the associations between PD-L1 protein expression and survival outcomes. A predictive nomogram model was constructed in accordance with the results of multivariate Cox model. Calibration analyses and decision curve analyses (DCA) were performed for the calibration of the nomogram model, and subsequently adopted to assess the accuracy and benefits of the nomogram model.ResultsA total of 104 breast cancer patients received NACT were enrolled into this study. According to semiquantitative scoring for IHC, patients were divided into: low PD-L1 group (61 cases) and high PD-L1 group (43 cases). Patients with high PD-L1 protein expression were associated with longer disease free survival (DFS) (mean: 48.21 months vs. 31.16 months; P=0.011) and overall survival (OS) (mean: 83.18 months vs. 63.31 months; P=0.019) than those with low PD-L1 protein expression. Univariate and multivariate analyses indicated that PD-L1, duration of neoadjuvant therapy, E-Cadherin, targeted therapy were the independent prognostic factors for patients’ DFS and OS. Nomogram based on these independent prognostic factors was used to evaluate the DFS and OS time. The calibration plots shown PD-L1 based nomogram predictions were basically consistent with actual observations for assessments of 1-, 3-, and 5-year DFS and OS time. The DCA curves indicated the PD-L1 based nomogram had better predictive clinical applications regarding prognostic assessments of 3- and 5-year DFS and OS, respectively.ConclusionHigh PD-L1 protein expression was associated with significantly better prognoses and longer DFS and OS in breast cancer patients. Furthermore, PD-L1 protein expression was found to be a significant prognostic factor for patients who received NACT.

Photothermal ablation of murine melanomas by Fe3O4 nanoparticle clusters.

Melanoma is one of the deadliest forms of cancer, for which therapeutic regimens are usually limited by the development of resistance. Here, we fabricated Fe3O4 nanoparticle clusters (NPCs), which have drawn widespread attention, and investigated their role in the treatment of melanoma by photothermal therapy (PTT). Scanning electron microscopy imaging shows that our synthesized NPCs are spherical with an average diameter of 329.2 nm. They are highly absorptive at the near-infrared wavelength of 808 nm and efficient at locally converting light into heat. In vitro experiments using light-field microscopy and cell viability assay showed that Fe3O4 NPCs, in conjunction with near-infrared irradiation, effectively ablated A375 melanoma cells by inducing overt apoptosis. Consistently, in vivo studies using BALB/c mice found that intratumoral administration of Fe3O4 NPCs and concomitant in situ exposure to near-infrared light significantly inhibited the growth of implanted tumor xenografts. Finally, we revealed, by experimental approaches including semi-quantitative PCR, western blot and immunohistochemistry, the heat shock protein HSP70 to be upregulated in response to PTT, suggesting this chaperone protein could be a plausible underlying mechanism for the observed therapeutic outcome. Altogether, our results highlight the promise of Fe3O4 NPCs as a new PTT option to treat melanoma.

Spontaneous Facet Joint Osteoarthritis in NFAT1-Mutant Mice: Age-Dependent Histopathologic Characteristics and Molecular Mechanisms.

Facet joint (FJ) osteoarthritis (FJOA) is a widely prevalent spinal disorder but its pathogenesis remains unclear, largely due to the difficulties in conducting longitudinal human studies and lack of spontaneous-FJOA animal models for mechanistic investigations. This study aimed to investigate whether spontaneous FJOA occurs in mice bearing mutant NFAT1 (nuclear factor of activated T cells 1) transcription factor. The lumbar FJs of 50 NFAT1-mutant mice and of 50 wild-type control mice, of both sexes, were examined by histopathology, quantitative gene expression analysis, semiquantitative immunohistochemistry, and a novel FJOA scoring system for semiquantitative assessment of the histopathologic changes at 2, 6, 12, and 18 months of age. Age-dependent and tissue-specific histopathologic and gene or protein expression changes were analyzed statistically. FJs in NFAT1-mutant mice displayed significantly increased expression of specific catabolic genes (p < 0.05) and proteins (p < 0.001) in cartilage and synovium as early as 2 months of age, followed by early osteoarthritic structural changes such as articular surface fissuring and chondro-osteophyte formation at 6 months. More severe cartilage lesions, osteophytes, subchondral bone changes, synovitis, and tissue-specific molecular alterations in FJs of NFAT1-mutant mice were observed at 12 and 18 months. Osteoarthritic structural changes were not detected in FJs of wild-type mice at any ages, although age-related cartilage degeneration was observed at 18 months. The novel FJOA scoring system had high intraobserver and interobserver reproducibility (correlation coefficients: r > 0.97). Whole-joint FJOA scoring showed significantly higher OA scores in FJs of NFAT1-mutant mice compared with wild-type mice at all time points (p = 0.0033 at 2 months, p = 0.0001 at 6 months, p < 0.0001 at 12 and 18 months). This study has identified the NFAT1-mutant mouse as a novel animal model of spontaneous FJOA with age-dependent and slowly progressing osteoarthritic features, developed the first FJOA scoring system, and elucidated the molecular mechanisms of NFAT1 mutation-induced FJOA. This murine FJOA model resembles the features of human FJOA and may provide new insights into the pathogenesis of and therapeutic strategies for FJOA in humans.

Abstract P1-08-10: Ki67 assessment based on international Ki67 working group recommendations and correlation with 21-gene assay results in a large integrated health care system: We might not be there yet

Abstract Background: While substantial evidence indicates that Ki67, a marker of proliferation, is strongly associated with breast cancer outcomes, its clinical utility has been limited given concerns about scoring inter-rater reliability and appropriate cut points. Nonetheless, Ki67 has been used in multiple clinical trials and results from POETIC indicated that low baseline Ki67 (ie, &amp;lt;10%) predicts good prognosis in postmenopausal women with hormone sensitive, early breast cancer. Further, the International Ki67 Working Group (IKWG) has developed website-based training materials to improve reproducibility of Ki67 scoring by immunohistochemistry (IHC) and recently considered the marker to be sufficiently validated to support treatment decisions in early ER+ breast cancer (≤ 5 no chemotherapy, ≥30 chemotherapy indicated). Our aims were to examine Ki67 scoring reproducibility following IKWG training and the extent to which low or high scores could accurately identify patients with low or high 21-gene assay Recurrence Scores (RS) who could selectively avoid this test. Methods: Setting and study population. The study was conducted within Kaiser Permanente Northern California (KPNC), an integrated health care system with over 4.4 million enrollees. We included a random sample of women aged 50+ years at diagnosis of node-negative, ER+PR+HER2- breast cancer with the 21-gene assay done on their surgical specimen from 2018-2020 (n=307). Ki67 staining, training and scoring. We retrieved archived core biopsy specimens, which were sent to NeoGenomics Laboratories, Inc for Ki67 staining and scoring by image analysis (IA) using the hot spot counting method. In addition, two KPNC pathologists specializing in semiquantitative IHC scoring underwent IKWG training and independently scored all slides using the global counting method, blinded to each other and to readings by AI; weighted Ki67 scores were calculated. Analysis. We examined inter-rater reliability across pathologists using intraclass correlation (ICC) and Kappa statistics. We also examined the percent of patients with low Ki67 scores (≤ 5, &amp;lt;10) by each pathologist and by AI who also had low RS (&amp;lt;26) and the percent who had high Ki67 scores (≥30) who also had high RS (≥26). Results: Approximately 83% of patients were ages 50-69 years (median 63), 61% were non-Hispanic white and 93% were stage 1A. The ICC for Ki67 scores (log-transformed) by the two pathologists was 0.82; using a dichotomous cut point of &amp;lt;10 vs ≥10%, the Kappa for the inter-rater reliability was 0.65. Among patients with Ki67 scores of &amp;lt;10% by IA (n=81), pathologist 1 (n=120) or pathologist 2 (n=111), the percent with a RS of &amp;lt;26 was 95.1% for IA, 95.8% for pathologist 1, and 94.6% for pathologist 2. Among patients with Ki67 scores ≤5, the percentages were 90.9%, 92.6% and 91.8% for IA, pathologist 1 and pathologist 2, respectively. Among patients with Ki67 scores ≥30 by AI (n=69), pathologist 1 (n=41) or pathologist 2 (n=35), the percent who had a RS ≥26 was 27.5% for IA, 41.5% for pathologist 1 and 51.4% for pathologist 2. Results are improved if we exclude all 50 patients with weak PR by IHC (1-10%); for example, among patients with Ki67 scores of &amp;lt;10%, the percent with a RS of &amp;lt;26 was 97.1% for IA, 98.1% for pathologist 1, and 97.9% for pathologist 2. Conclusion: Among women aged 50+ years with node-negative, ER+PR+HER2- breast cancer in our setting, approximately 5-10% of patients with Ki67 scores of ≤ 5% or &amp;lt;10% on core biopsies would have high RS (≥26) on surgical specimens and over 48% of cases with Ki67 scores ≥30 would have low RS (&amp;lt;26), which may be insufficiently accurate for avoiding the 21-gene or other multi-gene assays. Future studies are needed to examine whether restricting Ki67 testing to ER+HER2- patients with PR &amp;gt;10% would improve its clinical validity. Citation Format: Veronica C. Shim, Robin J Baker, Wen Jing, Sally S Agersborg, Thomas K Lee, Wamda Goreal, Ninah Achacoso, Catherine Lee, Marvella Villasenor, Amy Lin, Malathy Kapali, Laurel A Habel. Ki67 assessment based on international Ki67 working group recommendations and correlation with 21-gene assay results in a large integrated health care system: We might not be there yet [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-10.