Expression Of Basal Markers Research Articles

Fibrogenesis in Human Mucosa and Muscularis Precision-Cut Intestinal Slices.

In Crohn's Disease (CD), intestinal fibrosis is a prevalent yet unresolved complication arising from chronic and transmural inflammation. The histological assessment of CD intestines shows changes in tissue morphology in all the layers, including the mucosa and muscularis. This study aimed to determine the differences in fibrogenesis between mucosa and muscularis. Human precision-cut intestinal slices (hPCIS) were prepared from human intestine mucosa and muscularis and treated with TGF-β1 and/or PDGF-BB for 72 h. Gene and protein expression and matrix metalloproteinase (MMP) activity were determined. The basal gene expression of various fibrosis markers was higher in muscularis compared to mucosa hPCIS. During incubation, Pro-Collagen-1A1 secretion increased in muscularis but not in mucosa hPCIS. MMP gene expression increased during incubation in mucosa and muscularis hPCIS, except for MMP9, MMP12, and MMP13 in muscularis hPCIS. Incubation with TGF-β1 caused increased COL1A1 expression in the mucosa but not in muscularis hPCIS. In muscularis hPCIS, TGF-β1 treatment caused a decrease in MMP1 and CTSK expression, while MMP13 was increased. In the presence of TGF-β1, protease inhibitor expression was stable, except for SERPINE1, which was increased in muscularis hPCIS. We conclude that fibrogenesis is more pronounced in muscularis hPCIS compared to mucosa hPCIS, especially when stimulated with TGF-β1.

FGFR3 alterations (FGFRalt) in patients (pts) who develop locally advanced or metastatic urothelial cancer (mUC), and their association with tumor subtype and clinical outcomes in pts treated with erdafitinib (erda) vs. pembrolizumab (pembro).

4578 Background: Erda, an oral pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor is approved to treat adult pts with locally advanced or mUC with susceptible FGFR3alt, whose disease progressed on or after at least one line of prior systemic therapy. In superficial and surgically resectable UC, FGFRalt has been shown to be highly associated with luminal subtype characterized by expression of luminal markers, low expression of basal markers and with immune cold / poor immune infiltration. In a randomized open-label phase III THOR study (Cohort 2), erda showed improvement in objective response and progression-free survival (PFS) compared to pembro, with no significant improvement in overall survival (OS). Exploration of subtypes in a large cohort of mUC pts has not been done and so a molecular analysis was undertaken to further understand the clinical findings. Methods: All available tumors from pts enrolled in Cohort 2 ( FGFRalt positive; N = 201) and a subset of FGFR wildtype (N = 116) were used to perform whole transcriptome RNA sequencing, where 152 and 84, respectively, passed quality control. The consensus single-sample classifier was applied to the RNAseq data to determine molecular subtypes. Tumor subtypes was correlated with treatment response to erda or pembro, PFS and OS. Results: Molecular classification of tumors identified a significant proportion of luminal-papillary (LumP) subtype in the tumors harboring FGFRalt compared to FGFR WT (78.3% vs. 36.9%, p < 0.001) vs. other subtypes: basal/squamous (Ba/Sq; 11.2% vs. 31.0%), stroma-rich (6.6% vs. 10.7%), neuroendocrine-like (0% vs. 2.4%), luminal-unstable (3.3% vs. 17.9%) and luminal-unspecified (0.7% vs. 1.2%), respectively. Consistently, FGFRalt type showed differential association with subtypes: 3.2% of fusions and 14.1% of mutations were detected in Ba/Sq subtype while 74.2% and 79.3%, respectively, were detected in LumP. Clinical outcomes evaluated within LumP subset showed a significant improvement in ORR of erda-treated vs. pembro-treated pts (41.7 vs. 19.7%; p = 0.01), which was consistent with the intent to treat (ITT) population (40.0% vs. 21.6%). Numerical improvement were observed in PFS in the LumP subtype between erda vs. pembro (5.5 vs 2.7 months) compared with the ITT population (4.4 vs 2.7). However, the benefit of erda over pembro in the LumP subtype did not translate to OS (10.9 vs. 12.9 months), similar to the ITT population (10.9 vs. 11.1 months). Conclusions: A significant overlap was identified between genomic selection of FGFRalt and the LumP subtype. Clinical outcomes based on transcriptomic analyses were consistent with those observed in the ITT population. These findings warrant further understanding of LumP tumors and association to treatment response. Clinical trial information: NCT03390504 .

Abstract A011: Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition

Abstract Introduction: Epithelial changes can extend beyond the boundaries of an incipient tumor, leading to cancer recurrence even after effective excision of the primary tumor. This ‘field cancerization’ and consequent high recurrence rate is especially pronounced in bladder cancer. The purpose of this study is to characterize the urothelium in the context of ‘field cancerization’ in a mouse model of bladder cancer as well as in patients with bladder cancer and to determine if reversal of the field effect will prevent tumor formation. Results: We investigated field cancerization by single cell transcriptomics (scRNA-Seq) in a murine (male FVB mice) bladder carcinogenesis model using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and found that prior to overt tumor formation urothelial cells throughout the entire bladder take on a strongly basal phenotype, characterized by reduced expression of differentiation markers (i.e. Upk1a, Upk2, and Upk3a) and increased expression of basal markers (i.e. Krt14, Krt6a, Krt5). Using monocle, we applied single cells from a BBN treated mouse bladder library to a pseudotime trajectory generated from a control mouse bladder library in order to unbiasedly characterize urothelial differentiation in the non-tumor urothelium of BBN treated mouse bladders. scRNA-Seq analysis of non-tumor urothelium from bladder cancer patients revealed a similar ‘basalization’ among non-tumor urothelial cells. In order to apply our single cell findings to bulk RNA-seq data, we developed a basal index from differentially expressed genes in basal clusters versus non-basal clusters from healthy control human urothelium single cell libraries, and we applied this basal index to non-tumor bladder samples from the The Cancer Genome Atlas (TCGA). We found that among a small subset of patients with paired tumor and non-tumor urothelium in the bladder TCGA dataset, the basal index, when applied to the non-tumor urothelium, was prognostic of survival. Finally, we found that basalization in BBN treated mice could be reversed pharmacologically by epidermal growth factor (EGF) pathway antagonism using erlotinib and trametinib and that over a 4-month time course this reversal dramatically decreased tumor incidence and improved survival, suggesting a new, more effective approach using mechanism-based cancer drugs for the prevention of bladder cancer recurrence in patients. Conclusions: The non-tumor urothelium in BBN treated mouse bladders, prior to overt tumor formation, demonstrates a basal phenotype which we characterize with scRNA-Seq and pseudotime analysis. Similarly, in patients with bladder cancer we observe that the non-tumor urothelium also takes on a basal phenotype. This ‘basalization’ of the non-tumor urothelium may potentially be prognostic, and pharmacologic reversal of the basal phenotype in the non-tumor urothelium using EGF pathway inhibitors prevents tumors in BBN treated mice. Citation Format: Aaron M. Kershner, Kris B. Prado, Bernhard M. Kiss, Karim Mrouj, Lolita Penland, Wan-Jin Lu, Jiyun Choi, Edward Diaz, Rovin Lachmansingh, Bertha Chen, Chia-Sui Kao, Joseph C. Liao, Philip A. Beachy. Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A011.

Abstract A023: Stratification of bladder cancer patients with the use of flow cytometry is increasing the diagnostic potential of molecular classifiers

Abstract Recent research suggests the potential categorization of bladder cancer (BLCa) patients into molecular subtypes to aid in the selection of appropriate treatment strategies, be it conventional, targeted, or a combination thereof. However, the practical implementation of molecular classifiers has faced obstacles due to the intricate nature of gene regulation, tumor heterogeneity, and challenges related to tumor sampling caused by substantial stromal infiltration and tumor multifocality. Hence, there is a pressing need for more straightforward marker-based methodologies to assist in genomic and transcriptomic subtyping efforts. Our study addressed this gap by establishing and validating a panel of antibodies for application in flow cytometry (FCM) to enhance the stratification of BLCa patients. We curated a set of 17 FCM markers capable of delineating four cell clusters (epithelial, immune, endothelial, and stromal) and defining tumor classification (basal and luminal), immune infiltration, and hot-target expression on BLCa tissue samples. This FCM panel was deployed on ex vivo tissue samples treated with chemotherapy and targeted therapy agents. Initially tested on a cohort of 10 BLCa tissue samples, our validation of the FCM panel revealed correlations between marker expression and clinical-pathological features, such as the presence of metastasis and heightened immune and stromal infiltration. Notably, samples exhibiting lower claudin-4 levels displayed increased immune infiltration, while those with metastases at diagnosis showcased elevated basal marker expression. The translational potential of the FCM panel was demonstrated in ex vivo experiments where tissue slices from six patients were subjected to chemotherapy and targeted therapy agents, leading to observable shifts in marker expression post-treatment, particularly pronounced with chemotherapy. Noteworthy effects included reductions in epithelial cells expressing FGFR3 and EGFR following targeted therapy with erdafitinib or lapatinib, alongside alterations in the basal/luminal cell ratio indicated by increased CD44, CD90, and CD239 marker expressions. Our findings underscore the value of FCM in categorizing BLCa patients based on specific marker expression, supplementing conventional histological analyses to enhance cancer diagnosis, guide personalized therapeutic approaches, and monitor treatment efficacy. Citation Format: Martina Radic, Federico La Manna, Martina Minoli, Panagiotis Chouvardas, George N. Thalmann, Roland Seiler-Blarer, Marianna Kruithof-de Julio, Bernhard Kiss. Stratification of bladder cancer patients with the use of flow cytometry is increasing the diagnostic potential of molecular classifiers [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A023.

Morphologic patterns observed in prostate biopsy cases with discrepant grade group and molecular risk classification.

Molecular-based risk classifier tests are increasingly being utilized by urologists and radiation oncologists to guide clinical decision making. The Decipher prostate biopsy test is a 22-gene RNA biomarker assay designed to predict likelihood of high-grade disease at radical prostatectomy and risk of metastasis and mortality. The test provides a risk category of low, intermediate, or high. We investigated histologic features of biopsies in which the Grade Group (GG) and Decipher risk category (molecular risk) were discrepant. Our institutional urologic outcomes database was searched for men who underwent prostate biopsies with subsequent Decipher testing from 2016 to 2020. We defined discrepant GG and molecular risk as either GG1-2 with high Decipher risk category or GG ≥ 3 with low Decipher risk category. The biopsy slide on which Decipher testing was performed was re-reviewed for GG and various histologic features, including % Gleason pattern 4, types of Gleason pattern 4 and 5, other "high risk" features (e.g., complex papillary, ductal carcinoma, intraductal carcinoma [IDC]), and other unusual and often "difficult to grade" patterns (e.g., atrophic carcinoma, mucin rupture, pseudohyperplastic carcinoma, collagenous fibroplasia, foamy gland carcinoma, carcinoma with basal cell marker expression, carcinoma with prominent vacuoles, and stromal reaction). Follow-up data was also obtained from the electronic medical record. Of 178 men who underwent prostate biopsies and had Decipher testing performed, 41 (23%) had discrepant GG and molecular risk. Slides were available for review for 33/41 (80%). Of these 33 patients, 23 (70%) had GG1-2 (GG1 n = 5, GG2 n = 18) with high Decipher risk, and 10 (30%) had GG ≥ 3 with low Decipher risk. Of the 5 GG1 cases, one case was considered GG2 on re-review; no other high risk features were identified but each case showed at least one of the following "difficult to grade" patterns: 3 atrophic carcinoma, 1 collagenous fibroplasia, 1 carcinoma with mucin rupture, and 1 carcinoma with basal cell marker expression. Of the 18 GG2 high Decipher risk cases, 2 showed GG3 on re-review, 5 showed large cribriform and/or other high risk features, and 10 showed a "difficult to grade" pattern. Of the 10 GG ≥ 3 low Decipher risk cases, 5 had known high risk features including 2 with large cribriform, 1 with IDC, and 1 with Gleason pattern 5. In GG1-2 high Decipher risk cases, difficult to grade patterns were frequently seen in the absence of other known high risk morphologic features; whether these constitute true high risk cases requires further study. In the GG ≥ 3 low Decipher risk cases, aggressive histologic patterns such as large cribriform and IDC were observed in half (50%) of cases; therefore, the molecular classifier may not capture all high risk histologic patterns.

An insight into immunohistochemical profile of triple negative breast carcinoma in a tertiary care center

Introduction: Triple Negative Breast Cancer (TNBC) is a type of breast cancer that fails to express Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER2) in Immunohistochemical analysis. TNBC has a unique molecular profile, aggressive nature and lack of efficient targeted therapies. The aim of the study was to evaluate the expression of basal markers like Epidermal Growth Factor Receptor (EGFR) and Cytokeratin 5/6 (CK 5/6) and to correlate their expression with the clinico-pathological parameters, thus enabling the identification of the aggressive basal like phenotype of TNBC in resource-constrained settings. Methodology: The study was conducted at a South Indian tertiary care centre for 2 years. Data such as age of presentation, tumour measurements, margins and necrosis, numbers, levels and sizes of lymph nodes were collected. Data on histological type and grade, in situ component, lymphovascular and perineural invasion, stromal reaction and lymph node status were recorded. Primary Immunohistochemical studies were performed for the markers ER,PR and HER2.Patients with negative expression for all the three markers were identified. IHC studies for EGFR and Cytokeratin 5/6(CK5/6) were done on tissue blocks and the expression of these two markers were equated with the clinico-pathological parameters. Results: Among the 94 cases, 44 cases (46.8%) expressed CK5/6 and 46 (48.9%) expressed EGFR. A plausible association was noted between the expression of the two markers with age of the patient, tumour size, histological grade and tumour necrosis. Conclusion: Combination of the time honoured gross and histopathological examinations along with the application of basal markers like CK 5/6 and EGFR, helps in identifying the basal-like phenotype with dismal clinical outcome among the triple-negative breast cancers in resource-constrained settings. An insight into such an aggressive tumour, provided by these markers may be critical in designing personalized treatment strategies for such patients.

The Effect of Retinoic Acid on Arsenite-Transformed Malignant UROtsa Bladder Cancer Cells: In Vitro Model of Basal Muscle-Invasive Bladder Cancer.

Bladder cancer (BC) is the eighth most common cause of cancer death in the United States of America. BC is classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Genetically, MIBCs are categorized into the more aggressive basal subtype or less aggressive luminal subtype. All-trans retinoic acid (tretinoin), the ligand for the RAR-RXR retinoic acid receptor, is clinically used as a differentiation therapy in hematological malignancies. This study aims to determine the effects of retinoic acid on arsenite-transformed malignant urothelial cells (UROtsa As), serving as a model for basal muscle-invasive bladder cancer. We treated three independent isolates of arsenite-transformed malignant human urothelial UROtsa cells (UROtsa As) with tretinoin for 48 h. Cell viability, proliferation, and apoptosis were analyzed using crystal violet staining and flow cytometry. mRNA and protein level analyses were performed using RT-qPCR and the Simple Western™ platform, respectively. Tretinoin was found to reduce cell proliferation and urosphere formation, as well as decrease the expression of basal markers (KRT1, KRT5, KRT6, EGFR) and increase the expression of luminal differentiation markers (GATA3, FOXA1). Mechanistically, the antiproliferative effect of tretinoin was attributed to the downregulation of c-myc. Our results suggest that targeting the retinoic acid pathway can diminish the aggressive behavior of basal muscle-invasive urothelial cancer and may enhance patient survival.

The use of cultured human alveolar basal cells to mimic honeycomb formation in idiopathic pulmonary fibrosis

BackgroundHoneycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with a single-or stratified layer of basal cells (BC), or with a bronchiolar-like epithelium composed of basal-, ciliated- and secretory epithelial cells. By using cultured IPF patient-derived alveolar BC, we aimed to establish an in vitro- and in vivo model to mimic HC formation in IPF. We (1) optimized conditions to culture and propagate IPF patient-derived alveolar BC, (2) cultured the cells on an air liquid interface (ALI) or in a three dimensional (3D) organoid model, and (3) investigated the cells` behavior after instillation into bleomycin-challenged mice.MethodsAlveolar BC were cultured from peripheral IPF lung tissue and grown on tissue-culture treated plastic, an ALI, or in a 3D organoid model. Furthermore, cells were instilled into bleomycin-challenged NRG mice. Samples were analyzed by TaqMan RT-PCR, immunoblotting, immunocytochemistry/immunofluorescence (ICC/IF), or immunohistochemistry (IHC)/IF. Mann–Whitney tests were performed using GraphPad Prism software.ResultsCultured alveolar BC showed high expression of canonical basal cell markers (TP63, keratin (KRT)5, KRT14, KRT17), robust proliferation, and wound closure capacity. The cells could be cryopreserved and propagated for up to four passages without a significant loss of basal cell markers. When cultured on an ALI or in a 3D organoid model, alveolar BC differentiated to ciliated- and secretory epithelial cells. When instilled into bleomycin-challenged mice, human alveolar BC cells formed HC-like structures composed of human basal-, and secretory epithelial cells within the mouse parenchyma.ConclusionIPF patient-derived alveolar BC on an ALI, in 3D organoids or after instillation into bleomycin-challenged mice form HC-like structures that closely resemble HC within the IPF lung. These models therefore represent powerful tools to study honeycomb formation, and its potential therapeutic inhibition in IPF.

Maternal suboptimal selenium intake and low-level lead exposure affect offspring’s microglial immune profile and its reactivity to a subsequent inflammatory hit

Micronutrients such as selenium (Se) are essentials since prenatal life to support brain and cognitive development. Se deficiency, which affects up to 1 billion people worldwide, can interact with common adverse environmental challenges including (Pb), exacerbating their toxic effects. Exploiting our recently validated rat model of maternal Se restriction and developmental low Pb exposure, our aims were to investigate: (i) the early consequences of suboptimal Se intake and low-Pb exposure on neuroinflammation in neonates’ whole brains; (ii) the potential priming effect of suboptimal Se and low-Pb exposure on offspring’s glial reactivity to a further inflammatory hit. To these aims female rats were fed with suboptimal (0.04 mg/kg; Subopt) and optimal (0.15 mg/kg; Opt) Se dietary levels throughout pregnancy and lactation and exposed or not to environmentally relevant Pb dose in drinking water (12.5 µg/mL) since 4 weeks pre-mating. We found an overall higher basal expression of inflammatory markers in neonatal brains, as well as in purified microglia and organotypic hippocampal slice cultures, from the Subopt Se offspring. Subopt/Pb cultures were highly activated than Subopt cultures and showed a higher susceptibility to the inflammatory challenge lipopolysaccharide than cultures from the Opt groups. We demonstrate that even a mild Se deficiency and low-Pb exposure during brain development can influence the neuroinflammatory tone of microglia, exacerbate the toxic effects of Pb and prime microglial reactivity to subsequent inflammatory stimuli. These neuroinflammatory changes may be responsible, at least in part, for adverse neurodevelopmental outcomes.

Invasive urothelial carcinoma with chordoid features or abundant myxoid stroma: A reappraisal of morphologic spectrum and risk stratification based on molecular classification

Urothelial carcinoma (UC) with myxoid stroma or chordoid features is a rare diagnosis. We retrospectively collected data from 17 cases of diagnosed UC with myxoid stroma, mucin production, or chordoid features. We aimed to investigate the molecular subtypes of this neoplasm and to assess subtype correlations with clinical outcomes. Immunohistochemical (IHC) staining with a panel composed of markers for basal subtypes (CK5/6, CK14, and CD44) and luminal subtypes (GATA3, FOXA1, and CK20) was performed. Morphologically, all cases included an at least partial conventional UC component, with the first histologic pattern, named as “typical”, characterized by a small- or medium-sized tumor cell nest. The second histologic pattern, named as “chordoid”, was characterized by tumor cells with cording that mimic extra-skeletal myxoid chondrosarcoma or chordoma, and the third histologic pattern, named as “sarcomatoid”, was characterized by non-cohesive spindle tumor cells with a mucin-producing or myxoid stroma background. The “typical” cases showed [CK5/6- CK14- CD44-] [GATA3 + FOXA1 + CK20-] IHC results and was classified as lumina subtype. The “chordoid” cases showed [CK5/6 + CK14 + CD44-] [GATA3- FOXA1- CK20-] IHC results and was classified as basal subtype, and the “sarcomatoid” cases showed [CK5/6- CK14- CD44+] [GATA3- FOXA1- CK20-] IHC results and was “not classified”. All pT3 cases and all cases with lymph node (LN) metastasis belonged to the “sarcomatoid” pattern. All patients who had metastasis or died showed the “chordoid” or “sarcomatoid” morphology. Our findings suggest that UC with myxoid stroma/chordoid features shows characteristic expression of luminal and basal markers and different prognosis according to the morphologic pattern spectrum.

Urothelium-Specific Expression of Mutationally Activated Pik3ca Initiates Early Lesions of Noninvasive Bladder Cancer

Although approximately 70% of bladder cancers are noninvasive and have high recurrence rates, early-stage disease is understudied. The lack of models to validate the contribution of molecular drivers of bladder tumorigenesis is a significant issue. Although mutations in PIK3CA are frequent in human bladder cancer, an invivo model for understanding their contribution to bladder tumorigenesis is unavailable. Therefore, a Upk2-Cre/Pik3caH1047R mouse model expressing one or two R26-Pik3caH1047R alleles in a urothelium-specific manner was generated. Pik3caH1047R functionality was confirmed by quantifying Akt phosphorylation, and mice were characterized by assessing urothelial thickness, nuclear atypia, and expression of luminal and basal markers at 6 and 12 months of age. While at 6 months, Pik3caH1047R mice developed increased urothelial thickness and nuclear atypia, progressive disease was not observed at 12 months. Immunohistochemistry showed urothelium maintained luminal differentiation characterized by high forkhead box A1 (Foxa1) and peroxisome proliferator-activated receptor γ expression. Surprisingly, Pik3caH1047R mice subjected to low-dose carcinogen exposure [N-butyl-N-(4-hydroxybutyl)nitrosamine] exhibited no significant differences after exposure relative to mice without exposure. Furthermore, single-sample gene set enrichment analysis of invasive human tumors showed those with mutant PIK3CA did not exhibit significantly increased phosphatidylinositol 3-kinase/AKT pathway activity compared with wild-type PIK3CA tumors. Overall, these data suggest that Pik3caH1047R can elicit early tumorigenic changes in the urothelium, but progression to invasion may require additional genetic alterations.

Human Umbilical Cord Blood Endothelial Progenitor Cell-Derived Extracellular Vesicles Control Important Endothelial Cell Functions.

Circulating endothelial progenitor cells (EPCs) play a pivotal role in the repair of diseases in which angiogenesis is required. Although they are a potentially valuable cell therapy tool, their clinical use remains limited due to suboptimal storage conditions and, especially, long-term immune rejection. EPC-derived extracellular vesicles (EPC-EVs) may be an alternative to EPCs given their key role in cell-cell communication and expression of the same parental markers. Here, we investigated the regenerative effects of umbilical cord blood (CB) EPC-EVs on CB-EPCs in vitro. After amplification, EPCs were cultured in a medium containing an EVs-depleted serum (EV-free medium). Then, EVs were isolated from the conditioned medium with tangential flow filtration (TFF). The regenerative effects of EVs on cells were investigated by analyzing cell migration, wound healing, and tube formation. We also analyzed their effects on endothelial cell inflammation and Nitric Oxide (NO) production. We showed that adding different doses of EPC-EVs on EPCs does not alter the basal expression of the endothelial cell markers nor change their proliferative potential and NO production level. Furthermore, we demonstrated that EPC-EVs, when used at a higher dose than the physiological dose, create a mild inflammatory condition that activates EPCs and boosts their regenerative features. Our results reveal for the first time that EPC-EVs, when used at a high dose, enhance EPC regenerative functions without altering their endothelial identity.

Enhanced detection and characterization of targetable EGFR and ERBB2 exon20 insertion mutations in urothelial carcinoma.

e16500 Background: EGFR and ERBB2 exon 20 insertion (exon20ins) driver mutations are well described in non-small cell lung cancer due to their resistance to early generation EGFR tyrosine kinase inhibitors. This has led to the development and ultimate approval of exon20ins specific inhibitors, such as mobocertinib. Previous studies have reported EGFR and ERBB2 exon20ins mutations in 0.15%-1.91% of urothelial carcinoma (UC) patients, but this is likely an underrepresentation as indel variants are often under detected by commonly used NGS-variant analysis tools. Additionally, little is known about the biological consequences of EGFR and ERBB2 exon20ins mutations in UC. In this study, we aimed to: 1) characterize the prevalence of EGFR and ERBB2 exon20ins mutations in UC; 2) assess how EGFR and ERBB2 exon20ins mutations impact malignant transformation; and 3) evaluate the sensitivity of UC cell lines with the exon20ins to mobocertinib. Methods: UC patient datasets were realigned using the indel caller, ABRA2, then EGFR and ERBB2 exon20ins frequencies were calculated. Next, parental KT1970 urothelial cells expressing either EGFR WT, ERBB2 WT, or exon20ins mutation constructs (EGFRinsH [insH] or ERBB2insYVMA [YVMA]) were generated and in vitro transformation assays were performed. Anchorage-independent growth was assessed with soft agar assay. Engineered urothelial organoids with various exon20ins variants were created to assess for their ability to differentiate. Finally, sensitivity to erlotinib and mobocertinib was determined. Results: ABRA2 identified a prevalence of ERBB2 and EGFR Exon20ins mutations in 2.5% (7/275) of urothelial tumors in the IMVigor210 patient cohort. ERBB2 insYVMA expressing organoids had the highest basal marker (KRT5) expression in proliferation media which decreased in differentiation media. In the soft agar assay, YVMA cells had a 9.26 fold increase in colony formation (CF) compared to the ERBB2 WT counterpart and insH had a 6.02 fold increase in CF over the EGFR WT counterpart. The IC50 for mobocertinib (M) was significantly lower than erlotinib (E) in the exon20ins cell lines (insH M: 0.123 uM, E:10.69 uM / YVMA M: 0.57 uM, E: 13.54 uM). The difference in IC50 values for mobocertinib vs erlotinib in the WT cells was less pronounced. Conclusions: The prevalence of EFGR and ERBB2 exon20 insertion mutations is likely unrepresented in urothelial carcinoma by current variant calling pipelines. The ERBB2 YVMA mutation showed a more aggressive phenotype and possible basal pattern in vitro which may have implications in terms of prognosis and treatment considerations. Both EGFR and ERBB2 exon20ins urothelial cells were more sensitive to mobocertinib. This study provides evidence that exon20ins specific inhibitors could offer additional targeted therapy options for patients with urothelial cancer who have EGFRinsH and ERBB2insYVMA mutations.

Potency assessment of IFNγ-producing SARS-CoV-2-specific T cells from COVID-19 convalescent subjects.

The development of new therapies for COVID-19 high-risk patients remains necessary to prevent additional deaths. Here, we studied the phenotypical and functional characteristics of IFN-γ producing-SARS-CoV-2-specific T cells (SC2-STs), obtained from 12 COVID-19 convalescent donors, to determine their potency as an off-the-shelf T cell therapy product. We found that these cells present mainly an effector memory phenotype, characterized by the basal expression of cytotoxicity and activation markers, including granzyme B, perforin, CD38, and PD-1. We demonstrated that SC2-STs could be expanded and isolated in vitro, and they exhibited peptide-specific cytolytic and proliferative responses after antigenic re-challenge. Collectively, these data demonstrate that SC2-STs can be a suitable candidate for the manufacture of a T cell therapy product aimed to treat severe COVID-19.

Correlation between elevated tissue level of mercury, selenium, vanadium and barium and expression of luminal (GATA3) and basal (CYTOKERATIN5/6) markers in bladder cancer

Correlation between elevated tissue level of mercury, selenium, vanadium and barium and expression of luminal (GATA3) and basal (CYTOKERATIN5/6) markers in bladder cancer

Impact of exogenous hydrogen peroxide on osteogenic differentiation of broiler chicken compact bones derived mesenchymal stem cells.

The effects of hydrogen peroxide (H2O2) on the osteogenic differentiation of primary chicken mesenchymal stem cells (MSCs) were investigated. MSCs were subjected to an osteogenic program and exposed to various concentrations of H2O2 for 14 days. Results showed that high concentrations of H2O2 (200 and 400nM) significantly increased pro-apoptotic marker CASP8 expression and impaired osteogenic differentiation, as indicated by decreased mRNA expression levels of osteogenesis-related genes and reduced in vitro mineralization. In contrast, long-term H2O2 exposure promoted basal expression of adipogenic markers at the expense of osteogenesis in MSCs during osteogenic differentiation, and increased intracellular reactive oxygen species (ROS) production, as well as altered antioxidant enzyme gene expression. These findings suggest that long-term H2O2-induced ROS production impairs osteogenic differentiation in chicken MSCs under an osteogenic program.

Influence of luminal and basal subtype in prognosis of high-grade non muscle invasive urothelial carcinoma

BackgroundRecent studies have shown that the classification of high-grade urothelial carcinoma non-muscle invasive (HGBCNMI) based on molecular subtypes might be a valuable strategy to identify patients with a worse clinical prognosis. ObjectiveDetermine the effect of the luminal and basal molecular subtype determined by immunistochemical on prognosis in patients with HGBC in Mexican population. MethodsPhenotypes were evaluated by immunohistochemical staining of luminal (GATA3, FOXA1) and basal (CK5/6, CK14) markers in paraffin-embedded tissue samples from 45 patients with a diagnosis of HGBCNMI treated at Instituto Nacional de Cancerología-México (INCan) between 2009 and 2019. The association with prognosis was evaluated using Kaplan-Meier curves and multivariable-adjusted Cox models. ResultsHGBCNMI patients showed mean age of 58.77 years (SD: ±12.08 years). We identified expression of the luminal molecular subtype in 35 cases (77.78 %), and 10 cases (22.22 %) with “combined” expression of the molecular subtype (basal and luminal expression). The combined phenotype was statistically more frequent in metastatic cases (p-value = 0.028). In Kaplan-Meier curves, combined expression of luminal and basal molecular markers was associated with disease progression (p-value = 0.002, log-rank test). Cox regression models confirmed this association, which was not influenced by age (p-value = 0.007) or gender (p-value = 0.007). No association of phenotypes with overall survival (p-value = 0.860) or relapse (p-value = 0.5) was observed. ConclusionThe combined expression of immunohistochemical markers of the luminal and basal subtype might be considered as predictor for disease progression in patients with HGBCNMI in Mexican population.

To study the expression of cytokeratin 5 and 6 in triple negative breast carcinoma: An immunohistochemical study

Introduction: Breast cancer is the second most common cancer developing in women worldwide Triple Negative Breast Carcinomas (TNBC) are regarded as one of the most malignant phenotypes, Immunohisto-chemically these tumors are endocrine receptor negative, HER2 –receptor negative, with expression of basal markers - Cytokeratin 5 /6, carrying a poor prognosis. Effective evaluation of these tumor markers helps in efficient treatment of the patient with all these markers carrying a prognostic significance. The current study helps in identifying Cytokeratin 5/6 as a potential multifaceted biomarker and in identifying novel therapeutic agents for Triple Negative Breast Carcinomas.Materials and Methods: The present immunohistochemical study was conducted on 40 histologically proven cases of triple negative breast carcinoma (TNBC) which were further subjected to Cytokeratin 5/6 expression to determine prognostic and therapeutic value.Result: Cytokeratin 5/6 expression was seen in 16 cases of TNBC, out of 40 histologically proven cases of TNBC. The maximum number of patients of triple negative breast carcinoma were in the age group of 41-50 years comprising 40% of the total cases. % of the total cases. Maximum number of cases of triple negative breast carcinoma were of grade III, constituting 60% followed by grade II Conclusion: According to the current study, Cytokeratin 5/6 positivity has got direct relationship with the grade of the tumour, lymph nodal status and lymphovascular invasion in TNBC cases. TNBC have a grave clinical outcome and decreased 5 year survival rate. Thus, it is highlighted that all TNBC patients should be stained by CK 5/6 and if found positive for CK 5/6 expression it, implies basal like phenotype and aggressive intervention is required. Cytokeratin 5/6 carries a diagnostic as wells as therapeutic importance.

Liposome-based nanoparticles impact on regulatory and effector phenotypes of macrophages and T cells in multiple Sclerosis patients

Current available treatments of Multiple Sclerosis (MS) reduce neuroinflammation acting on different targets on the immune system, but potentially lead to severe side effects and have a limited efficacy in slowing the progression of the disease. Here, we evaluated in vitro the immunomodulatory potential of a new class of nanoparticles – liposomes, constituted by a double-layer of phosphatidylserine (PSCho/PS), and double-faced, with an outer layer of phosphatidylserine and an inner layer of phosphatidic acid (PSCho/PA), either alone or in the presence of the myelin basic protein (MBP) peptide (residues 85–99) (PSCho/PS-MBP and PSCho/PA-MBP). Results showed that PSCho/PS are equally and efficiently internalized by pro- and anti-inflammatory macrophages (M1 and M2 respectively), while PSCho/PA were internalized better by M2 than M1. PSCho/PS liposomes were able to inhibit the secretion of innate pro-inflammatory cytokine IL-1β. PSCho/PS liposomes expanded Tregs, reducing Th1 and Th17 cells, while PSCho/PA liposomes were unable to dampen pro-inflammatory T cells and to promote immune-regulatory phenotype (Treg). The ability of PSCho/PS liposomes to up-regulate Treg cells was more pronounced in MS patients with high basal expression of M2 markers. PSCho/PS liposomes were more effective in decreasing Th1 (but not Th17) cells in MS patients with a disease duration >3 months. On the other hand, down-modulation of Th17 cells was evident in MS patients with active, Gadolinium enhancing lesions at MRI and in MS patients with a high basal expression of M1-associated markers in the monocytes. The same findings were observed for the modulation of MBP-driven Th1/Th17/Treg responses.These observations suggest that early MS associate to a hard-wired pro-Th1 phenotype of M1 that is lost later during disease course. On the other hand, acute inflammatory events reflect a temporary decrease of M2 phenotype that however is amenable to restauration upon treatment with PSCho/PS liposomes. Thus, together these data indicate that monocytes/macrophages may play an important regulatory function during MS course and suggest a role for PSCho/PS and PSCho/PS-MBP as new therapeutic tools to dampen the pro-inflammatory immune responses and to promote its regulatory branch.

P63-expressing Prostate Adenocarcinoma: A Case Study of a Rare Subtype

Abstract Introduction/Objective Prostate acinar adenocarcinoma (PCa) is a well-researched,and often diagnosed entity. A combination of architectural and cytologic characteristics are used in accurately identifying these carcinomas. However, the diagnosis can be challenging when the foci are minute or the cancer presents with unusual features. Ancillary studies aid in these scenarios. The loss of basal cells in adenocarcinoma makes markers targeting these cells desirable. PIN4 cocktail immunohistochemical stain, which combines alpha-methylacyl-coA racemase (AMACR), that is overexpressed in prostate carcinoma, with basal markers high molecular weight cytokeratin and p63, is a popular ancillary study to assist diagnosing demanding cases. Herein, we present a rare, challenging subtype of prostate carcinoma expressing p63. Methods/Case Report A 66-year-old male, with a past medical history of treated HIV and hyperlipidemia, underwent screening biopsies for a PSA of 7.5 ng/mL. Out of 22 parts, three cores showed modest amounts of atypical infiltrating cells. The architecture comprised of ill-defined glands, individual cells and single files of cells. They possessed high nucleocytoplasmic ratio giving a hint of atrophy, with occasional spindling and prominent nucleoli. To add to the challenge, PIN4 immunostaining showed strong nuclear expression of a basal marker with negative AMACR. Due to unusual morphology and basal marker expression, a consideration of basal cell lesion was entertained. However, subsequent staining revealed negative expression of HMWCK, and positive expressions of NKX3.1 and p40. Overall, with this morphology and immunoprofile we diagnosed our case as prostate carcinoma with aberrant expression of p63. Results (if a Case Study enter NA) N/A Conclusion Grading these lesions is controversial as they are considered to carry a better prognosis over conventional acinar adenocarcinoma of similar grade. A distinct etiopathology is suspected as molecular tests for ERG and PTEN are without alterations. Further discussion and characterization of these lesions is warranted as they are infrequently encountered and prone to being overlooked due to their similarities to benign basal cells.