Differences In Biomarker Expression Research Articles

Insulin-like growth factor binding protein 2 predicts course of concomitant right-ventricular dilation and tricuspid regurgitation after transcatheter edge-to-edge mitral valve repair

Abstract Aims Right ventricular (RV) dilation and secondary tricuspid regurgitation (TR) are highly prevalent comorbidities in patients with mitral regurgitation and are associated with a worse prognosis. TR improvement after successful transcatheter edge-to-edge mitral valve repair (M-TEER) is reported regularly, however specific factors contributing to the recovery of RV function and TR after M-TEER are poorly understood. Particularly the role of serum biomarkers in this context is unclear. We aimed to identify specific biomarkers associated with the course of concomitant RV dilation and TR after M-TEER. Methods and Results We prospectively included 242 patients undergoing M-TEER at our center and obtained pre-procedural blood samples for biomarker analysis. A commercial multiplex protein assay (Cardiovascular III, Olink, Uppsala, Sweden) was used to quantify expression levels of 92 defined biomarkers. We then analyzed differences in biomarker expression between patients with and without right ventricular reverse remodeling (RVRR) as defined by improvement of concomitant TR by at least one grade and/or downsizing of the basal RV diameter of at least 10%. 242 patients were included in this analysis, 94 had no/mild concomitant TR at baseline, 148 had moderate/severe TR. RV diameters correlated with TR severity and were significantly larger in patients with moderate/severe TR. At baseline, expression of numerous cardiometabolic biomarkers was significantly higher in patients with moderate/severe TR. These included NT-proBNP (1.9-fold, p < 0.001), matrix metalloproteinase 2 (1.4-fold, p < 0.001) as well as insulin-like growth factor binding proteins 1 (1.7-fold, p < 0.001), -2 (1.3-fold, p = 0.001) and -7 (1.4-fold, p < 0.001). Follow-up analysis was performed in patients with initial moderate/severe TR. The course of TR and RV dimensions three months after M-TEER could be assessed in 99 patients (56 with moderate TR, 43 with severe TR). RVRR had occurred in 50 patients (50.5%). Patients without RVRR had a higher prevalence of chronic pulmonary disease (24.5 vs. 2.2%, p < 0.001). No further differences in clinical characteristics were found. Specifically, RV diameter, systolic pulmonary artery pressure (sPAP) and right-ventricular systolic function did not differ substantially between both groups. Strikingly however, the expression of insulin-like growth factor binding protein 2 (IGFBP-2) was significantly higher in patients, who did not develop RVRR (fold change 1.3 compared to patients with RVRR, p = 0.022). After adjustment for covariates IGFBP-2 was independently associated with absence of RVRR (Odds Ratio 2.078, 95% Confidence interval 1.108 – 3.897, p = 0.023). Conclusions In patients undergoing M-TEER with concomitant moderate or severe TR, numerous cardiometabolic biomarkers including IGFBP-2 are upregulated. Higher levels of IGFBP-2 at baseline are independently associated with persistent TR and/or RV dilation after M-TEER.

Multi-targeted nanoarrays for early broad-spectrum detection of lung cancer based on blood biopsy of tumor exosomes

Liquid biopsies utilizing tumor exosomes offer a noninvasive approach for cancer diagnosis. However, validation studies consistently report that in the early stages of cancer, the secretion of exosomes by cancer cells is relatively low, while bodily fluids exhibit a high abundance of other interfering biomolecules. Additionally, target mutations or differences in biomarker expression among various lung cancer subtypes may contribute to detection failures. In this study, we propose a targeted nanoarray-based early cancer diagnostic approach for multiple subtypes of lung cancer. The targeted nanoarray was constructed by modifying five targeting aptamers onto mesoporous silica nanoparticles through the conjugation between amino and carboxyl groups. The flow cytometry experiments demonstrated the specific recognition ability of the targeted nanoarray to tumor exosomes in PBS, even at biomarker expression levels as low as 1.5 %. Moreover, the TEM results indicated that the targeted nanoarray could isolate tumor exosomes in the blood of tumor-bearing mice. Furthermore, the targeted nanoarray could detect tumor exosomes in the blood of various lung cancer bearing mice, including at the early stages of cancer, which has just been established for 7 days. Overall, the targeted nanoarray represents a promising tool for the early detection of various subtypes of lung cancer.

A Prospective Study of Bevacizumab and Neoadjuvant Concurrent Chemoradiation in Locally Advanced Esophageal Squamous Cell Carcinoma: Paradoxical Increase in Circulating Vascular Endothelial Growth Factor-A and Effect on Outcome

In the prior prospective biomarker study, high serum vascular endothelial growth factor-A (VEGF-A) was associated with a poor prognosis. We conducted a prospective phase II trial of adding Bevacizumab, an anti-VEGF-A monoclonal antibody, to neoadjuvant concurrent chemoradiation (neoCCRT) for patients with locally advanced esophageal squamous cell carcinoma (LA-ESCC). This prospective biomarker study aims to evaluate the expressions of angiogenesis-associated circulating biomarkers before and after neoCCRT and compare clinical outcomes for patients receiving platinum/5-fluorouracil (PF) with or without Bevacizumab. Patients with biopsy-proven resectable non-T4 LA-ESCC were enrolled for the prospective phase II trial investigating PF-neoCCRT plus Bevacizumab (BPF group). A parallel patient cohort enrolled in a prospective biomarker study receiving PF-neoCCRT was included in the present analysis as the control group (PF group). Radiotherapy was delivered with 40 Gy in 20 fractions. All patients had restaging workups after enoCCRT and underwent radical esophagectomy if the disease remained resectable. Serums were collected before and after neoCCRT. The serum concentrations of angiogenesis-associated biomarkers were determined by the multiplex enzyme-linked immunosorbent assay. Survival analyses were performed by the Kaplan-Meier method. The t-test and log-rank test were used to compare differences in biomarker expression and survival between groups. From 2016 to 2019, 43 patients (BPF/PF group: 21/22) were enrolled in the study. Twenty patients in each group had serum samples available for biomarker analysis. 15 out of 21 patients in the BPF group and 20 out of 22 patients in the PF group underwent radical esophagectomy. Six patients in the BPF group and nine patients in the PF group achieved pathological complete responses. The median overall survival for the BPF and PF group was 20.8 months and not-reached, respectively (hazard ratio = 1.33, long rank p = 0.58). In the BPF group, the serum VEGF-A level was significantly increased from an average value of 446 pg/mL to 723 pg/mL after neoCCRT (p = 0.037), while its level was decreased from 815 ng/mL to 380 pg/mL in the PF group (p = 0.104). In addition, the expression value of circulating Angiopoietin-1 was not changed in the BPF group (before neoCCRT, mean value = 828 pg/mL; after neoCCRT, mean value 762 pg/mL, p = 0.67). In contrast, serum Angiopoietin-1 level was reduced from an average value of 659 pg/mL before neoCCRT to 271 pg/mL after neoCCRT (p = 0.002) in the PF group. The addition of Bevacizumab to PF-neoCCRT did not improve pathological or survival outcomes in patients with resectable LA-ESCC. Adding a single dose of Bevacizumab paradoxically increases circulating VEGF-A while maintaining the Angiopoietin-1 serum level after neoCCRT. Further investigation by using additional VEGF-A inhibition may be required to achieve sustained angiogenesis blocked for tumor control.

Biomarkers related to gas embolism: Gas score, pathology, and gene expression in a gas bubble disease model.

Fish exposed to water supersaturated with dissolved gas experience gas embolism similar to decompression sickness (DCS), known as gas bubble disease (GBD) in fish. GBD has been postulated as an alternative to traditional mammals' models on DCS. Gas embolism can cause mechanical and biochemical damage, generating pathophysiological responses. Increased expression of biomarkers of cell damage such as the heat shock protein (HSP) family, endothelin 1 (ET-1) or intercellular adhesion molecule 1 (ICAM-1) has been observed, being a possible target for further studies of gas embolism. The GBD model consisted of exposing fish to supersaturation in water with approximately 170% total dissolved gas (TDG) for 18 hours, producing severe gas embolism. This diagnosis was confirmed by a complete histopathological exam and the gas score method. HSP70 showed a statistically significant upregulation compared to the control in all the studied organs (p <0.02). Gills and heart showed upregulation of HSP90 with statistical significance (p = 0.015 and p = 0.02, respectively). In addition, HSP70 gene expression in gills was positively correlated with gas score (p = 0.033). These results suggest that gas embolism modify the expression of different biomarkers, with HSP70 being shown as a strong marker of this process. Furthermore, gas score is a useful tool to study the abundance of gas bubbles, although individual variability always remains present. These results support the validity of the GBD model in fish to study gas embolism in diseases such as DCS.

Noninvasive identification of molecular biomarkers of hepatocellular carcinoma in HCV-Egyptian patients

BackgroundThis study was performed to investigate the expression of different biomarkers in patients with hepatocellular carcinoma and its connection with detective biomarkers. To achieve this objective, seventy subjects were examined in this study, sub-grouped to forty HCC patients and thirty HCV-affected patients with matched thirty healthy individuals. The study involved several groups of participants who were matched based on their age and gender.MethodsThe expression pattern of biomarkers was monitored by quantitative polymerase chain reaction (qRT-PCR). Finally, we utilized a ROC curve to investigate the predictive accurateness of those distinct biomarkers as well as a traditional tumor marker, AFP, in detecting HCC cases.ResultsThe baseline biomarker expression levels were markedly greater in HCC patients than in those affected by HCV or healthy subjects. We stated that the sensitivity and the specificity of the different biomarkers alone did not improve than that of AFP alone. When comparing AFP with different biomarkers, the diagnostic validity improves only when combining with CK-1.ConclusionsOverall, our results indicate that CK-1 mRNA expression could help as a noninvasive tumor biomarker for HCC prognosis and diagnosis when combining with AFP.

CT measured pulmonary artery to ascending aorta ratio stratified by echocardiographically obtained systolic pulmonary artery pressure values for noninvasive detection of pulmonary hypertension in patients with severe aortic valve stenosis.

Transthoracic echocardiography (TTE) offers a measurement method for the determination of pulmonary hypertension (PH) in patients with severe aortic valve stenosis (AS) with determination of maximal tricuspid regurgitation velocity (TRVmax) and systolic pulmonary artery pressure (sPAP). Radiological parameters for noninvasive detection of PH, most importantly computed tomography (CT) based PA/AA-ratio = ratio of pulmonary artery diameter (PA) and ascending aorta diameter (AA), are also included in the latest ESC guidelines. The aim of the present study was to define cut-off values for PA/AA-ratio taking also into account cardiovascular biomarkers to determine criteria for noninvasive diagnosis of PH. 194 patients with severe AS undergoing transcatheter aortic valve replacement (TAVR) underwent pre-procedural TTE and CT with measurement of PA/AA-ratio. Additionally, common cardiovascular biomarkers were determined. TAVR patients with an sPAP ≥ 40mmHg or a TRVmax ≥ 2.9m/s had a PA/AA-ratio ≥ 0.80 in an AUROC analysis. The cut-off value of ≥ 0.80 resulted in a significantly higher mortality rate (log-rank test: p = 0.034) in these patients in a Kaplan-Meier analysis regarding 1-year survival after TAVR. Significant differences in biomarker expression between patients with a PA/AA-ratio ≥ 0.80 or < 0.80 occurred for BNP (p = 0.001), cTnI (p = 0.032), GDF-15 (p = 0.002) and H-FABP (p = 0.015). PA/AA-ratio ≥ 0.80 is a promising radiological parameter that can provide information about mortality in patients with severe AS undergoing TAVR; combined with biomarkers it may contribute to noninvasive detection of PH in patients with severe AS.

Biomarker Data from the Phase III KATHERINE Study of Adjuvant T-DM1 versus Trastuzumab for Residual Invasive Disease after Neoadjuvant Therapy for HER2-Positive Breast Cancer.

In KATHERINE, adjuvant T-DM1 reduced risk of disease recurrence or death by 50% compared with trastuzumab in patients with residual invasive breast cancer after neoadjuvant therapy (NAT) comprised of HER2-targeted therapy and chemotherapy. This analysis aimed to identify biomarkers of response and differences in biomarker expression before and after NAT. Exploratory analyses investigated the relationship between invasive disease-free survival (IDFS) and HER2 protein expression/gene amplification, PIK3CA hotspot mutations, and gene expression of HER2, PD-L1, CD8, predefined immune signatures, and Prediction Analysis of Microarray 50 intrinsic molecular subtypes, classified by Absolute Intrinsic Molecular Subtyping. HER2 expression on paired pre- and post-NAT samples was examined. T-DM1 appeared to improve IDFS versus trastuzumab across most biomarker subgroups, except the HER2 focal expression subgroup. High versus low HER2 gene expression in residual disease was associated with worse outcomes with trastuzumab [HR, 2.02; 95% confidence interval (CI), 1.32-3.11], but IDFS with T-DM1 was independent of HER2 expression level (HR, 1.01; 95% CI, 0.56-1.83). Low PD-L1 gene expression in residual disease was associated with worse outcomes with trastuzumab (HR, 0.66; 95% CI, 0.44-1.00), but not T-DM1 (HR, 1.05; 95% CI, 0.59-1.87). PIK3CA mutations were not prognostic. Increased variability in HER2 expression was observed in post-NAT versus paired pre-NAT samples. T-DM1 appears to overcome HER2 resistance. T-DM1 benefit does not appear dependent on immune activation, but these results do not rule out an influence of the tumor immune microenvironment on the degree of response.

Sex differences in circulating proteins of patients with rheumatoid arthritis: A cohort study.

Differences in proteomic profiles between men and women may provide insights into the biological pathways that contribute to known sex differences in rheumatoid arthritis (RA). Studies focusing on sex differences in circulating proteins in RA patients are scarce. Our objective was to investigate the sex differences in circulating proteins of RA patients. Cohort study enrolling 399 RA patients. Ninety-four circulating protein-biomarkers (92CVDIIOlink® +troponin-T+C-reactive protein) were measured. Clinical, demographic, and echocardiographic characteristics were compared between men and women. Sex differences in biomarker expression were assessed using regression modeling. In all, 306 (76.7%) patients were women. Compared with men, women had less visceral fat, smoked less, had diabetes and chronic obstructive pulmonary disease less frequently, and expressed more fatigue, anxiety, and depression. The association with cardiovascular outcomes did not differ between sexes. After adjusting for potential confounders, women expressed higher levels of circulating proteins related to adipokine signaling and vascular function (eg, leptin and vascular endothelial growth factor), whereas men expressed higher levels of circulating proteins related to extracellular matrix organization and inflammation (eg, matrix metalloproteinase-2 and C-reactive protein). These results were not found in patients without RA. Sex differences in circulating proteins reflect distinct pathways implicated in the pathogenesis of RA, including inflammation, adiposity, angiogenesis, and extracellular matrix organization. These findings may help further investigations into factors underlying sex-based differences and allow future studies focused on sex-specific personalized treatment approaches in RA. gov ID: NCT03960515.

Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis.

Investigating cellular and vesicular heterogeneity in breast cancer remains a challenge, which encourages the development of controllable in vitro systems that mimic the tumor microenvironment. Although three-dimensional cell culture better recapitulates the heterogeneity observed in tumor growth and extracellular vesicle (EV) biogenesis, the physiological relevance is often contrasted with the control offered by two-dimensional cell culture. Therefore, to challenge this misconception we developed a novel microfluidic system harboring highly tunable three-dimensional EV microbioreactors (EVμBRs) to model micrometastatic EV release in breast cancer while capitalizing on the convenient, low-volume, and sterile interface provided by microfluidics. The diameter and cellular occupancy of the EVμBRs could be precisely tailored to various configurations, supporting the formation of breast cancer tumor spheroids. To immobilize the EVμBRs within a microchannel and facilitate EV extraction, oxygen inhibition in free-radical polymerization was repurposed to rapidly generate two-layer hydrodynamic traps in situ using a digital-micromirror device (DMD)-based ultraviolet (UV) projection system. Breast cancer tumor spheroid-derived EVs were harvested with as little as 20 μL from the microfluidic system and quantified by single-EV immunofluorescence for CD63 and CD81. Despite the low-volume extraction, differences in biomarker expression and coexpression of the tetraspanins on single EVs were observed. Furthermore, the EVμBRs were capable of recapitulating heterogeneity at a cellular and vesicular degree, indicating the utility and robustness of the microfluidic system to investigate physiologically relevant EVs in breast cancer and other disease models.

A Study of Prognostic Factors in Young Patients With Non-HPV Oral Cancer in Central Europe.

The etiological factors of squamous cell carcinomas of the head and neck have been well known for a long time. It is also well known that the incidence of oral cancer diagnosed in younger patients is on the rise. Due to the young age of these patients, the increase in the number of these cases and the fact that many of them neither smoke nor drink alcohol it has been suggested that other factors might be at play in the carcinogenesis of oral cancer. Thus, along the classic etiological factors of smoking and alcohol abuse certain molecular marker anomalies and the human papilloma virus (HPV) have emerged as potential factors. The aim of the present study is to verify the potential prognostic factors and to map the differences in biomarker expression between the young and the old patient groups. In the present study the immunohistochemical profile of samples obtained from oral squamous cell carcinomas was studied and compared with various clinico-pathological parameters. In 88 samples the expressions of p16, p53, Ki67, EGFR were studied with a tissue microarray technique under standard reaction conditions as well as the detection and typing of HPV infection with the Full Spectrum HPV DNA method. The biomarker expression profile of young patients with oral squamous cell carcinoma was compared to that of older patients (above 50). A significant difference was found between the immunohistochemical profile of the young and old patient groups in p16, Ki67 expression. The overall survival and progression free survival were influenced by p16 expression in young age.

Quantitative Analysis of Multiplexed Immunofluorescence in T-Cell Lymphoma

IntroductionImmunohistochemistry provides biologically and clinically useful information on protein expression and cellular localization in cancer cells, but quantification of the expression by visual scores on light microscopy is subjective and suffers from poor reproducibility. There is increasing need for multiplex immunohistochemistry to study the co-localization of antigens in cell of interest. However, interpretation and quantification of co-localizing signals using conventional IHC is challenging. Novel methods in automated microscopy using machine learning algorithms have been utilized in epithelial cancers to quantitate the signals from immunohistochemistry. However, these have not been well tested in lymphoid malignancies, where IHC plays a critical role in the diagnosis and classification. Here we describe the application of quantitative analysis of immunofluorescence in lymphomas, using an example of a T- cell lymphoma, which can be challenging in terms of diagnosis and treatment.MethodsWe developed a multiplexed panel of immunofluorescent stains including CD4, CD8 and CD20 (T and B-lymphocyte markers) along with T-follicular helper markers (TFH) such as BCL6 and PD1. These were optimized and validated using benign tonsillar tissue to demonstrate appropriate cellular localization of each of these markers. (Figure1.) We then show the applicability of such a multiplexed panel in Angioimmunoblastic T-cell Lymphoma (AITL), a peripheral T-cell lymphoma characterized by cellular heterogeneity in the tumor microenvironment which often impedes IHC interpretation and quantification (figure not shown)Results and DiscussionIn AITL, we generated a tissue map which allows visualization of the spatial relationship of the different lymphocyte subsets and the quantification of TFH markers in tumor and non-tumor cell populations. We demonstrate the feasibility of an automated and quantitative imaging of this multiplexed panel of stains in formalin-fixed paraffin embedded tissue sections, which allows the study of the distribution of tumor cells in relationship to surrounding immune cells and the quantification of the expression of different biomarkers of interest in tumor compared to non-tumor cells in the microenvironment. Quantitative multiplex immunofluorescence and/or immunohistochemistry is likely to improve diagnostic accuracy and reproducibility compared to visual scoring in conventional single marker IHC. Furthermore, the ability to quantify the expression of multiple immune markers in tumor and surrounding immune cells and correlation with treatment response will play a vital role in the selection of patients for immunotherapy (such as anti PD1 or anti CD30 antibodies) which is becoming an increasingly important modality in lymphoma treatment.Figure legend. (Figure 1)Multiplexed composite immunofluorescence of CD20, CD8, CD4 and BCL6 in benign tonsil tissue (A). Unmixed images of CD20 (B, green, cell membrane), CD8 (C, magenta, cell membrane), CD4 (D, yellow, cell membrane), BCL6 (E, red, nuclear) and CD4/BCL6 (F) demonstrating the normal distribution of B and T cells and localization of BCL6-positive cells within germinal centres. The number of CD4-positive (G, red), BCL6-positive (H, green) and CD4/BCL6 double positive cells (I, yellow) are obtained after the images are segmented and scored using the InForm 2.0 software. [Display omitted] DisclosuresChng:Janssen China R&D: Research Funding.

Methods to Determine and Analyze the Cellular Spatial Distribution Extracted From Multiplex Immunofluorescence Data to Understand the Tumor Microenvironment.

Image analysis using multiplex immunofluorescence (mIF) to detect different proteins in a single tissue section has revolutionized immunohistochemical methods in recent years. With mIF, individual cell phenotypes, as well as different cell subpopulations and even rare cell populations, can be identified with extraordinary fidelity according to the expression of antibodies in an mIF panel. This technology therefore has an important role in translational oncology studies and probably will be incorporated in the clinic. The expression of different biomarkers of interest can be examined at the tissue or individual cell level using mIF, providing information about cell phenotypes, distribution of cells, and cell biological processes in tumor samples. At present, the main challenge in spatial analysis is choosing the most appropriate method for extracting meaningful information about cell distribution from mIF images for analysis. Thus, knowing how the spatial interaction between cells in the tumor encodes clinical information is important. Exploratory analysis of the location of the cell phenotypes using point patterns of distribution is used to calculate metrics summarizing the distances at which cells are processed and the interpretation of those distances. Various methods can be used to analyze cellular distribution in an mIF image, and several mathematical functions can be applied to identify the most elemental relationships between the spatial analysis of cells in the image and established patterns of cellular distribution in tumor samples. The aim of this review is to describe the characteristics of mIF image analysis at different levels, including spatial distribution of cell populations and cellular distribution patterns, that can increase understanding of the tumor microenvironment.

SV40T reprograms Schwann cells into stem-like cells that can re-differentiate into terminal nerve cells.

The specific effect of SV40T on neurocytes has seldom been investigated by the researchers. We transfected Schwann cells (SCs) that did not have differentiation ability with MPH 86 plasmid containing SV40T, in order to explore the effects of SV40T on Schwann cells. SCs were transfected with MPH 86 plasmid carrying the SV40T gene and cultured in different media, and also co-cultured with neural stem cells (NSCs). In our study, SCs overexpressing SV40T were defined as SV40T-SCs. The proliferation of these cells was detected by WST-1, and the expression of different biomarkers was analyzed by qPCR and immunohistochemistry. SV40T induced the characteristics of NSCs, such as the ability to grow in suspension, form spheroid colonies and proliferate rapidly, in the SCs, which were reversed by knocking out SV40T by the Flip-adenovirus. In addition, SV40T up-regulated the expressions of neural crest-associated markers Nestin, Pax3 and Slug, and down-regulated S100b as well as the markers of mature SCs MBP, GFAP and Olig1/2. These cells also expressed NSC markers like Nestin, Sox2, CD133 and SSEA-1, as well as early development markers of embryonic stem cells (ESCs) like BMP4, c-Myc, OCT4 and Gbx2. Co-culturing with NSCs induced differentiation of the SV40T-SCs into neuronal and glial cells. SV40T reprograms Schwann cells to stem-like cells at the stage of neural crest cells (NCCs) that can differentiate to neurocytes.

Markers of steroid receptor, kinase signalling pathways and Ki-67 expression in relation to tamoxifen sensitivity and resistance

Background: It remains clinically important to identify ER positive breast cancers likely to respond to tamoxifen (TAM) and so we aimed to select a group of biomarkers able to predict response. We also assessed whether data from different sample types [tumor microarrays (TMAs) and core biopsies] or tumor sites could be combined for biomarker studies. Methods: A total of 123 endocrine treatment naive patients with known ER and HER2 status treated with TAM had paraffin-embedded tumor tissue available either as TMAs (n=102) or core biopsies (n=21). TMA cores were collected from three different tumor sites, two central and one peripheral. Ten biomarkers were evaluated by immunohistochemistry, for % positivity and/or H-Score, comprising: ER, HER2, Ki67, phosphorylated forms of ER (Ser118), IGF1R, PRAS40, Akt & MAPK (ERK1/2), and PTEN & androgen receptor expression (AR). Each tumor was analysed for Akt1 E17K somatic mutation using BEAMing technology. Patient outcome was assessed by clinical benefit (CB) rate & survival analyses [time to progression (TTP) and time to death (TTD)]. Results: There was no significant difference in % positivity or H-Score between central & peripheral tumor sites for all biomarkers examined. After False Discovery Rate (FDR) correction differences (P<0.05) were observed between the two central samples only for HER2 & pER118 and pPRAS40. However, differences in biomarker expression were common between core biopsies and TMAs. Only 2/123 (1.6%) tumors had Akt1 E17K mutations. Univariate and multivariate analyses identified that lower levels of PTEN and higher levels of Ki-67 (% positivity) were predictive of poor outcome (TTP & TTD) following TAM. Higher ER. lower Ki-67 and AR/ER ratio <2 predicted increased CB rate. Conclusions: There were few differences in marker expression between TMAs from different intratumoral sites. More marked differences between TMAs and core biopsies suggest caution if combining such datasets. Loss of PTEN, a key regulator of the PI3K/Akt pathway, was the only RTK/kinase signaling biomarker related to poorer clinical outcome. PTEN along with ER & lower Ki-67 proved the most predictive markers for better outcome (TTP & TTD and/or CBR) following TAM treatment. Keywords: ER+ breast cancer; Akt pathway; tamoxifen

Hypercapnia Exacerbates the Blood-Brain Barrier Disruption Via Promoting HIF-1a Nuclear Translocation in the Astrocytes of the Hippocampus: Implication in Further Cognitive Impairment in Hypoxemic Adult Rats.

Hypercapnia in combination with hypoxemia is usually present in severe respiratory disease in the intensive care unit (ICU) and can lead to more severe cognitive dysfunction. Increasing evidence has indicated that the compromised blood–brain barrier (BBB) in the hippocampus in hypoxemia conditions can result in cognitive dysfunction. However, the role and underlying mechanism of hypercapnia in the BBB disruption remains poorly known. A rat model of hypercapnia was first established in this study by intubation and mechanical ventilation with a small-animal ventilator. After this, the cognitive function of the experimental rats was assessed by the Morris water maze test. The BBB permeability was evaluated by the Evans Blue (EB) test and brain water content (BWC). Western blot analysis was carried out to detect the protein expressions of total and nuclear hypoxia-inducible factor-1α (HIF-1α), matrixmetalloproteinase-9 (MMP-9) and Aquaporins-4 (AQP-4) in the hippocampus tissue. Double immunofluorescence further verified the protein expression of different biomarkers was localized in the astrocytes of the hippocampus. Hypercapnia alone did not disrupt the BBB, but it could further enhance the BBB permeability in hypoxemia. Concomitantly, up-regulation of nuclear HIF-1α, AQP-4, MMP-9 protein expression along with increased degradation of the occludin and claudin-5 proteins was found in the hypercapnia rat model, while the total HIF-1α remained unchanged. Interestingly, these changes were independent of the acidosis induced by hypercapnia. Of note, after premedication of 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α nuclear translocation), the disrupted BBB could be restored resulting in improvement of the cognitive impairment. Meanwhile, accumulation of nuclear HIF-1α, protein expression of AQP-4 and MMP-9 and protein degradation of the occludin and claudin-5 were decreased. Thus, our study demonstrated that hypercapnia can further disrupt the BBB through promoting HIF-1α nuclear translocation and up-regulation of AQP-4 and MMP-9 in hypoxemia. It is therefore suggested that the cascade of hypercapnia-induced nuclear HIF-1α protein translocation in hypoxia-activated astrocytes may be a potential target for ameliorating cognitive impairment.Electronic supplementary materialThe online version of this article (10.1007/s11064-020-03038-7) contains supplementary material, which is available to authorized users.

722 Type 2 helper T-cell (Th2) effects of crisaborole versus 3 topical corticosteroids (TCSs) with a range of potencies: An ex vivo human skin model study

The Th2 pathway plays a central role in the pathophysiology of atopic dermatitis (AD). Crisaborole ointment, 2%, is a nonsteroidal phosphodiesterase 4 inhibitor for the treatment of mild-to-moderate AD. An ex vivo human skin model with Th2 cytokine and T-cell stimulation was used to compare the T-cell and Th2 cytokine activation effects of crisaborole with TCSs of varying potencies (hydrocortisone cream, 1% [lowest], hydrocortisone butyrate cream, 0.1% [lower-medium], and betamethasone valerate cream, 0.1% [medium]). Test articles were applied topically to human skin explants from 4 healthy (ie, non-AD) donors (5 replicates each) 16 hours prior to Th2 activation via a proprietary Th2 stimulation cocktail. To determine the effect of TCS application on the Th2-mediated inflammatory state of the tissue, 4 AD-associated biomarkers were chosen. RNA was isolated 24 hours after stimulation and quantitative-PCR was used to measure the gene expression of 4 biomarkers (interleukin [IL]-13, IL-31, interferon [INF]-γ, and matrix metallopeptidase 12 [MMP12]) in the explants. Topical application of crisaborole or any of the 3 TCSs resulted in a significant reduction (>50%) in activity for all 4 AD-associated biomarkers. There were no differences in biomarker expression between crisaborole and the 3 TCSs except for a numerically greater reduction in MMP12 for crisaborole compared with hydrocortisone cream, 1%.

Immunohistochemical expression of podoplanin in patients with lichen planus: a cross sectional study

Event Abstract Back to Event Immunohistochemical expression of podoplanin in patients with lichen planus: a cross sectional study Ilaria Currò1*, Andrea Gabusi1, Dora Servidio1, Roberto Rossi1, Laura Felicetti1 and Davide Bartolomeo Gissi1 1 University of Bologna, Department of Biomedical and Neuromotor Sciences, Section of Oral Sciences, Italy Aim: The World Health Organization had included Lichen Planus (LP) among the risk conditions for malignant transformation, even if it is difficult to evaluate the real risk of malignant transformation in LP patients and it is difficult to identify a predictive marker of progression risk to Oral Squamous Cell Carcinoma (OSCC). Immunohistochemical analysis is a simple and reliable method which allows to evaluate the expression of a biomarker which may be correlated with an increased risk of malignant transformation in potentially malignant lesions of the oral cavity. Recently, few authors identified a relationship between dysplasia and podoplanin overexpression in patients with Oral Leukoplakia and proposed podoplanin as a predictive marker to identify Oral Leukoplakias likely to develop into OSCC. In the present cross-sectional study, we investigated a group of consecutive LP to evaluate a possible relationship between altered expression of podoplanin and the presence of dysplasia, and to compare the results with those obtained with other clinical and immunoistochemical variables. Materials and Methods. The population study consisted of 38 consecutive patients with a clinical and histological diagnosis of LP. The 38 patients were divided into 2 groups: 23 lichenoid lesions in which there was no dysplasia and 15 lichenoid lesions in which dysplasia was present with different degrees (11 LP with presence of mild dysplasia and 4 with presence of moderate dysplasia). Immunoistochemical expression of podoplanin, p53 and Ki67 was analyzed. Lesions with podoplanin expression in the suprabasal layer of the epithelium (≥ pattern 2 in Kawaguchi classification) were considered as positive, whereas the cut off for both p53 and Ki67 overexpression was set to 20% of positive cells. Statistical analysis: A multiple logistic regression model with stepwise selection was fitted to describe the relation between presence/absence of dysplasia (dependent variable) and the following independent variables: Ki67 (<20 vs >20), p53 (<20 vs > 20), podoplanin (<2 vs > 2), patient age (< 65 vs > 65), gender, smoking behavior, site, clinical appearance. A p value < .05 was considered statistically significant in all analyses. Results. The results of multiple logistic regression showed that podoplanin expression was the only variable significantly related to the presence of dysplasia (T=4.68; p < .01). Indeed, Podoplanin expression was observed in 5 of 23 cases of lichenoid lesions without dysplasia and in 12 of 15 cases of lichenoid lesions with dysplasia (Chi 12,465; p<.05). Statistical analysis didn’t find a significant relationship between the presence of dysplasia in LP and overexpression of ki67 (Chi 1,870; p=0,1715), overexpression of p53 (Chi 0,035; p=0,8510) and other clinical variables. Discussion. An altered expression of different biomarkers in LP patients may be uninformative about the risk of OSCC progression, because it might be influenced by tissue inflammation. The present study shows that podoplanin is a promising specific marker related to the presence of dysplasia in LP. If these preliminary results are confirmed in larger prospective cohort studies with an adequate follow up, immunohistochemical expression of podoplanin could be proposed in clinical practice as a marker to discriminate LPs at risk of developing cancer.

Microchannel device for proteomic analysis of migrating cancer cells

Tissue invasion and metastasis are leading causes of death among cancer patients due to cells escaping from the primary tumor and invading distant sites. To better understand these phenomena and develop efficient therapeutic regimens against different types of malignancies, there is a need for exclusive cellular and molecular examination of migrating cells. In this study, aggressive brain cancer cells, G55, migrating through confined microchannels were directly extracted and used for subsequent proteomic analysis via Western Blot and/or immunostaining quantification. The method was based on an engineered Polydimethylsiloxane microchannel platform that facilitated the exclusive extraction of migrating cells and their contents while preventing non-migrating (or proliferating—denoted as 2D) cell contamination. The migrating cells in physical confinement of the microchannels were exclusively examined for their protein expression. They were found with increased expression of Vimentin, approximately 2.5-fold higher than 2D cells. On the other hand, the migrating cells showed significantly decreased β3-Tubulin and Met signal compared to 2D cells. The differences in biomarker expression between migrating cells and non-migrating cells revealed by this study provided an insight into key features of cancer invasion and metastasis. The successful outcome of this research suggests improved targets for ceasing different types of malignancies.

Abstract OT1-04-02: Mifepristone treatment for breast cancer patients expressing levels of progesterone receptor isoform A (PRA) higher than those of isoform B (PRB)

Abstract Seventy percent of breast cancers express estrogen (ER) and progesterone receptors (PR) and respond to antiestrogen therapies. Emerging evidence from experimental studies and human epidemiology, points to a relevant role for progestins in breast carcinogenesis and cancer growth. Others and we have proposed that there is a role for antiprogestins in the therapeutic armamentarium, but the challenge remains to identify which patients would benefit from targeting the PR in addition to ER. Preclinical data indicates that antiprogestins block cell proliferation and increase apoptosis only in ER+ breast cancers expressing levels of PRA higher than those of PRB. The aim of this study is to evaluate the therapeutic effects of Mifepristone (MFP) on breast cancer patients selected by their PRA/PRB isoform ratio, for 14 days in between core biopsy and surgery (MIPRA trial ClinicalTrials.gov Identifier: NCT02651844). Methods: This is an open label, interventional with single group assignment study. We perform core biopsies on menopausal patients with clinically palpable breast cancers larger than 1.5 cm to confirm diagnosis. We will assess the PRA/PRB ratio by western blotting in frozen samples and total PR in formalin-fixed samples by immunohistochemistry (IHC). Twenty eligible PR+ patients (PR &amp;gt; 50 %) with PRA/PRB ≥1.5 who have signed consent forms, and meet the inclusion criteria will be recruited. Patients will be treated for 14 days with MFP p.o 200 mg. Surgery will be performed on day 15. Samples will be frozen for molecular studies or fixed for IHC. The primary outcome is the evaluation of Ki-67 staining pre- and post treatment. Secondary outcomes include comparatively expression of proliferation/apoptosis/PR signaling markers in core and surgical biopsy samples. Other pre-specified outcomes include molecular profiling, study of liquid biopsies, mammography, and ultrasound studies. Wilcoxon signed rank test will be used to evaluate differences in biomarker expression between core biopsy and surgical samples of each patient. Citation Format: Liguori M, Lanari C, Gass H, Rojas P, Elia A, Martinez Vazquez P, Burruchaga J, Gonzalez P, Caillet Bois I, Ventura C, San Martin G, Castets A, Lovisi S, Acosta Haab G, Lamb C, Fabris V, Novaro V, Molinolo A. Mifepristone treatment for breast cancer patients expressing levels of progesterone receptor isoform A (PRA) higher than those of isoform B (PRB) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT1-04-02.

Genetic Biomarker Prevalence Is Similar in Fecal Immunochemical Test Positive and Negative Colorectal Cancer Tissue

Fecal immunochemical test (FIT) screening detects most asymptomatic colorectal cancers. Combining FIT screening with stool-based genetic biomarkers increases sensitivity for cancer, but whether DNA biomarkers (biomarkers) differ for cancers detected versus missed by FIT screening has not been evaluated in a community-based population. To evaluate tissue biomarkers among Kaiser Permanente Northern California patients diagnosed with colorectal cancer within 2years after FIT screening. FIT-negative and FIT-positive colorectal cancer patients 50-77years of age were matched on age, sex, and cancer stage. Adequate DNA was isolated from paraffin-embedded specimens in 210 FIT-negative and 211 FIT-positive patients. Quantitative allele-specific real-time target and signal amplification assays were performed for 7 K-ras mutations and 10 aberrantly methylated DNA biomarkers (NDRG4, BMP3, SFMBT2_895, SFMBT2_896, SFMBT2_897, CHST2_7890, PDGFD, VAV3, DTX1, CHST2_7889). One or more biomarkers were found in 414 of 421 CRCs (98.3%). Biomarker expression was not associated with FIT status, with the exception of higher SFMBT2_897 expression in FIT-negative (194 of 210; 92.4%) than in FIT-positive cancers (180 of 211; 85.3%; p=0.02). There were no consistent differences in biomarker expression by FIT status within age, sex, stage, and cancer location subgroups. The biomarkers of a currently in-use multi-target stool DNA test (K-ras, NDRG4, and BMP3) and eight newly characterized methylated biomarkers were commonly expressed in tumor tissue specimens, independent of FIT result. Additional study using stool-based testing with these new biomarkers will allow assessment of sensitivity, specificity, and clinical utility.