ELISA-like QDB method to meet the emerging need of Her2 assessment for breast cancer patients.

Inherent issues of subjectivity and inconsistency have long plagued immunohistochemistry (IHC)-based Her2 assessment, leading to the repeated issuance of guidelines by the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) for its standardization for breast cancer patients. Yet, all these efforts may prove insufficient with the advent of Trastuzumab deruxtecan (T-Dxd), a drug with the promise to expand to tumors traditionally defined as Her2 negative (Her2-). In this study, we attempted to address these issues by exploring an ELISA-like quantitative dot blot (QDB) method as an alternative to IHC. The QDB method has been used to measure multiple protein biomarkers including ER, PR, Ki67, and cyclin D1 in breast cancer specimens. Using an independent cohort (cohort 2) of breast cancer formalin-fixed paraffin-embedded (FFPE) specimens, we validated cutoffs developed in cohort 1 (Yu et al., Scientific Reports 2020 10:10502) with overall 100% specificity (95% CI: 100-100) and 97.56% sensitivity (95% CI: 92.68-100) in cohort 2 against standard practice with the dichotomized absolutely quantitated values. Using the limit of detection (LOD) of the QDB method as the putative cutoff point, tumors with no Her2 expression were identified with the number comparable to those of IHC 0. Our results support further evaluation of the QDB method as an alternative to IHC to meet the emerging need of identifying tumors with low Her2 expression (Her2-low) in daily clinical practice.

Dramatic Plasmon Response to the Charge-Density-Wave Gap Development in 1T-TiSe_{2}.

1T-TiSe_{2} is one of the most studied charge density wave (CDW) systems, not only because of its peculiar properties related to the CDW transition, but also due to its status as a promising candidate of exciton insulator signaled by the proposed plasmon softening at the CDW wave vector. Using high-resolution electron energy loss spectroscopy, we report a systematic study of the temperature-dependent plasmon behaviors of 1T-TiSe_{2}. We unambiguously resolve the plasmon from phonon modes, revealing the existence of Landau damping to the plasmon at finite momentums, which does not support the plasmon softening picture for exciton condensation. Moreover, we discover that the plasmon lifetime at zero momentum responds dramatically to the band gap evolution associated with the CDW transition. The interband transitions near the Fermi energy in the normal phase are demonstrated to serve as a strong damping channel of plasmons, while such a channel in the CDW phase is suppressed due to the CDW gap opening, which results in the dramatic tunability of the plasmon in semimetals or small-gap semiconductors.

Validation of absolutely quantitated Ki67 and cyclinD1 protein levels for prognosis of Luminal-like breast cancer patients.

AIMS: To translate a clinical research finding into daily clinical practice requires well-controlled clinical trials. We have demonstrated the usage of absolute quantitation of Ki67 and cyclinD1 protein levels to improve prognosis of Luminal-like patients based on overall survival (OS) analysis of a cohort of 155 breast cancer specimens (cohort 1). However, this finding is considered the D level of evidence (LOE) to require subsequent validation before it may be used in daily clinical practice. To set the stage for future clinical trials, our findings were validated through OS analysis of an independent cohort (cohort 2) of 173 Luminal-like patients. METHODS: Both Ki67 and cyclinD1 levels were measured absolutely and quantitatively using the Quantitative Dot Blot (QDB) method in cohort 2. The proposed cutoffs for both biomarkers from cohort 1 were re-evaluated in cohort 2 and in the merged cohort of 1 and 2, respectively, through univariate, multivariate and Kaplan-Meier survival analysis. RESULTS: The proposed cutoffs of 2.31 nmol/g for Ki67 and 0.44 µmol/g for cyclinD1 were validated as effective cutoffs in cohort 2 and the merged cohort through OS analysis. The combined use of both biomarkers allowed us to identify patients with both biomarker levels below the cutoffs (59.3%) with10-year survival probability (SP) of 89%, in comparison to those above the cutoffs (8.3%) with 8 year SP of 28% through OS analysis in the merged cohort. CONCLUSIONS: This study validated our findings that absolute quantitation of Ki67 and cyclinD1 allows effective subtyping of luminal-like patients. It sets the stage for prospective or prospective-retrospective clinical studies.

Can nutrition interventions tackle the global insulin affordability via improving diabetes management and reducing insulin demand?

Diabetes, a global health concern, requires insulin therapy. As insulin demand and prices rise dramatically, insulin affordability has increasingly become an issue facing patients with diabetes worldwide. To cut insulin costs, many patients ration their supply, which may have dire health consequences. This particularly affects lower-income populations, who are often forced to choose between purchasing their medications or paying for other necessities. Nutrition might be one solution for this. This commentary aims to provide comprehensive insight with historical context into intersectional components of diabetes in the global arena through analyses of insulin affordability, coupled with the critical role of nutrition intervention after searching the PubMed for relevant articles. More studies in personalized nutrition, supplementations, and dietary behaviors may develop evidence-based nutrition interventions to control diabetes. We argue that alongside price regulation, a greater focus to nutrition to address issues of food insecurity and food assistance programs may help to improve insulin affordability.

3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways.

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A "seesaw" relationship between ER/PR and Her2 pathways was observed in ER-PR-Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER-PR-Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER-PR space. Conclusions: The unique distribution of specimens in ER-PR-Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

Combined Use of cyclinD1 and Ki67 for Prognosis of Luminal-Like Breast Cancer Patients.

BACKGROUND: Ki67 is a biomarker of proliferation to be used in immunohistochemistry (IHC)-based surrogate assay to determine the necessity of cytotoxic therapy for Luminal-like breast cancer patients. cyclinD1 is another frequently used biomarker of proliferation. A retrospective study was performed here to investigate if these two biomarkers may be combined to improve the prognosis of Luminal-like patients. METHODS: Both Ki67 and cyclinD1 protein levels were measured absolutely and quantitatively using Quantitative Dot Blot method in 143 Luminal-like specimens. Optimized cutoffs for these two biomarkers were developed to evaluate their prognostic roles using Kaplan-Meier overall survival (OS) analysis. RESULTS: cyclinD1 was found as an independent prognostic factor from Ki67 in univariate and multivariate OS analyses. At optimized cutoffs (cyclinD1 at 0.44 µmol/g and Ki67 at 2.31 nmol/g), the subgroup with both biomarkers below the cutoffs (n = 65) had 10-year survival probability at 90% in comparison to those with both biomarkers above the cutoffs (n = 18) with 8-year survival probability at 26% (log-rank test, p <0.0001). This finding was used to modify the surrogate assay using IHC-based cyclinD1 scores, with p-value decreased from 0.031 to 0.00061 or from 0.1 to 0.02, when the Ki67 score of 14 or 20% was used as cutoff, respectively, in the surrogate assay. CONCLUSION: The current study supports the prospective investigation of cyclinD1 relevance in the clinic.

Improving Prognosis of Surrogate Assay for Breast Cancer Patients by Absolute Quantitation of Ki67 Protein Levels Using Quantitative Dot Blot (QDB) Method.

BACKGROUND: Immunohistochemistry (IHC)-based surrogate assay is the prevailing method in daily clinical practice to determine the necessity of chemotherapy for Luminal-like breast cancer patients worldwide. It relies on Ki67 scores to separate Luminal A-like from Luminal B-like breast cancer subtypes. Yet, IHC-based Ki67 assessment is known to be plagued with subjectivity and inconsistency to undermine the performance of the surrogate assay. A novel method needs to be explored to improve the clinical utility of Ki67 in daily clinical practice. MATERIALS AND METHODS: The Ki67 protein levels in a cohort of 253 specimens were assessed with IHC and quantitative dot blot (QDB) methods, respectively, and used to assign these specimens into Luminal A-like and Luminal B-like subtypes accordingly. Their performances were compared with the Kaplan-Meier, univariate, and multivariate survival analyses of the overall survival (OS) of Luminal-like patients. RESULTS: The surrogate assay based on absolutely quantitated Ki67 levels (cutoff at 2.31 nmol/g) subtyped the Luminal-like patients more effectively than that based on Ki67 scores (cutoff at 14%) (Log rank test, p = 0.00052 vs. p = 0.031). It is also correlated better with OS in multivariate survival analysis [hazard ratio (HR) at 6.89 (95% CI: 2.66-17.84, p = 0.0001) vs. 2.14 (95% CI: 0.89-5.11, p = 0.087)]. CONCLUSIONS: Our study showed that the performance of the surrogate assay may be improved significantly by measuring Ki67 levels absolutely, quantitatively, and objectively using the QDB method.

Formation of dislocations via misfit strain across interfaces in epitaxial BaTiO3and SrIrO3heterostructures.

Dislocations often occur in thin films with large misfit strain as a result of strain energy accumulation and can drastically change the film properties. Here the structure and dislocations in oxide heterostructures with large misfit strain are investigated on atomic scale. When grown on SrTiO3(001), the dislocations in both the monolithic BaTiO3thin film and its superlattices with SrIrO3appear above a critical thickness around 6 nm. The edge component of the dislocations is seen in both cases with the Burgers vector ofa⟨100⟩. However, compared to monolithic BaTiO3, the dislocation density is slightly lower in BaTiO3/SrIrO3superlattices. In the superlattice, when considering the SrTiO3lattice constant as the reference, BaTiO3has a larger misfit strain comparing with SrIrO3. It is found that in both cases, the formation of dislocation is only affected by the critical thickness of the film with larger lattice misfit (BaTiO3), regardless of the existence of a strong octahedral tilt/rotation mismatch at BaTiO3/SrIrO3interface. Our findings suggest that it is possible to control the position of dislocations, an important step toward defect engineering.

Objective Quantitation of EGFR Protein Levels using Quantitative Dot Blot Method for the Prognosis of Gastric Cancer Patients.

PURPOSE: An underlying factor for the failure of several clinical trials of anti-epidermal growth factor receptor (EGFR) therapies is the lack of an effective method to identify patients who overexpress EGFR protein. The quantitative dot blot method (QDB) was used to measure EGFR protein levels objectively, absolutely, and quantitatively. Its feasibility was evaluated for the prognosis of overall survival (OS) of patients with gastric cancer. MATERIALS AND METHODS: Slices of 2×5 µm from formalin-fixed paraffin-embedded gastric cancer specimens were used to extract total tissue lysates for QDB measurement. Absolutely quantitated EGFR protein levels were used for the Kaplan-Meier OS analysis. RESULTS: EGFR protein levels ranged from 0 to 772.6 pmol/g (n=246) for all gastric cancer patients. A poor correlation was observed between quantitated EGFR levels and immunohistochemistry scores with ρ=0.024 and P=0.717 in Spearman's correlation analysis. EGFR was identified as an independent negative prognostic biomarker for gastric cancer patients only through absolute quantitation, with a hazard ratio of 1.92 (95% confidence interval, 1.05-3.53; P=0.034) in multivariate Cox regression OS analysis. A cutoff of 208 pmol/g was proposed to stratify patients with a 3-year survival probability of 44% for patients with EGFR levels above the cutoff versus 68% for those below the cutoff based on Kaplan-Meier OS analysis (log rank test, P=0.002). CONCLUSIONS: A QDB-based assay was developed for gastric cancer specimens to measure EGFR protein levels absolutely, quantitatively, and objectively. This assay should facilitate clinical trials aimed at evaluation of anti-EGFR therapies retrospectively and prospectively for gastric cancer.

Developing a growing cancer profile database based on quantitative analysis of protein biomarkers in formalin-fixed paraffin-embedded specimens.

Over a century of clinical practice has led to the accumulation of millions of archived formalin fixed paraffin embedded (FFPE) cancer specimens with detailed medical records worldwide. Absolute quantitation of clinical protein biomarkers in these FFPE specimens allows individual specimens to be profiled at the population level, with the absolute nature of the measurements enabling the continuous processing of archived FFPE specimens over the time. A continuously growing cancer patient profile database is proposed here to support "big data" profiling of these protein biomarkers alone or in combination, enabling next-generation retrospective-prospective analytics into the field of clinical diagnostics.

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.

Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC; and 96.9% with combined results from both IHC and FISH analyses. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries.

Interface-induced magnetic polar metal phase in complex oxides.

Polar metals are commonly defined as metals with polar structural distortions. Strict symmetry restrictions make them an extremely rare breed as the structural constraints favor insulating over metallic phase. Moreover, no polar metals are known to be magnetic. Here we report on the realization of a magnetic polar metal phase in a BaTiO3/SrRuO3/BaTiO3 heterostructure. Electron microscopy reveals polar lattice distortions in three-unit-cells thick SrRuO3 between BaTiO3 layers. Electrical transport and magnetization measurements reveal that this heterostructure possesses a metallic phase with high conductivity and ferromagnetic ordering with high saturation moment. The high conductivity in the SrRuO3 layer can be attributed to the effect of electrostatic carrier accumulation induced by the BaTiO3 layers. Density-functional-theory calculations provide insights into the origin of the observed properties of the thin SrRuO3 film. The present results pave a way to design materials with desired functionalities at oxide interfaces.

Raman interrogation of the ferroelectric phase transition in polar metal LiOsO3.

Ferroelectric (FE) distortions in a metallic material were believed to be experimentally inaccessible because itinerant electrons would screen the long-range Coulomb interactions that favor a polar structure. It has been suggested by Anderson and Blount [P. W. Anderson, E. I. Blount, Phys. Rev. Lett. 14, 217-219 (1965)] that a transition from paraelectric phase to FE phase is possible for a metal if, in the paraelectric phase, the electrons at the Fermi level are decoupled from the soft transverse optical phonons, which lead to ferroelectricity. Here, using Raman spectroscopy combined with magnetotransport measurements on a recently discovered FE metal LiOsO3, we demonstrate active interplay of itinerant electrons and the FE order: Itinerant electrons cause strong renormalization of the FE order parameter, leading to a more gradual transition in LiOsO3 than typical insulating FEs. In return, the FE order enhances the anisotropy of charge transport between parallel and perpendicular to the polarization direction. The temperature-dependent evolution of Raman active in-plane 3Eg phonon, which strongly couples to the polar-active out-of-the-plane A2u phonon mode in the high-temperature paraelectric state, exhibits a deviation in Raman shift from the expectation of the pseudospin-phonon model that is widely used to model many insulating FEs. The Curie-Weiss temperature (θ ≈ 97 K) obtained from the optical susceptibility is substantially lower than T s, suggesting a strong suppression of FE fluctuations. Both line width and Fano line shape of 3Eg Raman mode exhibit a strong electron-phonon coupling in the high-temperature paraelectric phase, which disappears in the FE phase, challenging Anderson/Blount's proposal for the formation of FE metals.

Atomic-scale determination of spontaneous magnetic reversal in oxide heterostructures.

Interfaces between transition metal oxides are known to exhibit emerging electronic and magnetic properties. Here we report intriguing magnetic phenomena for La2/3Sr1/3MnO3 films on an SrTiO3 (001) substrate (LSMO/STO), where the interface governs the macroscopic properties of the entire monolithic thin film. The interface is characterized on the atomic level utilizing scanning transmission electron microscopy and electron energy loss spectroscopy (STEM-EELS), and density functional theory (DFT) is employed to elucidate the physics. STEM-EELS reveals mixed interfacial stoichiometry, subtle lattice distortions, and oxidation-state changes. Magnetic measurements combined with DFT calculations demonstrate that a unique form of antiferromagnetic exchange coupling appears at the interface, generating a novel exchange spring-type interaction that results in a remarkable spontaneous magnetic reversal of the entire ferromagnetic film, and an inverted magnetic hysteresis, persisting above room temperature. Formal oxidation states derived from electron spectroscopy data expose the fact that interfacial oxidation states are not consistent with nominal charge counting. The present work demonstrates the necessity of atomically resolved electron microscopy and spectroscopy for interface studies. Theory demonstrates that interfacial nonstoichiometry is an essential ingredient, responsible for the observed physical properties. The DFT-calculated electrostatic potential is flat in both the LSMO and STO sides (no internal electric field) for both Sr-rich and stoichiometric interfaces, while the DFT-calculated charge density reveals no charge transfer/accumulation at the interface, indicating that oxidation-state changes do not necessarily reflect charge transfer and that the concept of polar mismatch is not applicable in metal-insulator polar-nonpolar interfaces.

Quantitative Dot Blot (QDB) as a universal platform for absolute quantification of tissue biomarkers.

To circumvent the limitations associated with sandwich ELISA for tissue biomarker quantitation, Quantitative Dot Blot method (QDB) was proposed using antibodies clinically validated for immunohistochemistry (IHC), as this method requires only one primary antibody in the analysis. The protein levels of four breast cancer tissue biomarkers, including Estrogen Receptor (ER), Progesterone Receptor (PR), Ki67 and Her2, were absolutely quantitated successfully in 190 frozen breast tissue biopsies, and the results were further verified with provided IHC results. We propose QDB method as an alternative platform to Sandwich ELISA for absolute quantitation of tissue biomarkers with significantly reduced developing effort and time.

Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB).

BACKGROUND: Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice. METHODS: In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry. RESULTS: In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC. CONCLUSION: In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.

Enhanced Superconducting State in FeSe/SrTiO_{3} by a Dynamic Interfacial Polaron Mechanism.

The observation of substantially enhanced superconductivity of single-layer FeSe films on SrTiO_{3} has stimulated intensive research interest. At present, conclusive experimental data on the corresponding electron-boson interaction is still missing. Here we use inelastic electron scattering spectroscopy and angle resolved photoemission spectroscopy to show that the electrons in these systems are dressed by the strongly polarized lattice distortions of the SrTiO_{3}, and the indispensable nonadiabatic nature of such a coupling leads to the formation of dynamic interfacial polarons. Furthermore, the collective motion of the polarons results in a polaronic plasmon mode, which is unambiguously correlated with the surface phonons of SrTiO_{3} in the presence of the FeSe films. A microscopic model is developed showing that the interfacial polaron-polaron interaction leads to the superconductivity enhancement.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB).

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.