c-MET immunohistochemistry for differentiating malignant mesothelioma from benign mesothelial proliferations.

The separation of benign from malignant mesothelial proliferations can be a difficult problem for the surgical pathologist. c-MET is a receptor tyrosine kinase that is overexpressed and detectable by immunohistochemistry in many malignancies, including malignant mesothelioma. Whether c-MET is also expressed in benign mesothelial reactions is unclear from the literature. To determine whether c-MET immunohistochemistry can separate benign from malignant mesothelial processes, we stained 2 tissue microarrays containing 33 reactive epithelioid mesothelial proliferations (E-RMPs), 23 reactive spindle cell mesothelial proliferations, 45 epithelioid malignant mesotheliomas (EMMs), and 26 sarcomatoid/desmoplastic mesotheliomas (SMMs) for c-MET and compared the results with immunohistochemistry for two established markers, BAP1 and methylthioadenosine phosphorylase (MTAP). Membrane staining for c-MET was evaluated using a 12-point H-score classified as negative (score = 0), trace (score = 1-3), moderate (score = 4-6), and strong (score = 8-12). Staining was seen in only 3 of 33 (all trace) E-RMPs compared with 36 of 45 (80%) EMMs (chi-square comparing reactive and malignant = 39.80, p = 1.2 × 10-8). The H-score was >3 (moderate or strong) in 24 of 45 (53%) EMMs. Addition of BAP1 staining to the c-MET-negative/trace EMM increased sensitivity to 75% (32/42), whereas similar addition of MTAP staining increased sensitivity to 77% (33/43). No benign spindle cell proliferations showed staining compared with 10 of 26 (38%) positive SMMs, but only 4 (15%) SMMs were classified as moderate or strong. We conclude that moderate/strong c-MET staining can be used to support a diagnosis of EMM vs an epithelial reactive proliferation. c-MET is too insensitive to use for detecting SMM.

HER2-HER3 Heterodimer Quantification by FRET-FLIM and Patient Subclass Analysis of the COIN Colorectal Trial.

BACKGROUND: The phase III MRC COIN trial showed no statistically significant benefit from adding the EGFR-target cetuximab to oxaliplatin-based chemotherapy in first-line treatment of advanced colorectal cancer. This study exploits additional information on HER2-HER3 dimerization to achieve patient stratification and reveal previously hidden subgroups of patients who had differing disease progression and treatment response. METHODS: HER2-HER3 dimerization was quantified by fluorescence lifetime imaging microscopy in primary tumor samples from 550 COIN trial patients receiving oxaliplatin and fluoropyrimidine chemotherapy with or without cetuximab. Bayesian latent class analysis and covariate reduction was performed to analyze the effects of HER2-HER3 dimer, RAS mutation, and cetuximab on progression-free survival and overall survival (OS). All statistical tests were two-sided. RESULTS: Latent class analysis on a cohort of 398 patients revealed two patient subclasses with differing prognoses (median OS = 1624 days [95% confidence interval [CI] = 1466 to 1816 days] vs 461 days [95% CI = 431 to 504 days]): Class 1 (15.6%) showed a benefit from cetuximab in OS (hazard ratio = 0.43, 95% CI = 0.25 to 0.76, P = .004). Class 2 showed an association of increased HER2-HER3 with better OS (hazard ratio = 0.64, 95% CI = 0.44 to 0.94, P = .02). A class prediction signature was formed and tested on an independent validation cohort (n = 152) validating the prognostic utility of the dimer assay. Similar subclasses were also discovered in full trial dataset (n = 1630) based on 10 baseline clinicopathological and genetic covariates. CONCLUSIONS: Our work suggests that the combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy. A larger prospective cohort will be required to confirm its utility in predicting the outcome of anti-EGFR treatment.

Randomized Phase II Trial of Seribantumab in Combination with Erlotinib in Patients with EGFR Wild-Type Non-Small Cell Lung Cancer.

BACKGROUND: Seribantumab (MM-121) is a fully human IgG2 monoclonal antibody that binds to human epidermal growth factor receptor 3 (HER3/ErbB3) to block heregulin (HRG/NRG)-mediated ErbB3 signaling and induce receptor downregulation. This open-label, randomized phase 1/2 study evaluated safety and efficacy of seribantumab plus erlotinib in advanced non-small cell lung cancer (NSCLC). Here, we report the activity of seribantumab plus erlotinib, versus erlotinib alone, in patients with EGFR wild-type tumors and describe the potential predictive power of HRG. MATERIALS AND METHODS: Patients with EGFR wild-type NSCLC were assigned randomly to receive seribantumab + erlotinib or erlotinib alone. Patients underwent pretreatment core needle biopsy and archived tumor samples were collected to support prespecified biomarker analyses. RESULTS: One hundred twenty-nine patients received seribantumab + erlotinib (n = 85) or erlotinib alone (n = 44). Median estimated progression-free survival (PFS) in the unselected intent-to-treat (ITT) population was 8.1 and 7.7 weeks in the experimental and control arm, respectively (hazard ratio [HR], 0.822; 95% confidence interval [CI], 0.37-1.828; p = 0.63), and median estimated overall survival was 27.3 and 40.3 weeks in the experimental and control arm, respectively (HR, 1.395; 95% CI, 0.846 to 2.301; p = .1898) In patients whose tumors had detectable HRG mRNA expression, treatment benefit was observed in the seribantumab + erlotinib combination (HR, 0.35; 95% CI, 0.16-0.76; p = .008). In contrast, in patients whose tumors were HRG negative, the HR was 2.15 (95% CI, 0.97-4.76; p = .059, HRG-by-treatment interaction, p value = .0016). CONCLUSION: The addition of seribantumab to erlotinib did not result in improved PFS in unselected patients. However, predefined retrospective exploratory analyses suggest that detectable HRG mRNA levels identified patients who might benefit from seribantumab. An ongoing clinical trial of seribantumab, in combination with docetaxel, is underway in patients with advanced NSCLC and high HRG mRNA expression (NCT02387216). IMPLICATIONS FOR PRACTICE: The poor prognosis of patients with non-small cell lung cancer (NSCLC) underscores the need for more effective treatment options, highlighting the unmet medical need in this patient population. The results of this study show that a novel biomarker, heregulin, may help to identify patients with advanced NSCLC who could benefit from treatment with seribantumab. On the basis of the observed safety profile and promising clinical efficacy, a prospective, randomized, open-label, international, multicenter phase II trial (SHERLOC, NCT02387216) is under way to investigate the efficacy and safety of seribantumab in combination with docetaxel in patients with heregulin-positive advanced adenocarcinoma.

Evaluation of antibody fragment properties for near-infrared fluorescence imaging of HER3-positive cancer xenografts.

In vivo imaging is influenced by the half-life, tissue penetration, biodistribution, and affinity of the imaging probe. Immunoglobulin G (IgG) is composed of discrete domains with known functions, providing a template for engineering antibody fragments with desired imaging properties. Here, we engineered antibody-based imaging probes, consisting of different combinations of antibody domains, labeled them with the near-infrared fluorescent dye IRDye800CW, and evaluated their in vivo imaging properties. Antibody-based imaging probes were based on an anti-HER3 antigen binding fragment (Fab) isolated using phage display. Methods: We constructed six anti-HER3 antibody-based imaging probes: a single chain variable fragment (scFv), Fab, diabody, scFv-CH3, scFv-Fc, and IgG. IRDye800CW-labeled, antibody-based probes were injected into nude mice bearing FaDu xenografts and their distribution to the xenograft, liver, and kidneys was evaluated. Results: These imaging probes bound to recombinant HER3 and to the HER3-positive cell line, FaDu. Small antibody fragments with molecular weight <60 kDa (scFv, diabody, and Fab) accumulated rapidly in the xenograft (maximum accumulation between 2-4 h post injection (hpi)) and cleared primarily through the kidneys. scFv-CH3 (80 kDa) had fast clearance and peaked in the xenograft between 2-3 hpi and cleared from xenograft in a rate comparable to Fab and diabody. IgG and scFv-Fc persisted in the xenografts for up to 72 hpi and distributed mainly to the xenograft and liver. The highest xenograft fluorescence signals were observed with IgG and scFv-Fc imaging probes and persisted for 2-3 days. Conclusion: These results highlight the utility of using antibody fragments to optimize clearance, tumor labeling, and biodistribution properties for developing anti-HER3 probes for image-guided surgery or PET imaging.

A Platform To Enhance Quantitative Single Molecule Localization Microscopy.

Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell lines, we detected distinct differences in receptor density and nano-organization upon treatment with therapeutic agents. This new platform can improve molecular quantification and can be developed to study the local protein environment of intact cells.

Localized Visualization and Autonomous Detection of Cell Surface Receptor Clusters Using DNA Proximity Circuit.

Cell surface receptors play an important role in mediating cell communication and are used as disease biomarkers and therapeutic targets. We present a one-pot molecular toolbox, which we term the split proximity circuit (SPC), for the autonomous detection and visualization of cell surface receptor clusters. Detection was powered by antibody recognition and a series of autonomous DNA hybridization to achieve localized, enzyme-free signal amplification. The system under study was the human epidermal growth factor receptor (HER) family, that is, HER2:HER2 homodimer and HER2:HER3 heterodimer, both in cell lysate and in situ on fixed whole cells. The detection and imaging of receptors were carried out using standard microplate scans and confocal microscopy, respectively. The circuit operated specifically with minimal leakages and successfully captured the receptor expression profiles on three cell types without any intermediate washing steps.

Soluble Neuregulin1 is strongly up-regulated in the rat model of Charcot-Marie-Tooth 1A disease.

Neuregulin1 (NRG1) is a growth factor playing a pivotal role in peripheral nerve development through the activation of the transmembrane co-receptors ErbB2-ErbB3. Soluble NRG1 isoforms, mainly secreted by Schwann cells, are strongly and transiently up-regulated after acute peripheral nerve injury, thus suggesting that they play a crucial role also in the response to nerve damage. Here we show that in the rat experimental model of the peripheral demyelinating neuropathy Charcot-Marie-Tooth 1A (CMT1A) the expression of the different NRG1 isoforms (soluble, type α and ß, type a and b) is strongly up-regulated, as well as the expression of NRG1 co-receptors ErbB2-ErbB3, thus showing that CMT1A nerves have a gene expression pattern highly reminiscent of injured nerves. Because it has been shown that high concentrations of soluble NRG1 negatively affect myelination, we suggest that soluble NRG1 over-expression might play a negative role in the pathogenesis of CMT1A disease, and that a therapeutic approach, aimed to interfere with NRG1 activity, might be beneficial for CMT1A patients. Further studies will be necessary to test this hypothesis in animal models and to evaluate NRG1 expression in human patients. Impact statement Charcot-Marie-Tooth1A (CMT1A) is one of the most frequent inherited neurological diseases, characterized by chronic demyelination of peripheral nerves, for which effective therapies are not yet available. It has been recently proposed that the treatment with soluble Neuregulin1 (NRG1), a growth factor released by Schwann cells immediately after acute nerve injury, might be effective in CMT1A treatment. However, the expression of the different isoforms of endogenous NRG1 in CMT1A nerves has not been yet investigated. In this preliminary study, we demonstrate that different isoforms of soluble NRG1 are strongly over-expressed in CMT1A nerves, thus suggesting that a therapeutic approach based on NRG1 treatment should be carefully reconsidered. If soluble NRG1 is over-expressed also in human CMT1A nerves, a therapeutic approach aimed to inhibit (instead of stimulate) the signal transduction pathways driven by NRG1 might be fruitfully developed. Further studies will be necessary to test these hypotheses.

Clinical significance of overexpression of NRG1 and its receptors, HER3 and HER4, in gastric cancer patients.

BACKGROUND: Neuregulin 1 (NRG1), a ligand for human epidermal growth factor (HER) 3 and HER4, can activates cell signaling pathways to promote carcinogenesis and metastasis. METHODS: To investigate the clinicopathologic significance of NRG1 and its receptors, immunohistochemistry was performed for NRG1, HER3, and HER4 in 502 consecutive gastric cancers (GCs). Furthermore, HER2, microsatellite instability (MSI), and Epstein-Barr virus (EBV) status were investigated. NRG1 gene copy number (GCN) was determined by dual-color fluorescence in situ hybridization (FISH) in 388 available GCs. RESULTS: NRG1 overexpression was observed in 141 (28.1%) GCs and closely correlated with HER3 (P = 0.034) and HER4 (P < 0.001) expression. NRG1 overexpression was significantly associated with aggressive features, including infiltrative tumor growth, lymphovascular, and neural invasion, high pathologic stage, and poor prognosis (all P < 0.05), but not associated with EBV, MSI, or HER2 status. Multivariate analysis identified NRG1 overexpression as an independent prognostic factor for survival (P = 0.040). HER3 and HER4 expressions were observed in 157 (31.3%) and 277 (55.2%), respectively. In contrast to NRG1, expression of these proteins was not associated with survival. NRG1 GCN gain (GCN ≥ 2.5) was detected in 14.7% patients, including two cases of amplification, and was moderately correlated with NRG1 overexpression (κ, 0.459; P < 0.001). CONCLUSIONS: Although our results indicate a lack of prognostic significance of HER3 and HER4 overexpression in GC, overexpression of their ligand, NRG1, was associated with aggressive clinical features and represented an independent unfavorable prognostic factor. Therefore, NRG1 is a potential prognostic and therapeutic biomarker in GC patients.

Evaluation of a radiocobalt-labelled affibody molecule for imaging of human epidermal growth factor receptor 3 expression.

The human epidermal growth factor receptor 3 (HER3) is involved in the development of cancer resistance towards tyrosine kinase-targeted therapies. Several HER3­targeting therapeutics are currently under clinical evaluation. Non-invasive imaging of HER3 expression could improve patient management. Affibody molecules are small engineered scaffold proteins demonstrating superior properties as targeting probes for molecular imaging compared with monoclonal antibodies. Feasibility of in vivo HER3 imaging using affibody molecules has been previously demonstrated. Preclinical studies have shown that the contrast when imaging using anti-HER3 affibody molecules can be improved over time. We aim to develop an agent for PET imaging of HER3 expression using the long-lived positron-emitting radionuclide cobalt-55 (55Co) (T1/2=17.5 h). A long-lived cobalt isotope 57Co was used as a surrogate for 55Co in this study. The anti-HER3 affibody molecule HEHEHE-ZHER3-NOTA was labelled with radiocobalt with high yield, purity and stability. Biodistribution of 57Co-HEHEHE-ZHER3-NOTA was measured in mice bearing DU145 (prostate carcinoma) and LS174T (colorectal carcinoma) xenografts at 3 and 24 h post injection (p.i.). Tumour-to-blood ratios significantly increased between 3 and 24 h p.i. (p<0.05). At 24 h p.i., tumour-to-blood ratios were 6 for DU145 and 8 for LS174T xenografts, respectively. HER3­expressing xenografts were clearly visualized in a preclinical imaging setting already 3 h p.i., and contrast further improved at 24 h p.i. In conclusion, the radiocobalt-labelled anti-HER3 affibody molecule, HEHEHE-ZHER3-NOTA, is a promising tracer for imaging of HER3 expression in tumours.

HER2-HER3 dimer quantification by FLIM-FRET predicts breast cancer metastatic relapse independently of HER2 IHC status.

Overexpression of HER2 is an important prognostic marker, and the only predictive biomarker of response to HER2-targeted therapies in invasive breast cancer. HER2-HER3 dimer has been shown to drive proliferation and tumor progression, and targeting of this dimer with pertuzumab alongside chemotherapy and trastuzumab, has shown significant clinical utility. The purpose of this study was to accurately quantify HER2-HER3 dimerisation in formalin fixed paraffin embedded (FFPE) breast cancer tissue as a novel prognostic biomarker.FFPE tissues were obtained from patients included in the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) study. HER2-HER3 dimerisation was quantified using an improved fluorescence lifetime imaging microscopy (FLIM) histology-based analysis. Analysis of 131 tissue microarray cores demonstrated that the extent of HER2-HER3 dimer formation as measured by Förster Resonance Energy Transfer (FRET) determined through FLIM predicts the likelihood of metastatic relapse up to 10 years after surgery (hazard ratio 3.91 (1.61-9.5), p = 0.003) independently of HER2 expression, in a multivariate model. Interestingly there was no correlation between the level of HER2 protein expressed and HER2-HER3 heterodimer formation. We used a mathematical model that takes into account the complex interactions in a network of all four HER proteins to explain this counterintuitive finding.Future utility of this technique may highlight a group of patients who do not overexpress HER2 protein but are nevertheless dependent on the HER2-HER3 heterodimer as driver of proliferation. This assay could, if validated in a group of patients treated with, for instance pertuzumab, be used as a predictive biomarker to predict for response to such targeted therapies.

MITF depletion elevates expression levels of ERBB3 receptor and its cognate ligand NRG1-beta in melanoma.

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway is frequently hyper-activated upon vemurafenib treatment of melanoma. We have here investigated the relationship between SRY-box 10 (SOX10), forkhead box 3 (FOXD3) and microphthalmia-associated transcription factor (MITF) in the regulation of the receptor tyrosine-protein kinase ERBB3, and its cognate ligand neuregulin 1-beta (NRG1-beta). We found that both NRG1-beta and ERBB3 mRNA levels were elevated as a consequence of MITF depletion, induced by either vemurafenib or MITF small interfering RNA (siRNA) treatment. Elevation of ERBB3 receptor expression after MITF depletion caused increased activation of the PI3K pathway in the presence of NRG1-beta ligand. Together, our results suggest that MITF may play a role in the development of acquired drug resistance through hyper-activation of the PI3K pathway.

HER3 expression is correlated to distally located and low-grade colon cancer.

UNLABELLED: Background HER3 is a member of the human epidermal growth factor receptor complex (EGFR, HER2, HER3 and HER4). It has been investigated as a prognostic biomarker in colorectal cancer but is sparingly studied in colon cancer. HER3 can affect cellular proliferation, differentiation and migration in oncogenesis through ligand binding and activation of intracellular signal pathways. Recently, we found that expression of cell surface HER3 can be detected at a high extent in primary colorectal tumors, lymph node and liver metastases and that it correlated with poor prognosis. This large, explorative, retrospective study evaluates the prognostic value of HER3 in colon cancer and the association of HER3 to tumor location. MATERIAL AND METHODS: Immunohistochemical detection with a monoclonal HER3 antibody in primary colon tumors of stage II and III, from 521 patients, was performed. Results HER3 was expressed at high levels in 67% of the colon tumors. High HER3 expression was associated with distal tumor location (p < 0.0001) and low-grade tumor (p < 0.0001). In the group of patients with distal colon cancer (230/521), HER3 expression correlated to shorter disease-free survival (DFS) (p = 0.03) in the univariate analysis and in the multivariate analysis, a hazard ratio of 0.56 (95% CI 0.31-0.99) (p = 0.047) was observed. Conclusion In this explorative, retrospective study, high HER3 expression in colon cancer was associated to distal colon location and low-grade tumor. High HER3 expression was of prognostic value according to DFS in distal colon cancer in univariate and multivariate analysis. We could not find a significant value of HER3 expression with respect to overall survival (OS).

Protein expression of HER2, 3, 4 in gastric cancer: correlation with clinical features and survival.

BACKGROUND AND AIMS: Despite significant improvements in targeted therapies for patients with advanced gastric cancer (GC), the prognosis of those patients remains poor. This study explores the expression and clinicopathological significance of human epidermal growth factor receptor 2, 3 and 4 (HER2, HER3, HER4) in GC, in order to find more prognostic biomarkers of GC and putative targets of therapy. METHODS: Immunohistochemistry was performed for HER2, HER3 and HER4 in 498 patients with GC using tissue microarray. Correlations between the receptor expression and clinicopathological features, as well as prognosis of the patients were statistically analysed. RESULTS: The high expression rates of HER2, HER3 and HER4 proteins in the patients were 8.6% (43/498), 20.7% (103/498) and 13.3% (66/498), respectively. High expression of HER2 and HER3 was correlated with proximal GC of the cardia (p<0.05). High expression of HER3 was associated with the tumour depth, tumour node metastasis (TNM) stage and lymph node metastasis (p<0.05). High expression of HER4 was associated with TNM stage (p<0.05) only. According to a regression model, high expression of HER3 was significantly associated with patients' poor survival (p=0.004). High expression rates of HER2, HER3 and HER4 were correlated with each other, but they were all associated merely with histologically intestinal-type adenocarcinoma of GC (p<0.05). CONCLUSIONS: HER3 is correlated with the malignant biological behaviour of GC. Expression of HER3 is a significant predictor of poor survival in GC. Therefore, the development of HER3-targeted agents may provide new possibilities in the treatment of GC.

Novel Peptidomimetics for Inhibition of HER2:HER3 Heterodimerization in HER2-Positive Breast Cancer.

The current approach to treating HER2-overexpressed breast cancer is the use of monoclonal antibodies or a combination of antibodies with traditional chemotherapeutic agents or kinase inhibitors. Our approach is to target clinically validated HER2 domain IV with peptidomimetics and inhibit the protein-protein interactions (PPI) of HERs. Unlike antibodies, peptidomimetics have advantages in terms of stability, modification, and molecular size. We have designed peptidomimetics (compounds 5 and 9) that bind to HER2 domain IV, inhibit protein-protein interactions, and decrease cell viability in breast cancer cells with HER2 overexpression. We have shown, using enzyme fragment complementation and proximity ligation assays, that peptidomimetics inhibit the PPI of HER2:HER3. Compounds 5 and 9 suppressed the tumor growth in a xenograft mouse model. Furthermore, we have shown that these compounds inhibit PPI of HER2:HER3 and phosphorylation of HER2 as compared to control in tissue samples derived from in vivo studies. The stability of the compounds was also investigated in mouse serum, and the compounds exhibited stability with a half-life of up to 3 h. These results suggest that the novel peptidomimetics we have developed target the extracellular domain of HER2 protein and inhibit HER2:HER3 interaction, providing a novel method to treat HER2-positive cancer.

A quantum dot-based microfluidic multi-window platform for quantifying the biomarkers of breast cancer cells.

Conventional molecular profiling methods using immunochemical assays have limits in terms of multiplexity and the quantification of biomarkers in investigation of cancer cells. In this paper, we demonstrate a quantum dot (QD)-based microfluidic multiple biomarker quantification (QD-MMBQ) method that enables labeling of more than eight proteins immunochemically on cell blocks within 1 h, in a quantitative manner. An internal reference, ß-actin, was used as a loading control to compensate for differences in not only the cell number but also in staining quality among specimens. Furthermore, the microfluidic blocking method exhibited less nonspecific binding of QDs than the conventional static blocking method.

Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.

Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications.

Electrochemical biosensor based on self-assembled monolayers modified with gold nanoparticles for detection of HER-3.

We have developed a new immunological biosensor for ultrasensitive quantification of human epidermal growth factor receptor-3(HER-3). In order to construct the biosensor, the gold electrode surface was layered with, hexanedithiol, gold nanoparticles, and cysteamine, respectively. Anti-HER-3 antibody was covalently attached to cysteamine by glutaraldehyde and used as a bioreceptor in a biosensor system for the first time by this study. Surface characterization was obtained by means of electrochemical impedance spectroscopy and voltammetry. The proposed biosensor showed a good analytical performance for the detection of HER-3 ranging from 0.2 to 1.4 pg mL(-1). Kramers-Kronig transform was performed on the experimental impedance data. Moreover, in an immunosensor system, the single frequency impedance technique was firstly used for characterization of interaction between HER-3 and anti-HER-3. Finally the presented biosensor was applied to artificial serum samples spiked with HER-3.

HER3, serious partner in crime: therapeutic approaches and potential biomarkers for effect of HER3-targeting.

The human epidermal growth factor receptor (HER) family members are targeted by a growing numbers of small molecules and monoclonal antibodies. Resistance against the epidermal growth factor receptor (EGFR) and HER2-targeting agents is a clinically relevant problem forcing research on optimizing targeting of the HER family. In view of its overexpression in tumors, and compensatory role in HER signaling, HER3 has gained much interest as a potential additional target within the HER family. It is the only member of the HER family lacking intrinsic tyrosine kinase activity and therefore its role in cancer has long been underestimated. Drugs that block HER3 or interfere with HER3 dimer signaling, including fully human anti-HER3 antibodies, bispecific antibodies and tyrosine kinase inhibitors (TKIs), are currently becoming available. Several compounds have already entered clinical trial. In the meantime potential biomarkers are tested such as tumor analysis of HER3 expression, functional assays for downstream effector molecules and molecular imaging techniques. This review describes the biology and relevance of HER3 in cancer, agents targeting HER3 and potential biomarkers for effect of HER3-targeting.

Ultrasensitive electrochemical detection of cancer associated biomarker HER3 based on anti-HER3 biosensor.

The development of a new impedimetric biosensor for the detection of HER3, based on self-assembled monolayers (SAMs) of 4-aminothiophenol on gold electrodes, is reported. Anti-HER3 was used as a biorecognition element for the first time in an impedimetric biosensor. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were applied to characterize the immobilization process and to detect HER3. To provide the best biosensor response all experimental parameters were optimized. In addition, Kramers-Kronigs transform was also performed on the immobilization and measurement processes successfully. The biosensor had a linear detection range of 0.4-2.4 pg/mL. The chrono-impedance technique to real time monitor the interaction between HER3 and anti-HER3 is also implemented. The biosensor has exhibited good repeatability and reproducibility. To demonstrate the feasibility of the biosensor in practical analysis, the artificial serum samples were experienced.

Prevalence, clinicopathologic characteristics, and molecular associations of EGFR exon 20 insertion mutations in East Asian patients with lung adenocarcinoma.

PURPOSE: To define the prevalence, clinicopathologic characteristics and molecular associations of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in East Asian lung adenocarcinoma patients. METHODS: A total of 1,086 lung adenocarcinomas were sequenced for EGFR mutations. EGFR and HER2 copy number variations; total and phosphorylated (p) protein expression of ErbB family members including EGFR, HER2, and HER3; phosphorylated protein expression of downstream signaling molecules including Akt and Erk; and clinicopathologic features in lung adenocarcinomas with EGFR exon 20 insertion mutations were all investigated. RESULTS: EGFR exon 20 insertion mutations were present in 2.9 % of lung adenocarcinomas and 4.7 % of all the EGFR mutations. Compared to those with classic activating EGFR mutations, lung adenocarcinomas with exon 20 insertion mutations were characterized by significantly younger age at diagnosis (P = 0.032 for exon 20 insertions vs. L858R) and shorter relapse-free survival [P = 0.045 for exon 20 insertions versus (vs) exon 19 deletions]. Molecularly, samples harboring exon 20 insertion mutations had lower expression of phosphorylated (p)-EGFR (P < 0.001) and HER3 (P = 0.016). In addition, higher expression of p-Akt (P = 0.007) and lower expression of p-Erk (P = 0.009) were observed in tumors with exon 20 insertion mutations. CONCLUSIONS: Lung adenocarcinomas with EGFR exon 20 insertion mutations were present in a substantial proportion. This subset showed distinct clinicopathologic features, less dependence on EGFR molecularly, and different pathway activation patterns compared to those with classic EGFR activating mutations.