Circulating ERBB3 levels are inversely associated with the risk of overweight-related hypertension: a cross-sectional study.

BACKGROUND: Hypertension and overweight are independent risk factors for cardiovascular disease, and overweight increase the risk of developing high blood pressure. ERBB3( also known as HER3) plays a considerable role in the development of cardiovascular diseases. However, the effect of ERBB3 levels in hypertensive overweight patients is unknown. The aim of this study was to assess the association between ERBB3 levels and hypertension in overweight Chinese patients. METHODS: We evaluated the height,weight, blood pressure, biochemical indicators, and ERBB3 levels in 128 Chinese adults aged 33-79 years. Plasma ERBB3 levels were assessed by the enzyme-linked immunosorbent assay, and body mass index(BMI) was calculated as body weight divided by height squared. Participants were allocated into three groups according to blood pressure and BMI: healthy control (CNT, n = 31; normotensive and non-overweight), hypertension (HT, n = 33; hypertension and non-overweight), and hypertension with overweight (HTO, n = 64; hypertension and overweight). Statistical significance was defined as a two-tailed P < 0.05. RESULTS: There was no significant difference in mean ERBB3 levels among the three groups, although a linear decrease from CNT (1.13 ± 0.36), HT (1.03 ± 0.36), to HTO (0.84 ± 0.26 ng/mL) was observed in men (P = 0.007). Among the drinking population, the ERBB3 level was significantly reduced in the HTO group as compared with those of the CNT and HT groups (0.76 ± 0.23 versus 1.18 ± 0.37 and 1.20 ± 0.30, respectively). ERBB3 levels were negatively correlated with diastolic blood pressure in men (r= - 0.293, P = 0.012), smoking (r= - 0.47, P = 0.004), and drinking (r = - 0.387, P = 0.008). BMI in men and among drinkers, and uric acid among drinkers were negatively correlated with ERBB3 levels. Multivariate conditional logistic regression showed that plasma ERBB3 levels were associated with a reduced risk of HTO in men [odds ratio (OR) 0.054; 95 % confidence interval (CI): 0.007-0.412) and drinkers (OR 0.002; 95 % CI: 0.000-0.101). CONCLUSIONS: ERBB3 may contribute to the pathogenesis of hypertension in overweight patients, with BMI, gender, and drinking all potentially modulating the process.

Multiplex plasma protein profiling identifies novel markers to discriminate patients with adenocarcinoma of the lung.

BACKGROUND: The overall prognosis of non-small cell lung cancer (NSCLC) is poor, and currently only patients with localized disease are potentially curable. Therefore, preferably non-invasively determined biomarkers that detect NSCLC patients at early stages of the disease are of high clinical relevance. The aim of this study was to identify and validate novel protein markers in plasma using the highly sensitive DNA-assisted multiplex proximity extension assay (PEA) to discriminate NSCLC from other lung diseases. METHODS: Plasma samples were collected from a total of 343 patients who underwent surgical resection for different lung diseases, including 144 patients with lung adenocarcinoma (LAC), 68 patients with non-malignant lung disease, 83 patients with lung metastasis of colorectal cancers and 48 patients with typical carcinoid. One microliter of plasma was analyzed using PEA, allowing detection and quantification of 92 established cancer related proteins. The concentrations of the plasma proteins were compared between disease groups. RESULTS: The comparison between LAC and benign samples revealed significantly different plasma levels for four proteins; CXCL17, CEACAM5, VEGFR2 and ERBB3 (adjusted p-value < 0.05). A multi-parameter classifier was developed to discriminate between samples from LAC patients and from patients with non-malignant lung conditions. With a bootstrap aggregated decision tree algorithm (TreeBagger), a sensitivity of 93% and specificity of 64% was achieved to detect LAC in this risk population. CONCLUSIONS: By applying the highly sensitive PEA, reliable protein profiles could be determined in microliter amounts of plasma. We further identified proteins that demonstrated different plasma concentration in defined disease groups and developed a signature that holds potential to be included in a screening assay for early lung cancer detection.

Specific antibodies and sensitive immunoassays for the human epidermal growth factor receptors (HER2, HER3, and HER4).

The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, <2.5 µg/L; human epidermal growth factor receptor 3, <2.8 µg/L; and human epidermal growth factor receptor 4, <1.8 µg/L. There were significant differences in human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2-positive and tissue human epidermal growth factor receptor 2-negative ( p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 ( p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with sensitive immunoassays for measuring serum levels of the respective targets and in monitoring established treatment.

Prognostic significance of serum ERBB3 and ERBB4 mRNA in lung adenocarcinoma patients.

Serum messenger RNA (mRNA) is an emerging prognostic tool for noninvasive malignant disease prognosis, and to study serum mRNA may have importance in the prognosis and detection of disease. This study aimed to evaluate the possible prognostic role of serum ERBB3 and ERBB4 mRNA expressions in lung adenocarcinoma patients. One hundred newly diagnosed lung adenocarcinoma patients and 100 age- and sex-matched healthy controls were included. Expression was analysed by quantitative real-time PCR and overall survival was analysed by Kaplan-Meier analysis. Serum ERBB3 and ERBB4 mRNA expressions was found to be significantly associated with distant metastases and TNM stages. It was observed that patients with distant metastases had 4.8- and 3.4-fold high ERBB3 and ERBB4 expression in contrast to patients without distant metastases, respectively. It was also found that ERBB3 and ERBB4 mRNA expression was 7.7-fold and 6.7-fold high in TNM stage IV compared to TNM stage I, respectively. Significantly, 2.6-fold increased serum ERBB4 mRNA expression was found in patients with pleural effusion compared to patients without pleural effusion (p = 0.005). Lung adenocarcinoma patients with ≤8- and >8-fold increased serum ERBB3 mRNA expression had 10.0 and 5.5 months of overall median survival while serum ERBB4 mRNA with ≤10- and >10-fold increased expression showed 11.4 and 5.0 months overall median survival, respectively. ERBB3 and ERBB4 together also found to be significantly associated with poor overall median survival. Patients with ≤8 + ≤10- and >8 + >10-fold expression showed 11.3 vs 4.8 months of overall median survival, respectively. In conclusion, serum ERBB3 and ERBB4 mRNA expressions may be a prognostic marker and monitoring of serum ERBB3 and ERBB4 mRNA can be one of the predictive factors for metastases and poor overall survival of lung adenocarcinoma patients.

HER1-4 protein concentrations in normal breast tissue from breast cancer patients are expressed by the same profile as in the malignant tissue.

BACKGROUND: The epidermal growth factor receptor HER2 is overexpressed or amplified in 25%-30% of patients with breast cancer. The mechanism behind HER2 amplification is unknown, but may be a patho-physiological phenomenon caused by continuous stimulation and activation of the HER1-4 system. We have mapped the protein concentrations of HER1-4 in breast cancer tissue, autologous reference tissue, normal breast tissue and serum samples, to see whether non-cancer cells from these patients express a protein profile indicating general activation. METHODS: Tissue samples from malignant and adjacent normal breast tissue (autologous reference tissue) were collected from 118 women consecutively admitted for surgical treatment of breast cancer. In addition, 26 samples of normal breast tissue were collected from healthy women having breast reduction surgery. The tissue samples were homogenized and the proteins extracted. The tissue and serum concentrations of HER1-4 were determined quantitatively using a commercially available enzyme linked immunosorbent assay (ELISA) method. RESULTS: HER1 was down regulated in cancer tissue when compared to autologous reference tissue (p=8 x 10(-6)), while HER2 (p<10(-7)) and HER3 (p=3 x 10(-5)) were up regulated. Comparing autologous reference tissue with normal tissue showed down regulation of HER1 (p=0.122) and up regulation of HER2 (p=10(-6)), HER3 (p<10(-7)) and HER4 (p<10(-7)). Furthermore, we observed that correlations between the receptor combinations HER1-2, HER1-3 and HER1-4 were maintained from normal breast tissue to autologous reference breast tissue, but were lost in cancer tissue. CONCLUSIONS: We suggest that these findings indicate that breast cancer is a systemic disease where the HER1-4 system in autologous reference tissue is continuously activated, thus favoring the subsequent development of cancer.

Soluble ErbB3 levels in bone marrow and plasma of men with prostate cancer.

PURPOSE: Prostate cancer tends to metastasize to bone and induce osteoblastic lesions. We identified a soluble form of ErbB3 (sErbB3), p45-sErbB3, in bone marrow supernatant from men with prostate cancer bone metastasis and showed that p45-sErbB3 enhances bone formation. We aimed to understand clinical implications of sErbB3 by establishing an ELISA to detect sErbB3 levels in bone marrow and plasma samples. EXPERIMENTAL DESIGN: We did ELISAs on marrow from 108 men [34 with androgen-dependent disease, 30 with androgen-independent disease (AI) but negative bone scan (AI/BS-), and 44 with AI and positive bone scan (AI/BS+)], sequential marrow from 5 men during treatment, plasma from 52 men before and after docetaxel treatment, and plasma from 95 men ages > or =70 years old without prostate cancer. RESULTS: Some men with clinically detectable bone metastasis had high sErbB3 levels. Within the AI/BS- group, higher sErbB3 levels seemed to yield lower rates of bone metastasis. In the AI/BS+ group, detectable bone metastases took longer to appear in men with higher sErbB3 levels than in men with lower sErbB3 levels (median, 82 versus 41 months). However, high sErbB3 levels did not confer survival benefit after metastasis development. Among men with metastatic progression in bone, docetaxel treatment reduced plasma sErbB3 (P < 0.0001) but did not affect bone-specific alkaline phosphatase (P = 0.206) or prostate-specific antigen (P = 0.906). sErbB3 was also detected in men without prostate cancer. CONCLUSIONS: The apparent correlation between higher sErbB3 levels and longer time to bone metastasis suggests that sErbB3 participates in progression in bone of prostate cancer.

Evaluation of Protein Levels of the Receptor Tyrosine Kinase ErbB3 in Serum.

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTK) consists of four members: EGFR1/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and HER4/ErbB4. Signaling through these receptors regulates many key cellular activities, such as cell division, migration, adhesion, differentiation, and apoptosis. The ErbB family has been shown to be overexpressed in different types of cancers and is a target of several inhibitors already in clinical trials. ErbB3 lacks a functional tyrosine kinase domain and therefore has not been as extensively studied as the other members of this family, but its importance in activating downstream pathways, such as the PI3K/Akt pathway, makes this RTK a worthy investigation target, especially in urothelial carcinoma where the PI3K/Akt pathway is vital for progression. In recent times, ErbB3 overexpression has been linked to drug resistance and progression of various diseases, especially cancer. ErbB3 levels in the serum were shown in many cases to be reflective of its role in disease progression, and therefore detection of serum ErbB3 levels during treatment may be of importance.Here we describe two methods for detecting ErbB3 protein in serum from patients who have undergone a clinical trial, utilizing two well-established methods in molecular biology-western blotting and ELISA, focusing on sample preparation and troubleshooting.

ERBB3: A potential serum biomarker for early detection and therapeutic target for devil facial tumour 1 (DFT1).

Devil Facial Tumour 1 (DFT1) is one of two transmissible neoplasms of Tasmanian devils (Sarcophilus harrisii) predominantly affecting their facial regions. DFT1's cellular origin is that of Schwann cell lineage where lesions are evident macroscopically late in the disease. Conversely, the pre-clinical timeframe from cellular transmission to appearance of DFT1 remains uncertain demonstrating the importance of an effective pre-clinical biomarker. We show that ERBB3, a marker expressed normally by the developing neural crest and Schwann cells, is immunohistohemically expressed by DFT1, therefore the potential of ERBB3 as a biomarker was explored. Under the hypothesis that serum ERBB3 levels may increase as DFT1 invades local and distant tissues our pilot study determined serum ERBB3 levels in normal Tasmanian devils and Tasmanian devils with DFT1. Compared to the baseline serum ERBB3 levels in unaffected Tasmanian devils, Tasmanian devils with DFT1 showed significant elevation of serum ERBB3 levels. Interestingly Tasmanian devils with cutaneous lymphoma (CL) also showed elevation of serum ERBB3 levels when compared to the baseline serum levels of Tasmanian devils without DFT1. Thus, elevated serum ERBB3 levels in otherwise healthy looking devils could predict possible DFT1 or CL in captive or wild devil populations and would have implications on the management, welfare and survival of Tasmanian devils. ERBB3 is also a therapeutic target and therefore the potential exists to consider modes of administration that may eradicate DFT1 from the wild.

Clinical Translation and Validation of a Predictive Biomarker for Patritumab, an Anti-human Epidermal Growth Factor Receptor 3 (HER3) Monoclonal Antibody, in Patients With Advanced Non-small Cell Lung Cancer.

BACKGROUND: During early clinical development, prospective identification of a predictive biomarker and validation of an assay method may not always be feasible. Dichotomizing a continuous biomarker measure to classify responders also leads to challenges. We present a case study of a prospective-retrospective approach for a continuous biomarker identified after patient enrollment but defined prospectively before the unblinding of data. An analysis of the strengths and weaknesses of this approach and the challenges encountered in its practical application are also provided. METHODS: HERALD (NCT02134015) was a double-blind, phase 2 study in patients with non-small cell lung cancer (NSCLC) randomized to erlotinib with placebo or with high or low doses of patritumab, a monoclonal antibody targeted against human epidermal growth factor receptor 3 (HER3). While the primary objective was to assess safety and progression-free survival (PFS), a secondary objective was to determine a single predictive biomarker hypothesis to identify subjects most likely to benefit from the addition of patritumab. Although not identified as the primary biomarker in the study protocol, on the basis of preclinical results from 2 independent laboratories, expression levels of the HER3 ligand heregulin (HRG) were prospectively declared the predictive biomarker before data unblinding but after subject enrollment. An assay to measure HRG mRNA was developed and validated. Other biomarkers, such as epidermal growth factor receptor (EGFR) mutation status, were also evaluated in an exploratory fashion. The cutoff value for high vs. low HRG mRNA levels was set at the median delta threshold cycle. A maximum likelihood analysis was performed to evaluate the provisional cutoff. The relationship of HRG values to PFS hazard ratios (HRs) was assessed as a measure of internal validation. Additional NSCLC samples were analyzed to characterize HRG mRNA distribution. RESULTS: The subgroup of patients with high HRG mRNA levels ("HRG-high") demonstrated clinical benefit from patritumab treatment with HRs of 0.37 (P = 0.0283) and 0.29 (P = 0.0027) in the high- and low-dose patritumab arms, respectively. However, only 102 of the 215 randomized patients (47.4%) had sufficient tumor samples for HRG mRNA measurement. Maximum likelihood analysis showed that the provisional cutoff was within the optimal range. In the placebo arm, the HRG-high subgroup demonstrated worse prognosis compared with HRG-low. A continuous relationship was observed between increased HRG mRNA levels and lower HR. Additional NSCLC samples (N = 300) demonstrated a similar unimodal distribution to that observed in this study, suggesting that the defined cutoff may be applicable to future NSCLC studies. CONCLUSIONS: The prospective-retrospective approach was successful in clinically validating a probable predictive biomarker. Post hoc in vitro studies and statistical analyses permitted further testing of the underlying assumptions. However, limitations of this analysis include the incomplete collection of adequate tumor tissue and a lack of stratification. In a phase 3 study, findings are being confirmed, and the HRG cutoff value is being further refined. CLINICALTRIALSGOV NUMBER: NCT02134015.

Measurement of cetuximab and panitumumab-unbound serum EGFR extracellular domain using an assay based on slow off-rate modified aptamer (SOMAmer) reagents.

BACKGROUND: Response to cetuximab (Erbitux®) and panitumumab (Vectibix®) varies among individuals, and even those who show response ultimately gain drug resistance. One possible etiologic factor is differential interaction between the drug and target. We describe the development of an assay based on Slow Off-rate Modified Aptamer (SOMAmer(™)) reagents that can distinguish drug-bound from unbound epidermal growth factor receptor (EGFR). METHODS: This quantitative assay uses a SOMAmer reagent specific for EGFR extracellular domain (ECD) as a capturing reagent. Captured SOMAmer is quantitated using PCR. Linearity and accuracy (recovery) of the assay were assessed using normal sera and purified EGFR ECD. RESULTS: This EGFR ECD assay showed linearity between 2.5 and 600 ng/mL. Average recovery was 101%. The assay detected EGFR but showed little cross-reactivity to other ErbB proteins: 0.4% for ErbB2, 6.9% for ErbB3, and 1.3% for ErbB4. Preincubation of normal serum with either cetuximab or panitumumab resulted in a dose-dependent decrease in EGFR ECD levels measured using the SOMAmer assay; preincubation did not affect measurement with an ELISA. CONCLUSIONS: This SOMAmer-based serum EGFR ECD assay accurately and specifically measures EGFR in serum. Detection of significant amounts of drug-unbound EGFR in patients undergoing cetuximab or panitumumab treatment could be an indicator of poor drug response. Further studies are needed to evaluate the utility of the assay as an indicator of drug efficacy or as a guide to dosing.