Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids.

Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the µ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCß3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.

Understanding patient-derived tumor organoid growth through an integrated imaging and mathematical modeling framework.

Patient-derived tumor organoids (PDTOs) are novel cellular models that maintain the genetic, phenotypic and structural features of patient tumor tissue and are useful for studying tumorigenesis and drug response. When integrated with advanced 3D imaging and analysis techniques, PDTOs can be used to establish physiologically relevant high-throughput and high-content drug screening platforms that support the development of patient-specific treatment strategies. However, in order to effectively leverage high-throughput PDTO observations for clinical predictions, it is critical to establish a quantitative understanding of the basic properties and variability of organoid growth dynamics. In this work, we introduced an innovative workflow for analyzing and understanding PDTO growth dynamics, by integrating a high-throughput imaging deep learning platform with mathematical modeling, incorporating flexible growth laws and variable dormancy times. We applied the workflow to colon cancer organoids and demonstrated that organoid growth is well-described by the Gompertz model of growth. Our analysis showed significant intrapatient heterogeneity in PDTO growth dynamics, with the initial exponential growth rate of an organoid following a lognormal distribution within each dataset. The level of intrapatient heterogeneity varied between patients, as did organoid growth rates and dormancy times of single seeded cells. Our work contributes to an emerging understanding of the basic growth characteristics of PDTOs, and it highlights the heterogeneity in organoid growth both within and between patients. These results pave the way for further modeling efforts aimed at predicting treatment response dynamics and drug resistance timing.

Uniform Synthesis of Bilayer Hydrogen Substituted Graphdiyne for Flexible Piezoresistive Applications.

Graphdiyne (GDY) has garnered significant attention as a cutting-edge 2D material owing to its distinctive electronic, optoelectronic, and mechanical properties, including high mobility, direct bandgap, and remarkable flexibility. One of the key challenges hindering the implementation of this material in flexible applications is its large area and uniform synthesis. The facile growth of centimeter-scale bilayer hydrogen substituted graphdiyne (Bi-HsGDY) on germanium (Ge) substrate is achieved using a low-temperature chemical vapor deposition (CVD) method. This material's field effect transistors (FET) showcase a high carrier mobility of 52.6 cm2 V-1 s-1 and an exceptionally low contact resistance of 10 Ω µm. By transferring the as-grown Bi-HsGDY onto a flexible substrate, a long-distance piezoresistive strain sensor is demonstrated, which exhibits a remarkable gauge factor of 43.34 with a fast response time of ≈275 ms. As a proof of concept, communication by means of Morse code is implemented using a Bi-HsGDY strain sensor. It is believed that these results are anticipated to open new horizons in realizing Bi-HsGDY for innovative flexible device applications.

Development of an Injectable, ECM-Derivative Embolic for the Treatment of Cerebral Saccular Aneurysms.

Cerebral aneurysms are a source of neurological morbidity and mortality, most often as a result of rupture. The most common approach for treating aneurysms involves endovascular embolization using nonbiodegradable medical devices, such as platinum coils. However, the need for retreatment due to the recanalization of coil-treated aneurysms highlights the importance of exploring alternative solutions. In this study, we propose an injectable extracellular matrix-derived embolic formed in situ by Michael addition of gelatin-thiol (Gel-SH) and hyaluronic acid vinyl sulfone (HA-VS) that may be delivered with a therapeutic agent (here, RADA-SP) to fill and remodel aneurysmal tissue without leaving behind permanent foreign bodies. The injectable embolic material demonstrated rapid gelation under physiological conditions, forming a highly porous structure and allowing for cellular infiltration. The injectable embolic exhibited thrombogenic behavior in vitro that was comparable to that of alginate injectables. Furthermore, in vivo studies in a murine carotid aneurysm model demonstrated the successful embolization of a saccular aneurysm and extensive cellular infiltration both with and without RADA-SP at 3 weeks, with some evidence of increased vascular or fibrosis markers with RADA-SP incorporation. The results indicate that the developed embolic has inherent potential for acutely filling cerebrovascular aneurysms and encouraging the cellular infiltration that would be necessary for stable, chronic remodeling.

Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus.

The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side effect of this widely used antimalarial drug. Here, we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and coexpress gastrin-releasing peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with the MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.

Controlled Bipolar Doping of One-Dimensional van der Waals Nb2Pd3Se8.

Tailoring the electrical properties of one-dimensional (1D) van der Waals (vdW) materials is desirable for their applications toward electronic devices by exploiting their unique characteristics. However, 1D vdW materials have not been extensively investigated for modulation of their electrical properties. Here we control doping levels and types of 1D vdW Nb2Pd3Se8 over a wide energy range by immersion in AuCl3 or ß-nicotinamide adenine dinucleotide (NADH) solutions, respectively. Through spectroscopic analyses and electrical characterizations, we confirm that the charges were effectively transferred to Nb2Pd3Se8, and the dopant concentration was adjusted to the immersion time. Furthermore, we make the axial p-n junction of 1D Nb2Pd3Se8 by a selective area p-doping using the AuCl3 solution, which exhibits rectifying behavior with an Iforward/Ireverse of 81 and an ideality factor of 1.2. Our findings could pave the way to more practical and functional electronic devices based on 1D vdW materials.

O-GlcNAcylation on LATS2 disrupts the Hippo pathway by inhibiting its activity.

The Hippo pathway controls organ size and tissue homeostasis by regulating cell proliferation and apoptosis. The LATS-mediated negative feedback loop prevents excessive activation of the effectors YAP/TAZ, maintaining homeostasis of the Hippo pathway. YAP and TAZ are hyperactivated in various cancer cells which lead to tumor growth. Aberrantly increased O-GlcNAcylation has recently emerged as a cause of hyperactivation of YAP in cancer cells. However, the mechanism, which induces hyperactivation of TAZ and blocks LATS-mediated negative feedback, remains to be elucidated in cancer cells. This study found that in breast cancer cells, abnormally increased O-GlcNAcylation hyperactivates YAP/TAZ and inhibits LATS2, a direct negative regulator of YAP/TAZ. LATS2 is one of the newly identified O-GlcNAcylated components in the MST-LATS kinase cascade. Here, we found that O-GlcNAcylation at LATS2 Thr436 interrupted its interaction with the MOB1 adaptor protein, which connects MST to LATS2, leading to activation of YAP/TAZ by suppressing LATS2 kinase activity. LATS2 is a core component in the LATS-mediated negative feedback loop. Thus, this study suggests that LATS2 O-GlcNAcylation is deeply involved in tumor growth by playing a critical role in dysregulation of the Hippo pathway in cancer cells.

Surface Passivation of Layered MoSe2 via van der Waals Stacking of Amorphous Hydrocarbon.

Development of efficient surface passivation methods for semiconductor devices is crucial to counter the degradation in their electrical performance owing to scattering or trapping of carriers in the channels induced by molecular adsorption from the ambient environment. However, conventional dielectric deposition involves the formation of additional interfacial defects associated with broken covalent bonds, resulting in accidental electrostatic doping or enhanced hysteretic behavior. In this study, centimeter-scaled van der Waals passivation of transition metal dichalcogenides (TMDCs) is demonstrated by stacking hydrocarbon (HC) dielectrics onto MoSe2 field-effect transistors (FETs), thereby enhancing the electric performance and stability of the device, accompanied with the suppression of chemical disorder at the HC/TMDCs interface. The stacking of HC onto MoSe2 FETs enhances the carrier mobility of MoSe2 FET by over 50% at the n-branch, and a significant decrease in hysteresis, owing to the screening of molecular adsorption. The electron mobility and hysteresis of the HC/MoSe2 FETs are verified to be nearly intact compared to those of the fabricated HC/MoSe2 FETs after exposure to ambient environment for 3 months. Consequently, the proposed design can act as a model for developing advanced nanoelectronics applications based on layered materials for mass production.

PDMS-Zwitterionic Hybrid for Facile, Antifouling Microfluidic Device Fabrication.

Poly(dimethylsiloxane) (PDMS) has been used in a wide range of biomedical devices and medical research due to its biostability, cytocompatibility, gas permeability, and optical properties. Yet, some properties of PDMS create critical limitations, particularly fouling through protein and cell adhesion. In this study, a diallyl-terminated sulfobetaine (SB-diallyl) molecule was synthesized and then directly mixed with a commercial PDMS base (Sylgard 184) and curing agent to produce a zwitterionic group-bearing PDMS (PDMS-SB) hybrid that does not require a complex or an additional surface modification process for the desired end product. In vitro examination of antifouling behavior following exposure to fresh ovine blood showed a significant reduction in platelet deposition for the PDMS-SB hybrid surface compared to that of a PDMS control (p < 0.05, n = 5). The manufacturability via soft lithography using the synthesized polymers was found to be comparable to that for unmodified PDMS. Bonding via O2 plasma treatment was confirmed, and the strength was measured and again found to be comparable to the control. PDMS-SB microfluidic devices were successfully fabricated and showed improved blood compatibility that could reduce channel occlusion due to clot formation relative to PDMS control devices. Further, gas (CO2) transfer through a PDMS-SB hybrid membrane was also tested with a proof-of-concept microchannel device and shown to be comparable to that through the PDMS control.

Pro-angiogenic Potential of Mesenchymal Stromal Cells Regulated by Matrix Stiffness and Anisotropy Mimicking Right Ventricles.

Capillary rarefaction is a hallmark of right ventricle (RV) failure. Mesenchymal stromal cell (MSC)-based therapy offers a potential treatment due to its pro-angiogenic function. However, the impact of RV tissue mechanics on MSC behavior is unclear, especially when referring to RV end-diastolic stiffness and mechanical anisotropy. In this study, we assessed MSC behavior on electrospun scaffolds with varied stiffness (normal vs failing RV) and anisotropy (isotropic vs anisotropic). In individual MSCs, we observed the highest vascular endothelial growth factor (VEGF) production and total tube length in the failing, isotropic group (2.00 ± 0.37, 1.53 ± 0.24), which was greater than the normal, isotropic group (0.70 ± 0.15, 0.55 ± 0.07; p < 0.05). The presence of anisotropy led to trends of increased VEGF production on normal groups (0.75 ± 0.09 vs 1.20 ± 0.17), but this effect was absent on failing groups. Our findings reveal synergistic effects of RV-like stiffness and anisotropy on MSC pro-angiogenic function and may guide MSC-based therapies for heart failure.

Wafer-Scale and Low-Temperature Growth of 1T-WS2 Film for Efficient and Stable Hydrogen Evolution Reaction.

The metallic 1T phase of WS2 (1T-WS2 ), which boosts the charge transfer between the electron source and active edge sites, can be used as an efficient electrocatalyst for the hydrogen evolution reaction (HER). As the semiconductor 2H phase of WS2 (2H-WS2 ) is inherently stable, methods for synthesizing 1T-WS2 are limited and complicated. Herein, a uniform wafer-scale 1T-WS2 film is prepared using a plasma-enhanced chemical vapor deposition (PE-CVD) system. The growth temperature is maintained at 150 °C enabling the direct synthesis of 1T-WS2 films on both rigid dielectric and flexible polymer substrates. Both the crystallinity and number of layers of the as-grown 1T-WS2 are verified by various spectroscopic and microscopic analyses. A distorted 1T structure with a 2a0 × a0 superlattice is observed using scanning transmission electron microscopy. An electrochemical analysis of the 1T-WS2 film demonstrates its similar catalytic activity and high durability as compared to those of previously reported untreated and planar 1T-WS2 films synthesized with CVD and hydrothermal methods. The 1T-WS2 does not transform to stable 2H-WS2 , even after a 700 h exposure to harsh catalytic conditions and 1000 cycles of HERs. This synthetic strategy can provide a facile method to synthesize uniform 1T-phase 2D materials for electrocatalysis applications.

Impact of Body Mass Index Change on the Prognosis of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Low body mass index (BMI) is an important prognostic factor in chronic obstructive pulmonary disease (COPD). However, the prognostic value of longitudinal BMI change in COPD has not been well studied. OBJECTIVE: We aimed to evaluate the association between longitudinal change of BMI and prognosis of COPD in Korean COPD cohort. METHODS: This study was conducted in a prospective Korean Obstructive Lung Disease (KOLD) cohort where COPD patients were recruited on an outpatient basis at 17 hospitals in South Korea. Annual BMI was measured over a period of 3 years or more. All patients were categorized into underweight (UW), normal weight (NW), and overweight (OW) groups by BMI. Clinical characteristics and outcomes including exacerbation and mortality were compared based on initial BMI grade and longitudinal change of BMI. RESULTS: This analysis included 537 COPD patients (mean age = 67.4 ± 7.9 years, male = 97.0%, mean BMI = 23.0 ± 3.1) of KOLD cohort. The proportions of UW, NW, and OW groups were 6.9% (n = 37), 68.9% (n = 370), and 24.2% (n = 130) respectively. The UW group showed lower forced expiratory volume in 1 s (FEV1) (p < 0.001), shorter 6-minute walk distance (p < 0.001), higher modified Medical Research Council score (p = 0.002), higher St. George Respiratory Questionnaire score (p < 0.001), higher emphysema index (p < 0.001) and air-trapping index (p < 0.001), and more frequent (p < 0.001) and severe exacerbations (p = 0.003). Multivariable analyses demonstrated that decrease of BMI (hazard ratio [HR] = 0.786, p = 0.038) and the descent of BMI group (HR = 3.167, p = 0.016) at 3-year follow-up along with age, initial BMI, post-bronchodilator FEV1, and severe exacerbations were significantly associated with mortality. CONCLUSIONS: This study demonstrated that BMI decrease during follow-up was independently associated with exacerbation and higher mortality of COPD, suggesting BMI reduction in COPD should be carefully managed.

MALDI-MSI of Immunotherapy: Mapping the EGFR-Targeting Antibody Cetuximab in 3D Colon-Cancer Cell Cultures.

Immunotherapies are treatments that use a patient's immune system to combat disease. One important type of immunotherapy employed in cancer treatments is the delivery of monoclonal antibodies to block growth receptors. In this manuscript, we develop a methodology that enables accurate and simple evaluation of antibody-type drug delivery using MALDI-MSI. To overcome the mass-range limitation that prevents the detection of large therapeutic antibodies, we used in situ reduction and alkylation to break disulfide bonds to generate smaller fragments. These smaller fragments are more readily ionized and detected by MALDI-MSI without loss of spatial information on the parent drug. As a proof of concept study, we evaluated the distribution of cetuximab in 3D colon cell cultures. Cetuximab is a monoclonal antibody that binds to the extracellular domain of epidermal-growth-factor receptor (EGFR), which is often overexpressed in colorectal cancer (CRC) and mediates cell differentiation, proliferation, migration, and angiogenesis. Cetuximab directly inhibits tumor growth and metastasis and induces apoptosis. By performing on-tissue reduction followed by MALDI-MSI analysis, we successfully mapped the time-dependent penetration and distribution of cetuximab in spheroids derived from two different colon-cancer cell lines (HT-29 and DLD-1). The localization patterns were further confirmed with IF staining of the drug. Changes in other biomolecules following drug treatment were also observed, including the elevation of ATP in spheroids. The developed method has also been applied to map cetuximab distribution in patient-derived colorectal-tumor organoids (CTOs). Overall, we believe this powerful label-free approach will be useful for visualizing the heterogeneous distribution of antibody drugs in tissues and tumors and will help to monitor and optimize their use in the clinic.

Convergence and extrusion are required for normal fusion of the mammalian secondary palate.

The fusion of two distinct prominences into one continuous structure is common during development and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. Using confocal live imaging, we directly observed the cellular processes underlying tissue fusion, using the secondary palatal shelves as a model. We find that convergence of a multi-layered epithelium into a single-layer epithelium is an essential early step, driven by cell intercalation, and is concurrent to orthogonal cell displacement and epithelial cell extrusion. Functional studies in mice indicate that this process requires an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non-muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development.

Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.

BACKGROUND: Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. METHODS: In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. RESULTS: The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). CONCLUSION: Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression.

Cross-inhibition of NMBR and GRPR signaling maintains normal histaminergic itch transmission.

We previously showed that gastrin-releasing peptide receptor (GRPR) in the spinal cord is important for mediating nonhistaminergic itch. Neuromedin B receptor (NMBR), the second member of the mammalian bombesin receptor family, is expressed in a largely nonoverlapping pattern with GRPR in the superficial spinal cord, and its role in itch transmission remains unclear. Here, we report that Nmbr knock-out (KO) mice exhibited normal scratching behavior in response to intradermal injection of pruritogens. However, mice lacking both Nmbr and Grpr (DKO mice) showed significant deficits in histaminergic itch. In contrast, the chloroquine (CQ)-evoked scratching behavior of DKO mice is not further reduced compared with Grpr KO mice. These results suggest that NMBR and GRPR could compensate for the loss of each other to maintain normal histamine-evoked itch, whereas GRPR is exclusively required for CQ-evoked scratching behavior. Interestingly, GRPR activity is enhanced in Nmbr KO mice despite the lack of upregulation of Grpr expression; so is NMBR in Grpr KO mice. We found that NMB acts exclusively through NMBR for itch transmission, whereas GRP can signal through both receptors, albeit to NMBR to a much lesser extent. Although NMBR and NMBR(+) neurons are dispensable for histaminergic itch, GRPR(+) neurons are likely to act downstream of NMBR(+) neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions. Together, we define the respective function of NMBR and GRPR in itch transmission, and reveal an unexpected relationship not only between the two receptors but also between the two populations of interneurons in itch signaling.

Detection of circulating tumor cell-specific markers in breast cancer patients using the quantitative RT-PCR assay.

BACKGROUND: Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within breast cancer tumors is used to determine the prognosis and select therapies, additional markers need to be identified. Circulating tumor cells (CTCs) are constituent cells that have detached from a primary tumor to circulate in the bloodstream. CTCs are considered the main source of breast cancer metastases; therefore, detection of CTCs could be a promising diagnostic method for metastatic breast cancer. METHODS: In this study, the CircleGen CTC RT-qDx assay was used to analyze the mRNA expression levels of six CTC-specific markers including EpCAM, CK19, HER2, Ki67, hTERT, and vimentin with a total of 692 peripheral whole blood samples from 221 breast cancer patients and 376 healthy individuals. RESULTS: This assay showed high specificity with multiple markers; none of the healthy controls were detected positive, whereas 21.7 and 14 % of breast cancer patients were positive for EpCAM and CK19, respectively. Of the 221 breast cancer patients, 84 (38 %), 46 (20.8 %), 83 (37.6 %), and 39 (17.6 %) were positively for HER2, Ki67, hTERT, and vimentin mRNA, respectively. Of the 84 patients who were HER2 positive, nine (4 %) were also positive for EpCAM, CK19, Ki67, hTERT, and vimentin. Of the 139 breast cancer patients who were HER2 negative, 65 (29.1 %) were negative for EpCAM, CK19, Ki67, hTERT, and vimentin. Furthermore, the EpCAM-positive population decreased from 21.5 to 8.3 % after completion of anti-tumor treatment (TP4). Similarly, the CK19, HER2, hTERT, and vimentin positives also decreased from 13.9 to 9.5 %, from 37.7 to 21.4 %, from 37.2 to 33.3 %, and from 17.5 to 14.3 %, respectively, after completion of anti-tumor treatment. In contrast, the Ki67 positives increased from 20.6 to 41.7 % after completion of anti-tumor treatment. CONCLUSIONS: mRNA overexpression of six CTC-specific markers was detected by the CircleGen CTC RT-qDx assay with high specificity, and the obtained mRNA expression levels of CTC-specific markers might provide useful criteria to select appropriate anti-tumor treatment for breast cancer patients.

Embryonic expression of EphA receptor genes in mice supports their candidacy for involvement in cleft lip and palate.

BACKGROUND: Eph receptors, comprising the A- and B-subfamilies, are the largest family of receptor tyrosine kinases in the mammalian genome, and their function is critical for morphogenesis in a variety of contexts. Whereas signaling through B-type Ephs has been demonstrated to play a role in cleft lip and palate (CL/P), the involvement of A-type Ephs has not been examined in this context notwithstanding a recent genome-wide association study that identified the EPHA3 locus as a candidate for non-syndromic CL/P. RESULTS: Here, we present a systematic analysis of the gene expression patterns for the nine EphA receptors at progressive stages of mouse development and find that EphA3, EphA4, and EphA7 exhibit restricted overlapping patterns of expression during palate development. We find that homozygous mutation of EphA3 or compound homozygous mutation of EphA3 and EphA4 in mice does not result in defective midfacial development, supporting the possibility of redundant function with EphA7. We also document previously undescribed expression patterns in other tissues of the craniofacial complex including the lacrimal duct and salivary glands. CONCLUSIONS: Together, these results are consistent with the hypothesis that mutations in EPHA family genes may cause CL/P and also suggest that functional redundancy between family members may be at play.

Evaluation of a quantitative RT-PCR assay to detect HER2 mRNA overexpression for diagnosis and selection of trastuzumab therapy in breast cancer tissue samples.

Breast cancer patients who have a positive result for HER2 overexpression are commonly treated with Herceptin, a HER2-targeted therapy. In the present study, the BrightGen HER2 RT-qDx (Syantra, Calgary, Canada), which is based on a one-tube nested RT-qPCR method that detects HER2 mRNA overexpression, was clinically evaluated in a total of 237 formalin-fixed paraffin-embedded (FFPE) tissue samples from breast cancer patients. Among the 38 HER2 positive samples, which were determined via IHC/FISH methods, 13 samples out of 16 (81.3%) that were IHC2+/FISH+ and 22 samples out of 22 (100%) that were IHC3+ have been decided positive for HER2 expression via the RT-qPCR method. The true positivity and false positivity results for the RT-qPCR were 92% (35/38) and 2% (1/65), respectively. The concordance between RT-qPCR and IHC results and RT-qPCR and IHC/FISH was 87.2% and 92.1%, respectively. Conclusively, the BrightGen HER2 RT-qDx may be a reliable and convenient method that can supplement traditional IHC and FISH methods for efficient use of trastuzumab.

Detection of circulating tumor cells in patients with breast cancer using the quantitative RT-PCR assay for monitoring of therapy efficacy.

Circulating tumor cells (CTCs) are an independent prognostic factor for patients with breast cancer. However, the role of CTCs in early breast cancer management is not yet clearly defined. The aim of this study was to isolate and characterize CTCs in blood sample of a breast cancer patient as a biomarker for monitoring treatments efficacy. In this study, 692 blood samples from 221 breast cancer patients and 376 healthy individuals was used to detect CTCs with multiple markers including epithelial cell adhesion molecule (EpCAM), cytokeratin (CK) 19, human epidermal growth factor (HER) 2, Ki67, human telomerase reverse transcriptase (hTERT), and vimentin using quantitative reverse transcription PCR (RT-qPCR). A total of 153 (69.2%) blood samples of 221 patients with breast cancer were found to be positive for at least one of the cancer-associated marker gene before treatment. After chemotherapy, no CTCs were found in 28 (33.3%) of the 84 blood samples analyzed for the presence of CTCs using the RT-qPCR, whereas 56 (66.7%) blood samples were still found to be positive for at least one of the markers. After completing the therapy, the CTC positivity rate decreased to 7 (20.6%) in the neoadjuvant group, whereas this increased to 7 (14%) cases in the adjuvant group. There was no statistically significant relationship between TNM stage and detection of CTC-related markers. Data from this study suggest that RT-qPCR assay for the detection of CTC markers might be useful in selecting appropriate therapeutics and for monitoring treatment efficacy in breast cancer patients.