Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples.

RATIONALE: Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. OBJECTIVES: Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. METHODS: Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. RESULTS: Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). CONCLUSIONS: ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status.

Daily rhythms of urotensin I and II gene expression and hormone secretion in the caudal neurosecretory system of the euryhaline flounder (Platichthys flesus).

The caudal neurosecretory system (CNSS) is a unique neuroendocrine structure for environmental adaptation in fish, and is the major site of expression and secretion of urotensin I (UI) and II (UII). This study examined daily changes in mRNA expression and the secretion profile of UI and UII in the CNSS. Daily rhythms were observed in mRNA level of CNSS UI, urophysis UI, plasma UII, glucose, potassium and sodium. No statistically significant (Cosinor, P>0.05) diel rhythmicity in mRNA level of CNSS UII, urophysis UII, cortisol, lactate, osmolality and chloride were detected. The calculated acrophase of sodium, cortisol, plasma UII, urophysis UII, urophysis UI and mRNA level of CNSS UI rhythms were recorded at 13:04 h, 13:39 h, 14:45 h, 15:27 h, 14:41 h and 14:39 h, respectively and a positive relationship was evident among them. The acrophase of glucose and potassium rhythms were recorded at 18:57 h and 22:35 h, respectively. The glucose levels increased progressively at the onset of the UII surge at 15:00 h and reached peak values at dusk. The results support the hypothesis that the CNSS may play a role in the control of co-ordinated daily changes in energy mobilization, nutritional behavior and osmoregulatory systems in euryhaline flounder. Our findings described for the first time the existence of daily rhythms of CNSS hormone expression and secretion in Platichthys flesus. These results reveal the importance of taking into account the time of day when assessing stress responses and evaluating UI and UII as physiological indicators of stress in this species.