Comprehensive biomarker analysis of long-term response to trastuzumab in patients with HER2-positive advanced gastric or gastroesophageal adenocarcinoma.

BACKGROUND: A subgroup of patients with HER2-positive metastatic gastric and gastroesophageal junction cancers shows long-term response under trastuzumab maintenance monotherapy. Obviously, HER2 status alone is not able to identify these patients. We performed this study to identify potential new prognostic biomarkers for this long-term responding patient group. PATIENTS AND METHODS: Tumour samples of 19 patients with HER2-positive metastatic gastric and gastroesophageal junction cancer who underwent trastuzumab treatment were retrospectively collected from multiple centres. Patients were divided into long-term responding (n = 7) or short-term responding group (n = 12) according to progression-free survival (PFS≥12 months vs. PFS < 12 months). Next-generation sequencing and microarray-based gene expression analysis were performed along with HER2 and PD-L1 immunohistochemistry. RESULTS: Long-term responding patients had significantly higher PD-L1 combined positive scores (CPS) and CPS correlated with longer progression-free survival. PD-L1 positivity (CPS ≥ 1) was further associated with an increased CD4+ memory T-cell score. The ERBB2 copy number as well as the tumour mutational burden could not discriminate between short-term and long-term responding patients. Genetic alterations and coamplifications in HER2 pathway associated genes such as EGFR, which were connected to trastuzumab resistance, were present in 10% of the patients and equally distributed between the groups. CONCLUSION: The study highlights the clinical relevance of PD-L1 testing also in the context of trastuzumab treatment and offers a biological rational by demonstrating elevated CD4+ memory T-cells scores in the PD-L1-positive group.

DNA-Methylation Analysis as a Tool for Thymoma Classification.

BACKGROUND: Thymomas are malignant thymic epithelial tumors that are difficult to diagnose due to their rarity and complex diagnostic criteria. They represent a morphologically heterogeneous class of tumors mainly defined by "organo-typical" architectural features and cellular composition. The diagnosis of thymoma is burdened with a high level of inter-observer variability and the problem that some type-specific morphological alterations are more on the continuum than clear-cut. Methylation pattern-based classification may help to increase diagnostic precision, particularly in borderline cases. METHODS AND RESULTS: We applied array-based DNA methylation analysis to a set of 113 thymomas with stringent histological annotation. Unsupervised clustering and t-SNE analysis of DNA methylation data clearly segregated thymoma samples mainly according to the current WHO classification into A, AB, B1, B2, B2/B3, B3, and micronodular thymoma with lymphoid stroma. However, methylation analyses separated the histological subgroups AB and B2 into two methylation classes: mono-/bi-phasic AB-thymomas and conventional/"B1-like" B2-thymomas. Copy number variation analysis demonstrated methylation class-specific patterns of chromosomal alterations. INTERPRETATION: Our study demonstrates that the current WHO classification is generally well reflected at the methylation level but suggests that B2- and AB-thymomas are (epi)genetically heterogeneous. Methylation-based classifications could help to refine diagnostic criteria for thymoma classification, improve reproducibility, and may affect treatment decisions.

nNOS-derived NO modulates force production and iNO-derived NO the excitability in C2C12-derived 3D tissue engineering skeletal muscle via different NO signaling pathways.

Nitric oxide (NO) is a bioactive gas produced by one of the three NO synthases: neuronal NOS (nNOS), inducible (iNOS), and endothelial NOS (eNOS). NO has a relevant modulatory role in muscle contraction; this takes place through two major signaling pathways: (i) activation of soluble guanylate cyclase and, thus, protein kinase G or (ii) nitrosylation of sulfur groups of cysteine. Although it has been suggested that nNOS-derived NO is the responsible isoform in muscle contraction, the roles of eNOS and iNOS and their signaling pathways have not yet been clarified. To elucidate the action of each pathway, we optimized the generation of myooids, an engineered skeletal muscle tissue based on the C2C12 cell line. In comparison with diaphragm strips from wild-type mice, 180 myooids were analyzed, which expressed all relevant excitation-contraction coupling proteins and both nNOS and iNOS isoforms. Along with the biochemical results, myooids treated with NO donor (SNAP) and unspecific NOS blocker (L-NAME) revealed a comparable NO modulatory effect on force production as was observed in the diaphragm strips. Under the effects of pharmacological tools, we analyzed the myooids in response to electrical stimulation of two possible signaling pathways and NO sources. The nNOS-derived NO exerted its negative effect on force production via the sGG-PKG pathway, while iNOS-derived NO increased the excitability in response to sub-threshold electrical stimulation. These results strengthen the hypotheses of previous reports on the mechanism of action of NO during force production, showed a novel function of iNOS-derived NO, and establish the myooid as a novel and robust alternative model for pathophysiological skeletal muscle research.

The heart arrhythmia-linked D130G calmodulin mutation causes premature inhibitory autophosphorylation of CaMKII.

The Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) is well known for transmitting Ca2+-signals, which leads to a multitude of physiological responses. Its functionality is believed to involve CaMKII holoenzyme dynamics where trans-autophosphorylation of the crucial phosphorylation site, T286 occurs. Phosphorylation of this site does not occur when stimulated exclusively with the arrhythmia associated D130G mutant form of CaM in vitro. Here, we present evidence that the loss-of-CaMKII function correlates with premature phosphorylation of its inhibitory phosphosite T306 in CaMKIIα and T307 in CaMKIIδ as this site was up to 20-fold more phosphorylated in the presence of D130G CaM compared to wildtype CaM. Indeed, changing this phosphosite to a non-phosphorylatable alanine reversed the inhibitory effect of D130G both in vitro and in live cell experiments. In addition, several phosphosites with so far undescribed functions directing the Ca2+-sensitivity of the CaMKII sensor were also affected by the presence of the D130G mutation implicating a role of several additional autophosphosites (besides T286 and T306/T307) so far not known to regulate CaMKII Ca2+ sensitivity. Furthermore, we show that introducing a D130G mutation in the CALM2 gene of the P19CL6 pluripotent mouse embryonic carcinoma cell line using CRISPR/Cas9 decreased the spontaneous beat frequency compared to wildtype cells when differentiated into cardiomyocytes supporting an alteration of cardiomyocyte physiology caused by this point mutation. In conclusion, our observations shed for the first time light on how the D130G CaM mutation interferes with the function of CaMKII and how it affects the beating frequency of cardiomyocyte-like cells.

Protein-specific localization of a rhodamine-based calcium-sensor in living cells.

A small synthetic calcium sensor that can be site-specifically coupled to proteins in living cells by utilizing the bio-orthogonal HaloTag labeling strategy is presented. We synthesized an iodo-derivatized BAPTA chelator with a tetramethyl rhodamine fluorophore that allows further modification by Sonogashira cross-coupling. The presented calcium sensitive dye shows a 200-fold increase in fluorescence upon calcium binding. The derivatization with an aliphatic linker bearing a terminal haloalkane-function by Sonogashira cross-coupling allows the localization of the calcium sensor to Halo fusion proteins which we successfully demonstrate in in vitro and in vivo experiments. The herein reported highly sensitive tetramethyl rhodamine based calcium indicator, which can be selectively localized to proteins, is a powerful tool to determine changes in calcium levels inside living cells with spatiotemporal resolution.