Assessment of the three-test genetic toxicology battery for groundwater metabolites.

The two-test in vitro battery for genotoxicity testing (Ames and micronucleus) has in the majority of cases replaced the three-test battery (as two-test plus mammalian cell gene mutation assay) for the routine testing of chemicals, pharmaceuticals, cosmetics, and agrochemical metabolites originating from food and feed as well as from water treatment. The guidance for testing agrochemical groundwater metabolites, however, still relies on the three-test battery. Data collated in this study from 18 plant protection and related materials highlights the disparity between the often negative Ames and in vitro chromosome aberration data and frequently positive in vitro mammalian cell gene mutation assays. Sixteen of the 18 collated materials with complete datasets were Ames negative, and overall had negative outcomes in in vitro chromosome damage tests (weight of evidence from multiple tests). Mammalian cell gene mutation assays (HPRT and/or mouse lymphoma assay (MLA)) were positive in at least one test for every material with this data. Where both MLA and HPRT tests were performed on the same material, the HPRT seemed to give fewer positive responses. In vivo follow-up tests included combinations of comet assays, unscheduled DNA synthesis, and transgenic rodent gene mutation assays, all gave negative outcomes. The inclusion of mammalian cell gene mutation assays in a three-test battery for groundwater metabolites is therefore not justified and leads to unnecessary in vivo follow-up testing.

Looking for more reliable biomarkers in breast cancer: Comparison between routine methods and RT-qPCR.

PURPOSE: Decades of quality control efforts have raised the standards of immunohistochemistry (IHC), the principle method used for biomarker testing in breast cancer; however, computational pathology and reverse transcription quantitative PCR (RT-qPCR) may also hold promise for additional substantial improvements. METHODS: Herein, we investigated discrepancies in the assessment of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and marker of proliferation Ki67 comparing routinely obtained IHC (and FISH) data (ORI) with the results of manual (REV) and semi-automated (DIA) re-evaluation of the original IHC slides and then with RNA expression data from the same tissue block using the MammaTyper® (MT) gene expression assay. RESULTS: Correlation for ER and PR was high between ORI IHC and the other three study methods (REV, DIA and RT-qPCR). For HER2, 10 out of 96 discrepant cases can be detected between ORI and REV that involved at least one call in the equivocal category (except for one case). For Ki67, 22 (29.1%) cases were categorized differently by either REV alone (n = 17), DIA alone (n = 15) or both (n = 10) and 28 cases (29.2%) for RT-qPCR. Most of the discrepant Ki67 cases changed from low to high between the original and following assessment and belonged to the intermediate Ki67 expression range (between 9 and 30%). CONCLUSIONS: Determination of the breast cancer biomarkers ER, PR, HER2 and Ki67 at the mRNA level shows high degree of correlation with IHC and compares well with correlations between original with subsequent independent manual or semi-automated IHC assessments. The use of methods with wider dynamic range and higher reproducibility such as RT-qPCR may offer more precise assessment of endocrine responsiveness, improve Ki67 standardization and help resolve HER2 cases that remain equivocal or ambiguous by IHC/FISH. In summary, our findings seem to configure RT-qPCR as a complementary method to be used in cases of either equivocal results or presenting, at the traditional determination assays, biomarkers expressions close to the cut-off values.

Impact of molecular subtypes on the prediction of distant recurrence in estrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2) negative breast cancer upon five years of endocrine therapy.

BACKGROUND: Current evidence suggests that patients with Luminal A early breast cancer can skip chemotherapy or extended endocrine therapy, but immunohistochemistry-based biomarker analysis for St Gallen subtyping may not be reproducible. We asked whether RT-qPCR can be used instead to address this clinical question. METHODS: RNA was extracted from tumor material derived from ER+/HER2- patients receiving adjuvant endocrine treatment for low-risk cancers and was semi-quantified by RT-qPCR with the MammaTyper®. St Gallen subtypes were based on the mRNA expression of ERBB2/HER2, ESR1/ER, PGR/PR and MKI67/Ki67 after dichotomizing at predefined cut-offs. Differences in distant disease-free survival (DDFS) were assessed by Kaplan Meier analysis and Cox regression. RESULTS: With a median follow up of 7.8 years, there were ten events in the group of 195 Luminal A-like tumors (5.1%) and 18 events in the remaining 127 tumors (14.1%), consisting mostly of Luminal B-like cases (N = 119). Luminal A-like had significantly better DDFS over the entire follow-up period (HR 0.35, 95% CIs 0.16-0.76, p = 0.0078) with a trend towards reduced probability of recurrences also in the late phase (> 5 years) (HR 0.20, p = 0.052). The survival advantage spanning the entire follow-up period persisted in the pN0 or pN0-N1 subgroups or after correcting for clinicopathological parameters. MKI67 alone significantly predicted for worse DDFS (HR 2.62, 95% CIs 1.24-5.56, p = 0.0088). CONCLUSIONS: St Gallen Luminal A-like tumors identified by RT-qPCR display markedly low rates of distant recurrence at ten years follow-up. Patients with such tumors could be spared chemotherapy due to the obviously unfavourable benefit/toxicity ratio.

Robustness of biomarker determination in breast cancer by RT-qPCR: impact of tumor cell content, DCIS and non-neoplastic breast tissue.

BACKGROUND: Tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) breast cancer specimens may affect the accuracy of reverse transcription quantitative real-time PCR (RT-qPCR). Herein, we tested the impact of tissue heterogeneity of breast cancer specimen on the RT-qPCR-based gene expression assay MammaTyper®. METHODS: MammaTyper® quantifies the mRNA expression of the four biomarkers ERBB2, ESR1, PGR, and MKI67. Based on pre-defined cut-off values, this molecular in vitro diagnostic assay permits binary marker classification and determination of breast cancer subtypes as defined by St Gallen 2013. In this study, we compared data from whole FFPE sections with data obtained in paired RNA samples after enrichment for invasive carcinoma via macro- or laser-capture micro-dissection. RESULTS: Compared to whole sections, removal of surrounding adipose tissue by macrodissection generated mean absolute 40-ddCq differences of 0.28-0.32 cycles for all four markers, with ≥90% concordant binary classifications. The mean raw marker Cq values in the adipose tissue were delayed by 6 to 7 cycles compared with the tumor-enriched sections, adding a trivial linear fold change of 1.0078 to 1.0156. Comparison of specimens enriched for invasive tumor with whole sections with as few as 20% tumor cell content resulted in mean absolute differences that remained on average below 0.59 Cq. The mean absolute difference between whole sections containing up to 60% ductal carcinoma in situ (DCIS) and specimens after dissection of DCIS was only 0.16-0.25 cycles, although there was a tendency for higher gene expression in DCIS. Observed variations were related to small size of samples and proximity of values to the limit of detection. CONCLUSION: Expression of ESR1, PGR, ERBB2 and MKI67 by MammaTyper® is robust in clinical FFPE samples. Assay performance was unaffected by adipose tissue and was stable in samples with as few as 20% tumor cell content and up to 60% DCIS.

Adipocyte Glucocorticoid Receptor Deficiency Attenuates Aging- and HFD-Induced Obesity and Impairs the Feeding-Fasting Transition.

Glucocorticoids (GCs) are important regulators of systemic energy metabolism, and aberrant GC action is linked to metabolic dysfunctions. Yet, the extent to which normal and pathophysiological energy metabolism depend on the GC receptor (GR) in adipocytes remains unclear. Here, we demonstrate that adipocyte GR deficiency in mice significantly impacts systemic metabolism in different energetic states. Plasma metabolomics and biochemical analyses revealed a marked global effect of GR deficiency on systemic metabolite abundance and, thus, substrate partitioning in fed and fasted states. This correlated with a decreased lipolytic capacity of GR-deficient adipocytes under postabsorptive and fasting conditions, resulting from impaired signal transduction from ß-adrenergic receptors to adenylate cyclase. Upon prolonged fasting, the impaired lipolytic response resulted in abnormal substrate utilization and lean mass wasting. Conversely, GR deficiency attenuated aging-/diet-associated obesity, adipocyte hypertrophy, and liver steatosis. Systemic glucose tolerance was improved in obese GR-deficient mice, which was associated with increased insulin signaling in muscle and adipose tissue. We conclude that the GR in adipocytes exerts central but diverging roles in the regulation of metabolic homeostasis depending on the energetic state. The adipocyte GR is indispensable for the feeding-fasting transition but also promotes adiposity and associated metabolic disorders in fat-fed and aged mice.

Molecular Actions of Glucocorticoids in Cartilage and Bone During Health, Disease, and Steroid Therapy.

Cartilage and bone are severely affected by glucocorticoids (GCs), steroid hormones that are frequently used to treat inflammatory diseases. Major complications associated with long-term steroid therapy include impairment of cartilaginous bone growth and GC-induced osteoporosis. Particularly in arthritis, GC application can increase joint and bone damage. Contrarily, endogenous GC release supports cartilage and bone integrity. In the last decade, substantial progress in the understanding of the molecular mechanisms of GC action has been gained through genome-wide binding studies of the GC receptor. These genomic approaches have revolutionized our understanding of gene regulation by ligand-induced transcription factors in general. Furthermore, specific inactivation of GC signaling and the GC receptor in bone and cartilage cells of rodent models has enabled the cell-specific effects of GCs in normal tissue homeostasis, inflammatory bone diseases, and GC-induced osteoporosis to be dissected. In this review, we summarize the current view of GC action in cartilage and bone. We further discuss future research directions in the context of new concepts for optimized steroid therapies with less detrimental effects on bone.

Sex differences of carotid intima-media thickness in healthy children and adolescents.

OBJECTIVES: In pediatric cardiovascular health prevention non-invasive diagnostics of subclinical atherosclerosis is essential for screenings and interventional purposes. Increased carotid artery intima-media thickness (IMT) has been reported in the presence of cardiovascular risk factors in childhood. However, little information exists regarding the age and sex distribution of IMT as stated in adults. This study investigates first, if IMT sonography is reliable and feasible; second, calculates sex- and age-specific percentiles; third, re-confirms IMT associations to cardiovascular risk parameters and analyses predictors for age-adjusted IMT. METHODS: 267 healthy pupils (age 6-17 years) were examined prospectively. Standardized IMT sonography and offline analysis were performed. Anthropometric data, BMI, body composition, blood pressure were taken; Spearman's rank correlation coefficient and multiple stepwise linear regression analyses were calculated. RESULTS: Intra-observer variability: CV was 2.42% (n=132; MD=0.012mm; r=0.849); inter-observer variability: CV was 1.71% (n=75; MD=0.013mm; r=0.780). Age- and sex-specific IMT percentiles for age groups 8/9 to 14/15 years were calculated. IMT values were higher in boys than in girls at the same age. Systolic blood pressure and IMT were positively related in boys (p<0.001, r=0.31) and girls (p=0.005, r=0.24). Predictors for age-adjusted IMT: systolic blood pressure was shown to be a predictor (r(2)=0.10, beta=0.31, p<0.001) in boys; weight emerged as a predictor (r(2)=0.19, beta=0.43, p<0.001) in girls. CONCLUSION: The main study benefit is the provision of IMT percentiles for both sexes for the age groups 8/9 to 14/15 years. The results suggest that sex-specific prevention should be given further attention in a comprehensive and multi-risk parameters approach.