Microenvironment‐induced restoration of cohesive growth associated with focal activation of P ‐cadherin expression in lobular breast carcinoma metastatic to the colon

Invasive lobular carcinoma (ILC) is a special breast cancer type characterized by noncohesive growth and E‐cadherin loss. Focal activation of P‐cadherin expression in tumor cells that are deficient for E‐cadherin occurs in a subset of ILCs. Switching from an E‐cadherin deficient to P‐cadherin proficient status (EPS) partially restores cell–cell adhesion leading to the formation of cohesive tubular elements. It is unknown what conditions control EPS. Here, we report on EPS in ILC metastases in the large bowel. We reviewed endoscopic colon biopsies and colectomy specimens from a 52‐year‐old female (index patient) and of 18 additional patients (reference series) diagnosed with metastatic ILC in the colon. EPS was assessed by immunohistochemistry for E‐cadherin and P‐cadherin. CDH1 /E‐cadherin mutations were determined by next‐generation sequencing. The index patient's colectomy showed transmural metastatic ILC harboring a CDH1 /E‐cadherin p.Q610* mutation. ILC cells displayed different growth patterns in different anatomic layers of the colon wall. In the tunica muscularis propria and the tela submucosa, ILC cells featured noncohesive growth and were E‐cadherin‐negative and P‐cadherin‐negative. However, ILC cells invading the mucosa formed cohesive tubular elements in the intercryptal stroma of the lamina propria mucosae. Inter‐cryptal ILC cells switched to a P‐cadherin‐positive phenotype in this microenvironmental niche. In the reference series, colon mucosa infiltration was evident in 13 of 18 patients, one of which showed intercryptal EPS and conversion to cohesive growth as described in the index patient. The large bowel is a common metastatic site in ILC. In endoscopic colon biopsies, the typical noncohesive growth of ILC may be concealed by microenvironment‐induced EPS and conversion to cohesive growth

An Extended ΔCT-Method Facilitating Normalisation with Multiple Reference Genes Suited for Quantitative RT-PCR Analyses of Human Hepatocyte-Like Cells

<div><p>Reference genes (RG) as sample internal controls for gene transcript level analyses by quantitative RT-PCR (RT-qPCR) must be stably expressed within the experimental range. A variety of <i>in vitro</i> cell culture settings with primary human hepatocytes, and Huh-7 and HepG2 cell lines, were used to determine candidate RG expression stability in RT-qPCR analyses. Employing GeNorm, BestKeeper and Normfinder algorithms, this study identifies <i>PSMB6, MDH1</i> and some more RG as sufficiently unregulated, thus expressed at stable levels, in hepatocyte-like cells <i>in vitro</i>. Inclusion of multiple RG, quenching occasional regulations of single RG, greatly stabilises gene expression level calculations from RT-qPCR data. To further enhance validity and reproducibility of relative RT-qPCR quantifications, the ΔCT calculation can be extended (e-ΔCT) by replacing the CT of a single RG in ΔCT with an averaged CT-value from multiple RG. The use of two or three RG - here identified suited for human hepatocyte-like cells - for normalisation with the straightforward e-ΔCT calculation, should improve reproducibility and robustness of comparative RT-qPCR-based gene expression analyses.</p></div

Breastfeeding and its prospective association with components of the GH-IGF-Axis, insulin resistance and body adiposity measures in young adulthood--insights from linear and quantile regression analysis.

BACKGROUND: Breastfeeding may lower chronic disease risk by long-term effects on hormonal status and adiposity, but the relations remain uncertain. OBJECTIVE: To prospectively investigate the association of breastfeeding with the growth hormone- (GH) insulin-like growth factor- (IGF) axis, insulin sensitivity, body composition and body fat distribution in younger adulthood (18-37 years). DESIGN: Data from 233 (54% female) participants of a German cohort, the Dortmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study, with prospective data on infant feeding were analyzed. Multivariable linear as well as quantile regression were performed with full breastfeeding (not: ≤ 2, short: 3-17, long: >17 weeks) as exposure and adult IGF-I, IGF binding proteins (IGFBP) -1, -2, -3, homeostasis model assessment of insulin resistance (HOMA-IR), fat mass index, fat-free mass index, and waist circumference as outcomes. RESULTS: After adjustment for early life and socio-economic factors, women who had been breastfed longer displayed higher adult IGFBP-2 (p(trend) = 0.02) and lower values of HOMA-IR (p(trend) = 0.004). Furthermore, in women breastfeeding duration was associated with a lower mean fat mass index (p(trend) = 0.01), fat-free mass index (p(trend) = 0.02) and waist circumference (p(trend) = 0.004) in young adulthood. However, there was no relation to IGF-I, IGFBP-1 and IGFBP-3 (all p(trend) > 0.05). Associations for IGFBP-2 and fat mass index were more pronounced at higher, for waist circumference at very low or high percentiles of the distribution. In men, there was no consistent relation of breastfeeding with any outcome. CONCLUSIONS: Our data suggest that breastfeeding may have long-term, favorable effects on extremes of adiposity and insulin metabolism in women, but not in men. In both sexes, breastfeeding does not seem to induce programming of the GH-IGF-axis

e-ΔCT-method and representative calculations of changes of target gene expression levels (expressed as fold change).

<p>(A) Calculation of e-ΔCT, an extension of the ΔCT-method by first calculating the mean CT-value of multiple RG included for each sample. (B) Depiction of data processing after geNorm-/e-ΔCT-calculations of sample-specific G-values to calculate fold changes and their significance (p-values) between two experimental settings, each consisting of three fully independent samples. (C) Example (Huh-7, without vs. with ActD) comparing fold changes in gene expression levels calculated by geNorm (grey) and e-ΔCT (black) using: GAPDH, the most common RG, the AS best ranking PSMB6, MDH1 and ACTB, and inclusion of two (PSMB6 and MDH1; P_M) or three (PSMB6, MDH1 and ACTB; P_M_A) RG. (D) Examples (primary hepatocytes, 0 vs. 24 hrs) comparing use of most stable RG of the subgroup (RG group, i.e. PH) vs. overall RG (RG all, i.e. AS) via e-ΔCT. *: p-values <0.05.</p

Evaluation and ranking of reference genes.

<p>(A) Overall ranking of 22 RG using geNorm, Bestkeeper and Normfinder algorithms and calculation of the cumulative ranking (column 4). (B) geNorm derived M-values as measures for the average pairwise expression stabilities within AS (column 1), and in the subgroups DD (2), CC (3) and PH (4). (C) Box-whisker plots of all CT-values of reference genes and target genes examined. Median (central horizontal line), the 25th and 75th quartile (boxes) and whiskers for the total CT-range are shown.</p

GH-IGF-axis and HOMA-IR in young adulthood according to breastfeeding duration in multivariable quantile regression models.

<p>Displayed are point estimates (95% CI) for IGF-I (A), IGFBP-1 (B), IGFBP-3 (C), IGFBP-2 (D) and HOMA-IR (E) differences between women and men breastfed for a long duration (i.e. >17 weeks) vs. those not breastfed (i.e. ≤2 weeks) for specific percentiles (10^th, 25^th, 50^th, 75^th and 90^th percentiles). Models included age in adulthood, maternal overweight (yes/no), paternal university degree (yes/no), firstborn status (yes/no), smoking in the household (yes/no) in the case of FMI and WC; in the case of FFMI: age in adulthood, maternal overweight (yes/no), paternal university degree (yes/no), birth weight and length (appropriate for gestational age yes/no), firstborn status (yes/no), smoking in the household (yes/no). DONALD Study, n = 228-232. * p<0.05 DONALD, Dortmund Nutritional and Anthropometric Longitudinally Designed; IGF-I, insulin-like growth factor 1, IGFBP, insulin-like growth factor binding protein, HOMA-IR, homeostasis model assessment for insulin resistance.</p

Body composition and body fat distribution in young adulthood according to breastfeeding duration in infancy, DONALD Study (n = 125 women, 108 men)^a.

a<p>Data are adjusted means (95% CI). Abbreviations used: DONALD, Dortmund Nutritional and Anthropometric Longitudinally Designed. Missing values: n = 1 for firstborn status.</p>b<p>Model 1: adjusted for age in adulthood.</p>c<p>Model 2 for FMI, WC: adjusted for age in adulthood, maternal overweight (yes/no), paternal university degree (yes/no), firstborn status (yes/no), smoking in the household (yes/no). Model 2 for FFMI: adjusted for age in adulthood, maternal overweight (yes/no), paternal university degree (yes/no), birth weight and length (appropriate for gestational age yes/no), firstborn status (yes/no), smoking in the household (yes/no).</p

Body composition measures in young adulthood according to breastfeeding duration in multivariable quantile regression models.

<p>Displayed are point estimates (95% CI) for FMI (A), FFMI (B) and WC (C) differences between women and men breastfed for a long duration (i.e. >17 weeks) vs. those not breastfed (i.e. ≤2 weeks) for specific percentiles (10^th, 25^th, 50^th, 75^th and 90^th percentiles) in multivariable quantile regression models. Models included age in adulthood, maternal overweight (yes/no), paternal university degree (yes/no), firstborn status (yes/no), smoking in the household (yes/no) in the case of FMI and WC, and age in adulthood, maternal overweight (yes/no), paternal university degree (yes/no), birth weight and length (appropriate for gestational age yes/no), firstborn status (yes/no), smoking in the household (yes/no) in the case of FFMI. DONALD Study, n = 232. * p<0.05 DONALD, Dortmund Nutritional and Anthropometric Longitudinally Designed; FFMI, fat-free mass index; FMI, fat mass index; WC, waist circumference.</p