Low Level of Her-2 Locus Amplification by Fluorescent In Situ Hybridization Does Not Correlate with Her-2 Protein Overexpression by Immunohistochemistry in Barrett's Esophagus

An accurate evaluation of the Her-2 status has important prognostic and therapeutic implications in many carcinomas. The aim of the study was to correlate Her-2 locus (17q11.2) amplification and chromosome 17 gains as assessed by fluorescent in situ hybridization (FISH) with Her-2 protein overexpression by immunohistochemistry (IHC) in patients with Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). We analyzed 34 patients with Her-2 amplification and/or chromosome 17gains using FISH on brush cytology specimens. Seven patients (21%) showed high Her-2 locus amplification (Her-2: Cep17 ≥ 5 : 1), 5 (15%) showed low Her-2 locus amplification (Her-2: Cep17 ≥ 2 < 5 : 1), and 22 (65%) displayed gains of chromosome 17 only. Further, we confirmed Her-2 amplification on corresponding biopsies that were taken at the same occasion as the cytologybrushings. Then, we compared the FISH results with IHC data obtained from the corresponding biopsies and showed that low level of Her-2 amplification does not correlate with Her-2 protein overexpression (score +3/+2; P = 1), in contrast to the high amplification level (P = .001). Thus, in our population of BE and EAC patients, low level of Her-2 amplification does not result in detectable level of Her-2 protein as assessed by IHC

Trastuzumab Mediated T-Cell Response against HER-2/Neu Overexpressing Esophageal Adenocarcinoma Depends on Intact Antigen Processing Machinery

BACKGROUND: Esophageal adenocarcinoma (EAC) is a highly aggressive disease with poor prognosis, which frequently exhibits HER-2 gene amplification. Trastuzumab, the humanized antibody against HER-2, has potent growth inhibitory effects on HER-2 overexpressing cancers. One effect of trastuzumab is that it causes HER-2 receptor internalization and degradation, enhancing presentation of HER-2 epitopes on MHC-Class I molecules. This enhances the ability of HER-2 specific cytotoxic T lymphocytes (CTLs) to recognize and kill cancer cells. Novel strategies targeting the HER-2 receptor either directly by trastuzumab and/or indirectly by inducing a CTL response against HER-2 epitopes with, for instance, DC immunotherapy and consequently combining these strategies might prove to be very effective. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report that trastuzumab has potent growth inhibitory effects on two HER-2 overexpressing EAC cell lines OE33 and OE19. However, we found that trastuzumab and HER-2 specific CTLs act synergistically in inducing tumor lysis in OE33 but not in OE19. We discovered that in OE19 this deficient response is due to a down-regulation of the Transporter Associated with Antigen Processing-2 (TAP-2). TAP-2 is an important member of the Antigen Processing Machinery (APM), and is one of the essential elements for loading antigens on MHC class I molecules. Importantly, we demonstrated that by inducing re-expression of TAP-2 in OE19 with INF-γ treatment or by incubating the cells with INF-γ producing CTLs, the specific anti HER-2 CTL tumor lysis response and synergistic effect with trastuzumab can be restored. CONCLUSION: An inefficient response of HER-2 overexpressing EAC to trastuzumab and/or DC immunotherapy can be due to a down-regulated TAP-2 expression and thus a deficient APM. Future studies combining trastuzumab with IFN-γ and/or immune-therapies inducing potent anti HER-2 CTL responses could lead to an effective combinatorial strategy for successful treatment of HER-2 overexpressing but APM defective cancer

EP-1179: What the gamma? The correlation between QA and clinical risk estimates for prostate RapidArc plans

Influenza virus infection can be accompanied by life-threatening immune pathology most likely due to excessive antiviral responses. Inhibitory immune receptors may restrain such overactive immune responses. To study the role of the inhibitory immune receptor CD200R and its ligand CD200 during influenza infection, we challenged wild-type and CD200(-/-) mice with influenza virus. We found that CD200(-/-) mice in comparison to wild-type controls when inoculated with influenza virus developed more severe disease, associated with increased lung infiltration and lung endothelium damage. CD200(-/-) mice did develop adequate adaptive immune responses and were able to control viral load, suggesting that the severe disease was caused by a lack of control of the immune response. Interestingly, development of disease was completely prevented by depletion of T cells before infection, despite dramatically increased viral load, indicating that T cells are essential for the development of disease symptoms. Our data show that lack of CD200-CD200R signaling increases immune pathology during influenza infection, which can be reduced by T cell depletion. The Journal of Immunology, 2009, 183: 1990-1996

Dynamic clonal equilibrium and predetermined cancer risk in Barrett's oesophagus

abstract: Surveillance of Barrett’s oesophagus allows us to study the evolutionary dynamics of a human neoplasm over time. Here we use multicolour fluorescence in situ hybridization on brush cytology specimens, from two time points with a median interval of 37 months in 195 non-dysplastic Barrett's patients, and a third time point in a subset of 90 patients at a median interval of 36 months, to study clonal evolution at single-cell resolution. Baseline genetic diversity predicts progression and remains in a stable dynamic equilibrium over time. Clonal expansions are rare, being detected once every 36.8 patient years, and growing at an average rate of 1.58 cm[superscript 2] (95% CI: 0.09–4.06) per year, often involving the p16 locus. This suggests a lack of strong clonal selection in Barrett’s and that the malignant potential of ‘benign’ Barrett’s lesions is predetermined, with important implications for surveillance programs.The final version of this article, as published in Nature Communications, can be viewed online at: https://www.nature.com/articles/ncomms1215

Mitochondrial complex 1 activity measured by spectrophotometry is reduced across all brain regions in ageing and more specifically in neurodegeneration

Mitochondrial function, in particular complex 1 of the electron transport chain (ETC), has been shown to decrease during normal ageing and in neurodegenerative disease. However, there is some debate concerning which area of the brain has the greatest complex 1 activity. It is important to identify the pattern of activity in order to be able to gauge the effect of age or disease related changes. We determined complex 1 activity spectrophotometrically in the cortex, brainstem and cerebellum of middle aged mice (70–71 weeks), a cerebellar ataxic neurodegeneration model (pcd5J) and young wild type controls. We share our updated protocol on the measurements of complex1 activity and find that mitochondrial fractions isolated from frozen tissues can be measured for robust activity. We show that complex 1 activity is clearly highest in the cortex when compared with brainstem and cerebellum (p<0.003). Cerebellum and brainstem mitochondria exhibit similar levels of complex 1 activity in wild type brains. In the aged brain we see similar levels of complex 1 activity in all three-brain regions. The specific activity of complex 1 measured in the aged cortex is significantly decreased when compared with controls (p<0.0001). Both the cerebellum and brainstem mitochondria also show significantly reduced activity with ageing (p<0.05). The mouse model of ataxia predictably has a lower complex 1 activity in the cerebellum, and although reductions are measured in the cortex and brain stem, the remaining activity is higher than in the aged brains. We present clear evidence that complex 1 activity decreases across the brain with age and much more specifically in the cerebellum of the pcd5j mouse. Mitochondrial impairment can be a region specific phenomenon in disease, but in ageing appears to affect the entire brain, abolishing the pattern of higher activity in cortical regions

Developmental exposures to common environmental contaminants, DEHP and lead, alter adult brain and blood hydroxymethylation in mice

Introduction: The developing epigenome changes rapidly, potentially making it more sensitive to toxicant exposures. DNA modifications, including methylation and hydroxymethylation, are important parts of the epigenome that may be affected by environmental exposures. However, most studies do not differentiate between these two DNA modifications, possibly masking significant effects.Methods: To investigate the relationship between DNA hydroxymethylation and developmental exposure to common contaminants, a collaborative, NIEHS-sponsored consortium, TaRGET II, initiated longitudinal mouse studies of developmental exposure to human-relevant levels of the phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP), and the metal lead (Pb). Exposures to 25 mg DEHP/kg of food (approximately 5 mg DEHP/kg body weight) or 32 ppm Pb-acetate in drinking water were administered to nulliparous adult female mice. Exposure began 2 weeks before breeding and continued throughout pregnancy and lactation, until offspring were 21 days old. At 5 months, perinatally exposed offspring blood and cortex tissue were collected, for a total of 25 male mice and 17 female mice (n = 5–7 per tissue and exposure). DNA was extracted and hydroxymethylation was measured using hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq). Differential peak and pathway analysis was conducted comparing across exposure groups, tissue types, and animal sex, using an FDR cutoff of 0.15.Results: DEHP-exposed females had two genomic regions with lower hydroxymethylation in blood and no differences in cortex hydroxymethylation. For DEHP-exposed males, ten regions in blood (six higher and four lower) and 246 regions (242 higher and four lower) and four pathways in cortex were identified. Pb-exposed females had no statistically significant differences in blood or cortex hydroxymethylation compared to controls. Pb-exposed males, however, had 385 regions (all higher) and six pathways altered in cortex, but no differential hydroxymethylation was identified in blood.Discussion: Overall, perinatal exposure to human-relevant levels of two common toxicants showed differences in adult DNA hydroxymethylation that was specific to sex, exposure type, and tissue, but male cortex was most susceptible to hydroxymethylation differences by exposure. Future assessments should focus on understanding if these findings indicate potential biomarkers of exposure or are related to functional long-term health effects

TGFbeta induces apoptosis and EMT in primary mouse hepatocytes independently of p53, p21Cip1 or Rb status

Melville Trust for the Care and Cure of Cancer to SP and SS.Background: TGF beta has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT). Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21(Cip1) and pRB, three critical regulators of the G1/S transition are variably involved in TGF beta-induced cell cycle arrest in hepatocytes. As these proteins are also involved in the regulation of apoptosis in many circumstances, we investigated their contribution to other relevant TGF beta-induced effects, namely apoptosis and EMT, and examined how the various processes were interrelated. Methods: Primary mouse hepatocytes deficient in p53, p21 and/or Rb, singly or in combination were treated with TGF beta for 24 to 96 hours. Apoptosis was quantified according to morphology and by immunostaining for cleavedcapsase 3. Epithelial and mesenchymal marker expression was studied using immunocytochemistry and real time PCR. Results: We found that TGF beta similarly induced morphological changes regardless of genotype and independently of proliferation index or sensitivity to inhibition of proliferation by TGF beta. Morphological changes were accompanied by decrease in E-cadherin and increased Snail expression but the mesenchymal markers (N-cadherin, SMA alpha and Vimentin) studied remained unchanged. TGF beta induced high levels of apoptosis in p53-/-, Rb-/-, p21(cip1)-/- and control hepatocytes although with slight differences in kinetics. This was unrelated to proliferation or changes in morphology and loss of cell-cell adhesion. However, hepatocytes deficient in both p53 and p21(cip1)were less sensitive to TGF beta-induced apoptosis. Conclusion: Although p53, p21(Cip1) and pRb are well known regulators of both proliferation and apoptosis in response to a multitude of stresses, we conclude that they are critical for TGF beta-driven inhibition of hepatocytes proliferation, but only slightly modulate TGF beta-induced apoptosis. This effect may depend on other parameters such as proliferation and the presence of other regulatory proteins as suggested by the consequences of p53, p21(Cip1) double deficiency. Similarly, p53, p21(Cip1) and pRB deficiency had no effect on the morphological changes and loss of cell adhesion which is thought to be critical for metastasis. This indicates that possible association of these genes with metastasis potential would be unlikely to involve TGF beta-induced EMT.Publisher PDFPeer reviewe