Nanostructuring lithium niobate substrates by focused ion beam milling

We report on two novel ways for patterning Lithium Niobate (LN) at submicronic scale by means of focused ion beam (FIB) bombardment. The first method consists of direct FIB milling on LiNbO3 and the second one is a combination of FIB milling on a deposited metallic layer and subsequent RIE (Reactive Ion Etching) etching. FIB images show in both cases homogeneous structures with well reproduced periodicity. These methods open the way to the fabrication of photonic crystals on LiNbO3 substrates

LiNbO3 acousto-optical and electro-optical micromodulators

We report on acousto-optical (AO) and electro-optical (EO) LiNbO3 modulators with an active length of only 11 µm. The miniature devices are based on photonic crystal (PhC) structures that are controlled by an external effect (DC electric field or Surface Acoustic Waves). Two processes are presented for realizing the PhCs despite the resistance of the material to etching. The first method is based on direct FIB writing and can yield the fabrication of holes with depth of 32 m and diameter of 12 m or less. The second method consists in FIB patterning of a mask which is deposited on the substrate. This process is followed by proton exchange (PE) and reactive ion etching (RIE). Thus, structures with a diameter of 400 nm and an aspect ratio of 3:1 have been fabricated. The methods have been applied to the fabrication of EO and AO micromodulators showing a driving voltage of 13,5 V and a driving electric power of 20 mW respectively. These developments open the way to dense integration of dynamic optical functionalities

AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines

BACKGROUND: The poly Q polymorphism in AIB1 (amplified in breast cancer) gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification. METHODS: The polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR. RESULTS: Significant amplifications (5–23 folds) of AIB1 gene were found in 2 out of 9 (22%) ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB-330). The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1) and resistance to 4-hydroxy tamoxifen (4-OH TAM) (LCC2 and R27), ICI 182,780 (LCC9) or 4-OH TAM, KEO and LY 117018 (LY-2), AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (<20%) extra poly Q encoding sequence patterns that were derived from the original allele, presumably due to somatic instability. Although all MCF-7 cells and their variants had the same predominant poly Q encoding sequence pattern of (CAG)(3)CAA(CAG)(9)(CAACAG)(3)(CAACAGCAG)(2)CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines. CONCLUSION: These data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification

Expression and subcellular localization of cyclin D1 protein in epithelial ovarian tumour cells

The expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (n = 72), the proportion displaying exclusively cytoplasmic localization of protein was higher in those with serous compared with non-serous histology (P = 0.004, odds ratio 4.8, 95% confidence interval 1.4–19.1). Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCNDI) was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification. © 1999 Cancer Research Campaig

Genetic profiling of chromosome 1 in breast cancer: mapping of regions of gains and losses and identification of candidate genes on 1q

Chromosome 1 is involved in quantitative anomalies in 50–60% of breast tumours. However, the structure of these anomalies and the identity of the affected genes remain to be determined. To characterise these anomalies and define their consequences on gene expression, we undertook a study combining array-CGH analysis and expression profiling using specialised arrays. Array-CGH data showed that 1p was predominantly involved in losses and 1q almost exclusively in gains. Noticeably, high magnitude amplification was infrequent. In an attempt to fine map regions of copy number changes, we defined 19 shortest regions of overlap (SROs) for gains (one at 1p and 18 at 1q) and of 20 SROs for losses (all at 1p). These SROs, whose sizes ranged from 170 kb to 3.2 Mb, represented the smallest genomic intervals possible based on the resolution of our array. The elevated incidence of gains at 1q, added to the well-established concordance between DNA copy increase and augmented RNA expression, made us focus on gene expression changes at this chromosomal arm. To identify candidate oncogenes, we studied the RNA expression profiles of 307 genes located at 1q using a home-made built cDNA array. We identified 30 candidate genes showing significant overexpression correlated to copy number increase. In order to substantiate their involvement, RNA expression levels of these candidate genes were measured by quantitative (Q)-RT–PCR in a panel of 25 breast cancer cell lines previously typed by array-CGH. Q–PCR showed that 11 genes were significantly overexpressed in the presence of a genomic gain in these cell lines, and 20 overexpressed when compared to normal breast

Frequency, prognostic impact, and subtype association of 8p12, 8q24, 11q13, 12p13, 17q12, and 20q13 amplifications in breast cancers

BACKGROUND: Oncogene amplification and overexpression occur in tumor cells. Amplification status may provide diagnostic and prognostic information and may lead to new treatment strategies. Chromosomal regions 8p12, 8q24, 11q13, 17q12 and 20q13 are recurrently amplified in breast cancers. METHODS: To assess the frequencies and clinical impact of amplifications, we analyzed 547 invasive breast tumors organized in a tissue microarray (TMA) by fluorescence in situ hybridization (FISH) and calculated correlations with histoclinical features and prognosis. BAC probes were designed for: (i) two 8p12 subregions centered on RAB11FIP1 and FGFR1 loci, respectively; (ii) 11q13 region centered on CCND1; (iii) 12p13 region spanning NOL1; and (iv) three 20q13 subregions centered on MYBL2, ZNF217 and AURKA, respectively. Regions 8q24 and 17q12 were analyzed with MYC and ERBB2 commercial probes, respectively. RESULTS: We observed amplification of 8p12 (amplified at RAB11FIP1 and/or FGFR1) in 22.8%, 8q24 in 6.1%, 11q13 in 19.6%, 12p13 in 4.1%, 17q12 in 9.9%, 20q13(Z )(amplified at ZNF217 only) in 9.9%, and 20q13(Co )(co-amplification of two or three 20q13 loci) in 8.5% of cases. The 8q24, 12p13, and 17q12 amplifications were correlated with high grade. The most frequent single amplifications were 8p12 (9.8%), 8q24 (3.3%) and 12p13 (3.3%), 20q13(Z )and 20q13(Co )(1.6%) regions. The 17q12 and 11q13 regions were never found amplified alone. The most frequent co-amplification was 8p12/11q13. Amplifications of 8p12 and 17q12 were associated with poor outcome. Amplification of 12p13 was associated with basal molecular subtype. CONCLUSION: Our results establish the frequencies, prognostic impacts and subtype associations of various amplifications and co-amplifications in breast cancers

PIK3CA mutations are common in lobular carcinoma in situ, but are not a biomarker of progression

Sample and data collection were funded by Cancer Research UK. Analysis was funded by Breast Cancer Now, the Rosetrees Trust, Guys & St Thomas’ Charity (CanHelp) and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London

Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression

The progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) marks a critical step in the evolution of breast cancer. There is some evidence to suggest that dynamic interactions between the neoplastic cells and the tumour microenvironment play an important role. Using the whole-genome cDNA-mediated annealing, selection, extension and ligation assay (WG-DASL, Illumina), we performed gene expression profiling on 87 formalin-fixed paraffin-embedded (FFPE) samples from 17 patients consisting of matched IDC, DCIS and three types of stroma: IDC-S ( 10 mm from IDC or DCIS). Differential gene expression analysis was validated by quantitative real time-PCR, immunohistochemistry and immunofluorescence. The expression of several genes was down-regulated in stroma from cancer patients relative to normal stroma from reduction mammoplasties. In contrast, neoplastic epithelium underwent more gene expression changes during progression, including down regulation of SFRP1. In particular, we observed that molecules related to extracellular matrix (ECM) remodelling (e.g. COL11A1, COL5A2 and MMP13) were differentially expressed between DCIS and IDC. COL11A1 was overexpressed in IDC relative to DCIS and was expressed by both the epithelial and stromal compartments but was enriched in invading neoplastic epithelial cells. The contributions of both the epithelial and stromal compartments to the clinically important scenario of progression from DCIS to IDC. Gene expression profiles, we identified differential expression of genes related to ECM remodelling, and specifically the elevated expression of genes such as COL11A1, COL5A2 and MMP13 in epithelial cells of IDC. We propose that these expression changes could be involved in facilitating the transition from in situ disease to invasive cancer and may thus mark a critical point in disease development