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Array comparative genomic hybridization analysis of flat epithelial atypia (DIN1a) and lobular intraepithelial neoplasia

ABSTRACT: Low grade flat ductal intraepithelial neoplasia (DIN1a, flat epithelial atypia) is one of the earliest morphologically recognizable neoplastic lesions of the breast. Frequently, it occurs in association with lobular intraepithelial neoplasia (LIN). The aim of this study was to elucidate chromosomal aberrations in these early neoplastic breast lesions using array comparative genomic hybridization (CGH) analysis. Laser capture microdissection of 12 archival formalin-fixed, paraffin-embedded specimens harbouring both foci of DIN1a as well as LIN was performed. All analyzed cases of DIN1a and LIN showed chromosomal gains and losses. The aberration encountered most often was loss on 16q in 7 DIN1a (70%) and 10 LIN (91%) cases. Regarding changes in chromosome 1, four DIN1a (40%) and 7 LIN (64%) cases showed a gain on 1q. The results of our study show concurrent chromosomal aberrations of 1q gains and 16q losses in several cases with coexisting LIN and low grade flat DIN. These aberrations are known to be common in low grade invasive ductal carcinomas as well as more advanced (conventional) types of low grade DIN (low grade ductal carcinoma in-situ). Our results raise the possibility of similar molecular-genetic pathways in most of the cases with coexisting LIN and low grade flat DIN. In this study, 11 cases of classic lobular intraepithelial neoplasias and 10 cases with several areas of low grade flat DIN (DIN1a or flat epithelial atypia) were analyzed by array CGH. Sections of formalin-fixed paraffin-embedded (FFPE) specimens cut at 7-8µm were mounted on special foil-coated slides (Molecular Devices, San Diego, USA). The sections were then deparaffinized with Xylene, processed in decreasing concentrations of ethanol and stained with haematoxylin. Lasercapture microdissection for both lesions was performed at multiple sites using Veritas Arcturus. For reference-DNA, female mammary tissue without histomorphological changes obtained from reduction mammoplasty specimens was procecessed and laser-microdissected as explained above. Cells were digested in 10µl TE, pH 9, and 0.5µl proteinase K (20mg/ml) for 48h at 55°C. After inactivation of proteinase K at 99°C for 10min, the digest was stored at -20°C. Without any further purification, the complete digest was used for whole genome amplification by means of the WGA (Whole Genome Amplification) kit from Sigma following the manufacturer’s recommendations. Array CGH was performed as described previously. In brief, two µg of amplified tumor and reference DNA were labelled by random priming (BioPrime® Total Genomic Labeling System, Invitrogen, Carlsbad, CA) with Alexa Fluor® 3 and Alexa Fluor® 5, respectively, and hybridized onto a tiling path BAC array, consisting of the human 32k BAC Re-Array Set (BACPAC Resources Center; http://bacpac.chori.org/pHumanMinSet.htm; DNA kindly provided by Pieter de Jong) and a 1Mb Resolution BAC set (clones kindly provided by Nigel Carter, Wellcome Trust Sanger Centre). All protocols are provided in detail on our website (http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/) and more information concerning this platform have been submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/; GPL5114). For the analysis and visualization of array CGH data, our software-package CGH-PRO was employed. No background subtraction was applied. Raw data were normalized by “Subgrid LOWESS”. For the assessment of copy number gains and losses, we used circular binary segmentation in combination with log2 ratio thresholds of 0.15 and -0.15, respectively.

ORGANISM(S): Homo sapiens

SUBMITTER: Reinhard Ullmann

PROVIDER: E-GEOD-18187 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Lobular intraepithelial neoplasia grade 3 (LIN) is a recently recognized variant of intraepithelial lobular neoplasia of the breast composed of either uniform, generally small, cells (classic) with massive lobular distension, pleomorphic cells, signet-ring cells, or any cell type with necrosis. In contrast to classic forms of LIN, there is no consensus on therapeutic strategies for LIN3. In part this is due to the paucity of molecular data that could assist in defining the relationship of LIN3 to classic LIN and carcinomas. In this study we have employed array CGH to determine the patterns of chromosomal aberrations in 9 LIN3 lesions. By comparison to array CGH data of 13 classic LIN lesions, we demonstrate that LIN and LIN3 share several recurrent changes, in particular gains of 1q and losses of 16q. Both aberrations are known to appear early in tumorigenesis and to be associated with good prognosis. However, apart from this overlap, there were a number of karyotypic features that were observed exclusively in LIN3. Clearly, this lesion was characterized by a significantly higher number of DNA copy number changes (9 vs. 31 on average), a considerable complexity of chromosomal rearrangements with up to 16 breakpoints in one chromosome and overlapping high copy amplifications. The amplicons encompassed a number of known oncogenes such as ERBB2, CyclinD1 and FGF3 that have already been reported to be overexpressed in breast carcinomas. Our data suggest that, at the genetic level, LIN3 represents a highly advanced lesion with considerable resemblance to carcinomas and, therefore, reflects a lesion on the verge of invasion, and might represent the transition state from an intraepithelial neoplasm to breast carcinoma. In this study, nine cases of pleomorphic/necrotic LIN lesions as well as one classic LIN and the concurrent invasive lobular carcinoma (ILC) were analyzed by array CGH. Sections of formalin-fixed paraffin-embedded (FFPE) specimens cut at 7-8Âµm were mounted on special foil-coated slides (Molecular Devices, San Diego, USA). The sections were then deparaffinized with Xylene, processed in decreasing concentrations of ethanol and stained with haematoxylin. Lasercapture microdissection for both lesions was performed at multiple sites using Veritas Arcturus. For reference-DNA, female mammary tissue without histomorphological changes obtained from reduction mammoplasty specimens was procecessed and laser-microdissected as explained above. Cells were digested in 10Âµl TE, pH 9, and 0.5Âµl proteinase K (20mg/ml) for 48h at 55Â°C. After inactivation of proteinase K at 99Â°C for 10min, the digest was stored at -20Â°C. Without any further purification, the complete digest was used for whole genome amplification by means of the WGA (Whole Genome Amplification) kit from Sigma following the manufacturerâs recommendations. Array CGH was performed as described previously. In brief, two Âµg of amplified tumor and reference DNA were labelled by random priming (BioPrimeÂ® Total Genomic Labeling System, Invitrogen, Carlsbad, CA) with Alexa FluorÂ® 3 and Alexa FluorÂ® 5, respectively, and hybridized onto a tiling path BAC array, consisting of the human 32k BAC Re-Array Set (BACPAC Resources Center; http://bacpac.chori.org/pHumanMinSet.htm; DNA kindly provided by Pieter de Jong) and a 1Mb Resolution BAC set (clones kindly provided by Nigel Carter, Wellcome Trust Sanger Centre). All protocols are provided in detail on our website (http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/) and more information concerning this platform have been submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/; GLP: 5114). For the analysis and visualization of array CGH data, our software-package CGH-PRO was employed. No background subtraction was applied. Raw data were normalized by âSubgrid LOWESSâ. For the assessment of copy number gains and losses, we used circular binary segmentation in combination with log2 ratio thresholds of 0.15 and -0.15, respectively. High copy amplifications were defined by focal appearance and a log2 ratio exceeding 0.45. In order to statistically investigate the similarity of aberration profiles we applied Pearson's product-moment correlation to cbs ratios of each of the corresponding arrays.

Project description:Copy number variations (CNVs) account for a substantial proportion of human genomic variation, and have been shown to cause neurodevelopmental disorders. We sought to determine the relevance of CNVs to the aetiology of schizophrenia. Whole genome, high resolution, tiling path BAC array comparative genomic hybridization (array CGH) was employed to test DNA from 91 individuals with DSM-IV schizophrenia. Common DNA copy number changes that are unlikely to be directly pathogenic in schizophrenia were identified by comparison to a reference dataset of 372 control individuals analysed in our laboratory, and a screen against the Database of Genomic Variants. The remaining aberrations were tested for inheritance from the parents, and validated with Affymetrix 250K SNP arrays or 244K Agilent oligo-arrays. Thirteen aberrations satisfied our criteria. Two of them are very likely to be pathogenic. A deletion at 2p16.3 disrupts NRXN1 and was present in an affected sibling. A de novo duplication at 15q13.1 spans APBA2. The proteins of these two genes interact directly and play a role in synaptic development and function. Both genes have been affected by CNVs in other neurodevelopmental disorders. Keywords: Array CGH We undertook a systematic search for CNVs in patients with schizophrenia using high resolution, whole genome tiling path BAC arrays. We selected 45 male and 48 female unrelated proband-parent trios from our sample of ~600 Bulgarian SZ trios recruited as described previously. In all cases IQ was > 70. The mean age of probands was 33.8 years (SD = 10.1, range 13-57 years). The mean age at onset of psychotic symptoms was 22.1 years (SD = 6.6, range 11-44 years). Sonicated patient and reference DNA was labelled by random priming (Bioprime Array CGH, Invitrogen, Carlsbad, CA) with Cy3 and Cy5 (Amersham Biosciences, Piscataway, NJ), respectively, and hybridized onto a tiling path BAC array, consisting of ~36,000 BAC clones obtained from several sources as described on our website (http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/). All protocols are also provided on that website. For the analysis and visualization of array CGH data, our software-package CGHPRO was employed. For the assessment of copy number gains and losses, we used conservative log2 ratio thresholds of 0.3 and -0.3, respectively. Deviant signal intensity ratios involving three or more neighboring BAC clones were considered to be potentially pathogenic, unless they were covered by more than one known DNA copy number variant, as listed in the Database of Genomic Variants. No dye swap was done. Array CGH analysis was successful in 91 cases.

Project description:Congenital Hypothyroidism occurs in 1:3500 live births and is therefore the most common congenital endocrine disorder. A spectrum of defective thyroid morphology, termed thyroid dysgenesis, represents 80% of permanent CH cases. Although several candidate genes have been implicated in thyroid development, comprehensive screens failed to detect mutation carriers in a significant number of patients with non-syndromic TD. Due to the sporadic occurrence of TD, de novo chromosomal rearrangements are conceivably representing one of the molecular mechanisms participating in its aetiology. Recently, the use of array CGH technique has provided the ability to map these variations genomewide with high resolution. We performed an array CGH screen of 74 TD patients to determine the role of copy number variants (CNV) in the aetiology of the disease. We identified novel CNVs in 8.75% of all patients that have not been described as frequent variations in the healthy population. Affected patients presented with athyreosis or thyroid hypoplasia and in one case with associated heart malformation. We selected 74 patients with thyroid dysgenesis for array CGH analysis. All individuals were detected in neonatal screening programs and abnormal thyroid gland morphology was subsequently confirmed by ultrasound examination. Intragenic mutations in NKX2-1, FOXE1 and NKX2.5 had been previously excluded in phenotype characteristic individuals. PAX8 mutations were excluded in all patients with hypoplastic thyroids by direct sequencing of the coding exons 1-11. The study was approved by the local ethics committee. Genomic DNA of all subjects as well as of healthy controls was isolated from peripheral blood leucocytes using the Qiagen DNA blood mini kit (Qiagen, Hilden, Germany). Array-comparative genomic hybridization was carried as described previously {Erdogan, 2006 #142; Pinkel, 1998 #151}. In brief, sonicated patient- and control DNA was labeled by random priming with Cy3-dUTP and Cy5-dUTP (Bioprime Array CGH, Invitrogen, Carlsbad, CA), respectively, and hybridized onto a submegabase resolution tiling path BAC array, consisting of ~ 36 000 BAC clones obtained from several sources as described elsewhere {Fiegler, 2003 #198; Ishkanian, 2004 #196; Krzywinski, 2004 #197} . Step-by-step protocols are also provided at http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/. Arrays were scanned with the G2565BA Agilent Microarray Scanner System (resolution 10 µm; PMT 100 % for Cy3/Cy5, respectively) (Agilent Inc. Santa Clara, CA) and analyzed using GENEPIX Pro 5.0 Software. Analysis and visualization of array CGH data were performed with our software package CGHPRO {Chen, 2005 #143}. For the assessment of copy number gains and losses, we used conservative log2 ratio thresholds of 0.3 and -0.3, respectively. Deviant signal intensity ratios involving three or more consecutive BAC clones were considered to be potentially pathogenic, unless they were covered by more than one known DNA copy number variant, as listed in the Database of Genomic Variants (http://projects.tcag.ca/variation/) or covered by > 50% of their length at least once in our reference set of 600 samples. Potentially pathogenic CNVs were verified by array CGH on a 244k oligonucleotide array from Agilent following the manufacturer’s instructions (Protocol-No. G4410-90010). Confirmed CNVs were tested for inheritance by co-hybridization of parental DNA on BAC arrays as described above. All chromosome coordinates are referring to the UCSC Genome Browser Assembly May 2004 (hg17/ NCBI Build 35; available at: http://genome.ucsc.edu/cgi-bin/hgGateway?hgsid=99195739&clade=vertebrate&org=Human&db=hg17). Cytoscape {Shannon, 2003 #201} was used for the elucidation of potential interactions between genes within the intervals of interest.

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Array comparative genomic hybridization analysis of flat epithelial atypia (DIN1a) and lobular intraepithelial neoplasia

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