Project description:DNA copy number analysis of myxoinflammatory fibroblastic sarcomas, and related lesions, using 32k BAC array CGH. All four cases analyzed with array CGH showed amplification of the region 86.30-87.74 Mb in 3p11.1-12.1. No additional aberration was detected in more than two tumors, except for the deletion distal to the amplification on chromosome 3. Case 1 showed deletion affecting the region from the centromere of chromosome 1 to the 5'-end of TGFBR3. Homozygous deletion was found in one case (case 6) affecting the region 21.08-22.15 Mb on chromosome arm 9p harboring the CDKN2A and CDKN2B genes. DNA copy numbers in cases 1 and 6-8 were analyzed using tiling microarrays containing more than 32 000 partly overlapping BAC clones, generating complete coverage of the human genome (JÃ¶nsson et al., 2007, Genes Chromosomes Cancer 46: 543-58.). The arrays were produced at the Swegene DNA Microarray Resource Center, Department of Oncology, Lund University (http://swegene.onk.lu.se), as previously described (JÃ¶nsson et al., 2007), using BAC clones mapped to the hg17 genome build. Extraction, labeling and hybridization of genomic DNA from freshly frozen tumor biopsies, as well as pretreatment and washing of slides were performed as described (Heidenblad et al., 2006, Oncogene 25: 7106-16). As a control for normal copy number, a DNA pool derived from multiple healthy male donors was used (Promega, Madison, WI, USA). Primary array CGH data were collected using the GenePix Pro 4.0 (Axon Instruments Inc., Foster City, CA, USA). The quantified data matrix was deposited into the web-based database BioArray Software Environment (BASE) (Saal et al., 2002, Genome Biol 3: software0003.1-0003.6), and data analyses were done as described (Hallor et al., 2008, Br J Cancer 98: 434-42). In brief, following background correction the log(2) ratios were calculated for each spot, and unreliable features and spots not showing signal-to-noise ratios ?5 for both channels were eliminated. Normalization of data was performed using pin-based Lowess normalization and single outlier probes were removed. Thereafter, the log(2) ratios for each sample were segmented, followed by elimination of segments less than 500 kb in size. Copy number alterations were determined by comparing the segmented log(2) ratios to gain/loss thresholds obtained by an adaptive scaling method (Staaf et al., 2007, BMC Genomics 8: 382).

Project description:Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. Fine mapping of such genomic changes, and evaluation of associated expression changes, are expected to provide reagents for diagnosis, insights into understanding the disease, and open up avenues for novel therapeutic intervention. We made an effort to search for genes on chromosome 8 with altered copy number and expression. A high resolution (0.1Mb) bacterial artificial chromosome (BAC) array of chromosome 8 that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC DNA. Hybridization of DNA from a breast cancer cell line (SKBR3) with two known amplified regions (8q21 and 8q24) resulted in resolving this region into 6 distinct amplicons, in addition identified 3 deleted regions. The boundaries and the peak of each region were verified by FISH, and the extent of amplification/deletion for each region was validated by qPCR. Using these BAC arrays, CGH was performed with a total of eight breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis. Two consensus regions in 8q24 included a 1.2 Mb region (125.3-126.5 Mb) and a 1.0 Mb region (128.1-129.1 Mb) that were amplified in 7/8 cell lines, and in the 8q21 region there was a smaller 0.88 Mb region (86.62-87.50 Mb) amplified in 4/8 cell lines. A global expression analysis was performed for all these cell lines using a high-density oligonucleotide array to identify the genes whose expression correlated with amplification/deletion. Representative genes from 5 commonly amplified regions (REXO1L1, TMEM55A, TRMT12, MTSS1, EIF2C2, MYC) and 2 deleted regions (DPYSL2, NRG1 and NDRG1) were verified by qPCR and RT-qPCR for genomic and expression changes. Validation by RT-qPCR using RNA from 30 breast tumors showed that the TRMT12 (125.5 Mb) gene were overexpressed in many of the tumors. TRMT12 is homolog of a yeast gene encoding tRNA methyltransferase involved in biosythesis of a modified base in a tRNA. It would be interesting to explore the role of TRMT12 and the RNA processing pathway in tumorigenesis. Keywords: Comparative Genomic Hybridization (CGH), Breast cancer cell lines, Expression, BAC array, Oligo expression array

Project description:In this study, we extend array CGH technology by making the accurate detection of segmental aneusomies possible from a single lymphoblast and fibroblast following Phi29 DNA polymerase amplification Array CGH experiments were performed on two segmental aneusomic cell lines derived from patients. As a proof of principle, an interstitial 4q-deletion (46, XX, del(4)(q13.1q22.3)) fibroblast cell line and an unbalanced reciprocal translocation involving chromosomes 14 and X (46, XX, der (X) t(X;14)(q21.3;q23.1)) EBV cell line were used. Array CGH experiments were performed using gDNA from the two cell lines to define the exact size of each rearrangement. For the 4q-deletion cell line, the size of the deleted region was 34 Mb corresponding to 39 clones spotted on the array (from RP11-340A13 to RP11-44P19). For the EBV cell line, the size of the 14q-duplication was 47 Mb corresponding to 63 clones (from RP11-62H20 to CTC-820M16) and the Xq-deletion was 58 Mb corresponding to 70 clones (from RP3-380C13 to RP11-218L14). For each cell line, three single cells were amplified. Following DNA amplification, all cells showed the expected DNA yields (n=6; ï?³ = 2.36 Âµg ï?± 0.12). Sex-mismatch array CGH experiments were conducted on amplified DNA samples obtained from the 4q-deletion cell line. Using the chromosome specific threshold obtained from our 18 experiments, sex chromosome and autosome ploidy levels were accurately identified with no false negative and no false positive results. Averaging the 39 clones within the chromosome 4q-deleted region enabled the accurate detection of the deletions. For each of the three amplified single-cell DNA of the unbalanced reciprocal translocation involving chromosomes 14 and X, male and female gDNA were used as references. Sex chromosome and autosome ploidy levels were accurately identified. Averaging intensity ratios of the 63 clones within the chromosome 14 duplicated region enabled the accurate detection of the duplications in the three replicate experiments. When, respectively, male or female gDNA were used as a reference, the average log2 mean ratio of the Xq-deleted region was â??0.04 or â??0.93, close to the theoretical expected value of 0 or minus 1. Interestingly, the log2 mean ratios of chromosomes 1 to 22 were highly similar when the same single cell amplified DNA was used for the two experiments using respectively female and male DNA as a reference. Hence, array CGH intensity ratio profiles were very reproducible.

Project description:Background: The amplification event occurring at chromosome locus 11q13, reported in several different cancers, includes a number of potential oncogenes. We have previously reported amplification of one such oncogene, CCND1, to be correlated with an adverse effect of tamoxifen in premenopausal breast cancer patients. Overexpression of cyclin D1 protein however, confers tamoxifen resistance but not a tamoxifen induced adverse effect. Potentially, co-amplification of an additional 11q13 gene, with a resulting protein overexpression, is required to cause an agonistic effect. Moreover, during 11q13 amplification a deletion of the distal 11q region has been described. In order to assess the potential impact of the deletion we have examined a selected marker for this event. Methods: Array CGH analysis was employed to identify and confirm changes in the gene expression of a number of different genes mapping to the 11q chromosomal region, associated with CCND1 amplification. The subsequent protein expression of these candidate genes was then examined in a clinical material of 500 primary breast cancers from premenopausal patients randomized to either tamoxifen or no adjuvant treatment. The protein expression was also compared to the CGH data in a subset of 56 breast cancer samples. Results: Cortactin and FADD overexpression was linked to CCND1 amplification, determined by FISH, but was not associated with a diminished effect of tamoxifen. However, deletion of distal chromosome 11q, defined as downregulation of the marker Chk1, was associated with an impaired tamoxifen response, and interestingly, also with low proliferative breast cancer of low grade. For Pak1 and cyclin D1 the protein expression corresponded to the gene expression data from the CGH analysis. Conclusions: The results indicate that many 11q13 associated gene products are overexpressed in conjunction with cyclin D1 but not linked to an agonistic effect of tamoxifen. Finally, the deletion of distal 11q, linked to 11q13 amplification, might be an important event affecting breast cancer outcome and tamoxifen response. Keywords: comparative genomic hybridisation

Project description:Background: The amplification event occurring at chromosome locus 11q13, reported in several different cancers, includes a number of potential oncogenes. We have previously reported amplification of one such oncogene, CCND1, to be correlated with an adverse effect of tamoxifen in premenopausal breast cancer patients. Overexpression of cyclin D1 protein however, confers tamoxifen resistance but not a tamoxifen induced adverse effect. Potentially, co-amplification of an additional 11q13 gene, with a resulting protein overexpression, is required to cause an agonistic effect. Moreover, during 11q13 amplification a deletion of the distal 11q region has been described. In order to assess the potential impact of the deletion we have examined a selected marker for this event. Methods: Array CGH analysis was employed to identify and confirm changes in the gene expression of a number of different genes mapping to the 11q chromosomal region, associated with CCND1 amplification. The subsequent protein expression of these candidate genes was then examined in a clinical material of 500 primary breast cancers from premenopausal patients randomized to either tamoxifen or no adjuvant treatment. The protein expression was also compared to the CGH data in a subset of 56 breast cancer samples. Results: Cortactin and FADD overexpression was linked to CCND1 amplification, determined by FISH, but was not associated with a diminished effect of tamoxifen. However, deletion of distal chromosome 11q, defined as downregulation of the marker Chk1, was associated with an impaired tamoxifen response, and interestingly, also with low proliferative breast cancer of low grade. For Pak1 and cyclin D1 the protein expression corresponded to the gene expression data from the CGH analysis. Conclusions: The results indicate that many 11q13 associated gene products are overexpressed in conjunction with cyclin D1 but not linked to an agonistic effect of tamoxifen. Finally, the deletion of distal 11q, linked to 11q13 amplification, might be an important event affecting breast cancer outcome and tamoxifen response. Keywords: comparative genomic hybridisation 56 samples with 11q13 amplification have been analysed and compared with protein expression results from immunohistochemical analyses.

Project description:Global gene expression analysis was performed in cases 1, 2, 5, 6 and 8 as well as in five pooled myxofibrosarcomas using the Affymetrix GeneChip Human Gene 1.0 ST Arrays (Affymetrix Inc., Santa Clara, CA, USA). Unsupervised hierarchical clustering analysis, using Pearson correlation and average linkage clustering, separated the control pool and case 6 from the rest of the cases. Cases 1 and 5, both harboring t(1;10)(p22;q24), were in turn segregated from cases 2 and 8. Tumors with t(1;10)(p22;q24) thus seemed to have a more distinct expression pattern than tumors affected by amplification of regions on chromosome 3. Tumors with t(1;10) showed increased expression levels of NPM3 and FGF8 compared with tumors without such a translocation, including the control pool. No other gene in the 5 Mb region proximal to the breakpoint on chromosome 10 displayed a more pronounced difference between the two groups. Of the genes located in the commonly amplified region on chromosome 3, VGLL3 and CHMP2B showed a higher expression in tumors affected by the amplification. Cases 1, 2, 5, 6 and 8 were analyzed using the GeneChip Human Gene 1.0 ST Arrays (Affymetrix Inc, Santa Clara, CA, USA). As a control, a pool of five myxofibrosarcomas was used. Total RNA was extracted from frozen tumor biopsies using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy (Qiagen, Valencia, CA, USA) according to the manufacturers’ instructions. RNA quality and concentration were measured using an Agilent 2100 bioanalyzer and Nanodrop ND-1000, respectively. cDNA was generated with the GeneChip® Whole Transcript (WT) cDNA Synthesis and Amplification Kit (Affymetrix) using 300 ng total RNA. Amplified cDNA was fragmented and end labeled using the GeneChip® WT Terminal Labelling Kit (Affymetrix). Subsequently, the fragmented and biotinylated cDNA was hybridized to the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix). The arrays were washed and stained on a GeneChip® Fluidics Station 450 (Affymetrix) according to the manufacturer’s recommendations. Scanning was carried out with the GeneChip® Scanner 3000 and image analysis was performed using GeneChip® Operating Software (GCOS, Affymetrix). Expression data was normalized, background corrected and summarized using the RMA algorithm implemented in the Affymetrix Expression Console™ version 1.0 software. Clustering was performed with MeV 4.1.01 software (http://www.tm4.org/mev.html), using unsupervised hierarchical clustering analysis on the basis of Pearson correlation and average linkage clustering.

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DNA copy number analysis of myxoinflammatory fibroblastic sarcoma, and morphologically similar lesions

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