Integrated RNA-seq and DNase-seq analyses identify phenotype-specific BMP4 signaling in breast cancer.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) plays an important role in cancer pathogenesis. In breast cancer, it reduces proliferation and increases migration in a cell line-dependent manner. To characterize the transcriptional mediators of these phenotypes, we performed RNA-seq and DNase-seq analyses after BMP4 treatment in MDA-MB-231 and T-47D breast cancer cells that respond to BMP4 with enhanced migration and decreased cell growth, respectively. RESULTS: The RNA-seq data revealed gene expression changes that were consistent with the in vitro phenotypes of the cell lines, particularly in MDA-MB-231, where migration-related processes were enriched. These results were confirmed when enrichment of BMP4-induced open chromatin regions was analyzed. Interestingly, the chromatin in transcription start sites of differentially expressed genes was already open in unstimulated cells, thus enabling rapid recruitment of transcription factors to the promoters as a response to stimulation. Further analysis and functional validation identified MBD2, CBFB, and HIF1A as downstream regulators of BMP4 signaling. Silencing of these transcription factors revealed that MBD2 was a consistent activator of target genes in both cell lines, CBFB an activator in cells with reduced proliferation phenotype, and HIF1A a repressor in cells with induced migration phenotype. CONCLUSIONS: Integrating RNA-seq and DNase-seq data showed that the phenotypic responses to BMP4 in breast cancer cell lines are reflected in transcriptomic and chromatin levels. We identified and experimentally validated downstream regulators of BMP4 signaling that relate to the different in vitro phenotypes and thus demonstrate that the downstream BMP4 response is regulated in a cell type-specific manner.

Finnish Fanconi anemia mutations and hereditary predisposition to breast and prostate cancer.

Mutations in downstream Fanconi anemia (FA) pathway genes, BRCA2, PALB2, BRIP1 and RAD51C, explain part of the hereditary breast cancer susceptibility, but the contribution of other FA genes has remained questionable. Due to FA's rarity, the finding of recurrent deleterious FA mutations among breast cancer families is challenging. The use of founder populations, such as the Finns, could provide some advantage in this. Here, we have resolved complementation groups and causative mutations of five FA patients, representing the first mutation confirmed FA cases in Finland. These patients belonged to complementation groups FA-A (n = 3), FA-G (n = 1) and FA-I (n = 1). The prevalence of the six FA causing mutations was then studied in breast (n = 1840) and prostate (n = 565) cancer cohorts, and in matched controls (n = 1176 females, n = 469 males). All mutations were recurrent, but no significant association with cancer susceptibility was observed for any: the prevalence of FANCI c.2957_2969del and c.3041G>A mutations was even highest in healthy males (1.7%). This strengthens the exclusive role of downstream genes in cancer predisposition. From a clinical point of view, current results provide fundamental information of the mutations to be tested first in all suspected FA cases in Finland.

Tissue microarrays for high-throughput molecular profiling of tumor specimens.

Many genes and signalling pathways controlling cell proliferation, death and differentiation, as well as genomic integrity, are involved in cancer development. New techniques, such as serial analysis of gene expression and cDNA microarrays, have enabled measurement of the expression of thousands of genes in a single experiment, revealing many new, potentially important cancer genes. These genome screening tools can comprehensively survey one tumor at a time; however, analysis of hundreds of specimens from patients in different stages of disease is needed to establish the diagnostic, prognostic and therapeutic importance of each of the emerging cancer gene candidates. Here we have developed an array-based high-throughput technique that facilitates gene expression and copy number surveys of very large numbers of tumors. As many as 1000 cylindrical tissue biopsies from individual tumors can be distributed in a single tumor tissue microarray. Sections of the microarray provide targets for parallel in situ detection of DNA, RNA and protein targets in each specimen on the array, and consecutive sections allow the rapid analysis of hundreds of molecular markers in the same set of specimens. Our detection of six gene amplifications as well as p53 and estrogen receptor expression in breast cancer demonstrates the power of this technique for defining new subgroups of tumors.

Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.

Comparative genomic hybridization produces a map of DNA sequence copy number as a function of chromosomal location throughout the entire genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes. Analysis of tumor cell lines and primary bladder tumors identified 16 different regions of amplification, many in loci not previously known to be amplified.

Bone morphogenetic protein 7 expression associates with bone metastasis in breast carcinomas.

BACKGROUND: We recently showed that bone morphogenetic protein 7 (BMP7) is overexpressed in primary breast tumors. Here we explored the clinical significance of BMP7 expression in breast cancer. MATERIALS AND METHODS: This study included 483 breast cancer patients with complete clinicopathological information and up to 15 years of follow-up. Samples contained 241 lobular carcinomas, 242 ductal carcinomas, and 40 local recurrences. BMP7 protein expression was determined using immunohistochemistry. RESULTS: BMP7 was expressed in 47% of the primary tumor samples and 13% of the local recurrences. The primary tumors expressed BMP7 more often than the corresponding local recurrences (P = 0.004). BMP7 expression was dependent on the tumor subtype; 57% of the lobular carcinomas but only 37% of the ductal carcinomas were BMP7 positive (P = 0.0001). BMP7 expression was associated with accelerated bone metastasis formation (P = 0.040), especially in ductal carcinomas (P = 0.033), and multivariate analysis confirmed that BMP7 is an independent prognostic indicator for early bone metastasis development (P = 0.032). CONCLUSION: BMP7 is clearly associated with bone metastasis formation and thus might have clinical utility in identification of patients with increased risk of bone metastasis. This is the first time that bone inducing factor BMP7 has been linked to the bone metastasis process in breast cancer.

Genome screening by comparative genomic hybridization.

Comparative genomic hybridization (CGH) provides a molecular cytogenetic approach for genome-wide scanning of differences in DNA sequence copy number. The technique is now attracting wide-spread interest, especially among cancer researchers. The rapidly expanding database of CGH publications already covers about 1500 tumors and is beginning to reveal genetic abnormalities that are characteristic of certain tumor types or stages of tumor progression. Six novel gene amplifications, as well as a locus for a cancer-predisposition syndrome, have been discovered based on CGH data. CGH has now been established as a first-line screening technique for cancer researchers and will serve as a basis for ongoing efforts to develop high-resolution next-generation genome scanning, such as the microarray technology.

Contrast agent enhanced pQCT of articular cartilage.

The delayed gadolinium enhanced MRI of cartilage (dGEMRIC) technique is the only non-invasive means to estimate proteoglycan (PG) content in articular cartilage. In dGEMRIC, the anionic paramagnetic contrast agent gadopentetate distributes in inverse relation to negatively charged PGs, leading to a linear relation between T1,Gd and spatial PG content in tissue. In the present study, for the first time, contrast agent enhanced peripheral quantitative computed tomography (pQCT) was applied, analogously to dGEMRIC, for the quantitative detection of spatial PG content in cartilage. The suitability of two anionic radiographic contrast agents, gadopentetate and ioxaglate, to detect enzymatically induced PG depletion in articular cartilage was investigated. First, the interrelationships of x-ray absorption, as measured with pQCT, and the contrast agent solution concentration were investigated. Optimal contrast agent concentrations for the following experiments were selected. Second, diffusion rates for both contrast agents were investigated in intact (n=3) and trypsin-degraded (n=3) bovine patellar cartilage. The contrast agent concentration of the cartilaginous layer was measured prior to and 2-27 h after immersion. Optimal immersion time for the further experiments was selected. Third, the suitability of gadopentetate and ioxaglate enhanced pQCT to detect the enzymatically induced specific PG depletion was investigated by determining the contrast agent concentrations and uronic acid and water contents in digested and intact osteochondral samples (n=16). After trypsin-induced PG loss (-70%, p<0.05) the penetration of gadopentetate and ioxaglate increased (p<0.05) by 34% and 48%, respectively. Gadopentetate and ioxaglate concentrations both showed strong correlation (r=-0.95, r=-0.94, p<0.01, respectively) with the uronic acid content. To conclude, contrast agent enhanced pQCT provides a technique to quantify PG content in normal and experimentally degraded articular cartilage in vitro. As high resolution imaging of e.g. the knee joint is possible with pQCT, the present technique may be further developed for in vivo quantification of PG depletion in osteoarthritic cartilage. However, careful in vitro and in vivo characterization of diffusion mechanics and optimal contrast agent concentrations are needed before diagnostic applications are feasible.

Acoustic properties of trabecular bone--relationships to tissue composition.

In osteoporosis, changes in tissue composition and structure reduce bone strength and expose it to fractures. The current primary diagnostic technique, i.e., dual energy X-ray absorptiometry, measures areal bone mineral density (BMD) but provides no direct information on trabecular structure or organic composition. Although still poorly characterized, ultrasound techniques may bring about information on bone composition and structure. In this study, relationships of 2.25-MHz ultrasound speed, attenuation, reflection and backscattering with composition of human trabecular bone (n=26) were characterized experimentally, as well as by using numerical analyses. We also determined composition of the trabecular sample (fat and water content, bone volume fraction) and that of the calcified matrix (mineral, proteoglycan and collagen content of trabeculae). In experimental analyses, bone volume fraction and mineral content of the calcified matrix were the only determinants of BMD. Further, bone volume fraction served as the strongest determinant of ultrasound parameters (r=0.51-0.87). In numerical simulations, density and mechanical properties of the calcified matrix systematically affected ultrasound speed, attenuation, reflection and backscattering. However, partial correlation coefficients revealed only low associations(|r|

Nordic collaborative study of the BARD1 Cys557Ser allele in 3956 patients with cancer: enrichment in familial BRCA1/BRCA2 mutation-negative breast cancer but not in other malignancies.

BACKGROUND: BARD1 was originally identified as a BRCA1-interacting protein but has also been described in tumour-suppressive functions independent of BRCA1. Several studies have indicated that the BARD1 gene is a potential target for germline changes predisposing to breast and ovarian cancer. The C-terminal Cys557Ser change has previously been uncovered to associate with an increased risk of breast cancer and was recently shown to result in defective apoptotic activities. AIM AND METHODS: Conformation-sensitive gel electrophoresis, minisequencing, TaqMan assays, denaturing high-performance liquid chromatography analysis and DNA sequencing were used to investigate the prevalence of the Cys557Ser allele in a large Nordic case-control study cohort consisting of 2906 patients with breast or ovarian cancer, 734 with prostate cancer, 188 with colorectal cancer, 128 men with breast cancer, and 3591 controls from Finland, Iceland, Denmark, Sweden and Norway. RESULTS: The frequency of the BARD1 Cys557Ser variant seemed to increase among patients from families with breast or ovarian cancer lacking BRCA1 or BRCA2 mutations: a significant difference was obtained compared with controls (6.8% v 2.7%; p<0.001; odds ratio (OR) 2.6; 95% confidence interval (CI) 1.7 to 4.0) and with patients from BRCA1/BRCA2 mutation-positive families (6.8% v 2.2%; p = 0.01; OR 3.2; 95% CI 1.2 to 8.3). In contrast, no major association with male breast, ovarian, colorectal or prostate cancer was observed. Additionally, a novel BARD1 allele resulting in Ser558Pro was identified in familial breast cancer cases. CONCLUSION: These results provide further evidence that BARD1 Cys557Ser confers a slightly increased risk of breast cancer in women.

Activation of multiple cancer-associated genes at the ERBB2 amplicon in breast cancer.

During the past decade the role of the ERBB2 (neu/HER2) oncogene as an important predictor of patient outcome and response to various therapies in breast cancer has been clearly established. This association of ERBB2 aberrations with more aggressive disease and poor clinical outcome, together with the high prevalence of such alterations in breast cancer, has also made ERBB2 an attractive target for therapy. A specific antibody-based therapy, Herceptin, directed against the extracellular domain of the ERBB2 receptor tyrosine kinase, was recently developed and several clinical trials have shown the therapeutic efficacy of this drug against ERBB2-positive breast cancer. However, a relatively large fraction of patients does not benefit from Herceptin treatment, indicating that other factors beyond ERBB2 itself must influence therapy response in ERBB2-positive tumors. It is well known that amplification of the 17q12-q21 region is the most common mechanism for ERBB2 activation in breast cancer and that it leads to simultaneous activation of several other genes. These co-amplified and co-activated genes may have an impact on disease progression and the clinical behavior of ERBB2-positive tumors and thus represent important targets of research. In this paper we discuss the current knowledge on the structure of the ERBB2 amplicon, the genes involved, and their possible contribution to breast cancer pathogenesis.

Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis.

BACKGROUND: Studies by comparative genomic hybridization (CGH) have shown that chromosomal region 17q23 is amplified in up to 20% of primary breast cancers. We used microarray analyses to measure the expression levels of genes in this region and to explore their prognostic importance. METHODS: A microarray that contained 4209 complementary DNA (cDNA) clones was used to identify genes that are overexpressed in the MCF-7 breast cancer cell line as compared with normal mammary tissue. Fluorescence in situ hybridization was used to analyze the copy number of one overexpressed gene, ribosomal protein S6 kinase (S6K), and to localize it to the 17q23 region. Northern and western blot analyses were used to measure S6K gene and protein expression, and an enzymatic assay was used to measure S6K activity. Tumor tissue microarray analysis was used to study amplification of S6K and the HER-2 oncogene, another 17q-linked gene, and the relationship between amplification and prognosis was analyzed. The Kaplan-Meier method was used for data analysis, and the log-rank test was used for statistical analysis. All P values are two-sided. RESULTS: S6K was amplified and highly overexpressed in MCF-7 cells relative to normal mammary epithelium, and protein expression and enzyme activity were increased. S6K was amplified in 59 (8.8%) of 668 primary breast tumors, and a statistically significant association between amplification and poor prognosis (P =.0021) was observed. Amplification of both S6K and HER-2 implied particularly poor survival (P =.0001). CONCLUSIONS: The combination of CGH information with cDNA and tissue microarray analyses can be used to identify amplified and overexpressed genes and to evaluate the clinical implications of such genes and genomic rearrangements. S6K is likely to be one of the genes at 17q23 that is amplified during oncogenesis and may adversely affect the prognosis of patients with this amplification.

New amplified and highly expressed genes discovered in the ERBB2 amplicon in breast cancer by cDNA microarrays.

Amplification of the ERBB2 oncogene at 17q12 has been well documented in breast cancer and has been shown to contribute to a poor clinical outcome. However, systematic surveys of copy number and expression levels of all genes within the 17q12 region have not been performed. Here, we used cDNA and comparative genomic hybridization microarray technologies to undertake a broad survey of genes involved in the 17q12 amplification in breast cancer. A chromosomal region-specific cDNA microarray containing 217 expressed sequence tag (EST) clones from 17q12 was constructed and used for parallel analysis of gene copy numbers and expression levels in seven breast cancer cell lines allowing direct identification of genes whose expression is elevated because of an increase in copy number in this chromosomal region. The copy number and expression survey identified 12 transcripts that showed a consistent pattern of increased copy number and expression in three or more of the 17q12-amplified cell lines. As expected, these included ERBB2 as well as the GRB7 and MLN64 genes previously shown to be coamplified with ERBB2. In addition, five other known genes and four uncharacterized ESTs were also found to be consistently activated by amplification in these breast cancer cell lines. Amplicon mapping by fluorescence in situ hybridization revealed a minimal common region of amplification containing four highly expressed genes, ERBB2, GRB7, MLN64, and an uncharacterized EST 48582. Furthermore, several other genes, although not located in the minimal common region of amplification, showed a correlated pattern of amplification and expression indicating that they might play a role in breast cancers with the 17q12 amplification. In conclusion, parallel analysis of gene copy number and expression levels by cDNA microarray can be used to directly identify candidate target genes involved in amplifications. Our results show that the 17q12 amplification in breast cancer leads to the simultaneous elevation of expression levels of several genes.

Multiple genes at 17q23 undergo amplification and overexpression in breast cancer.

Studies by comparative genomic hybridization imply that amplification of the chromosomal region 17q22-q24 is common in breast cancer. Here, amplification and expression levels of six known genes located at 17q23 were examined in breast cancer cell lines. Four of them (RAD51C, S6K, PAT1, and TBX2) were found to be highly amplified and overexpressed. To investigate the involvement of these genes in vivo, fluorescence in situ hybridization analysis of a tissue microarray containing 372 primary breast cancers was used. S6K, PAT1, and TBX2 were coamplified in about 10% of tumors, whereas RADS1C amplification was seen in only 3% of tumors. Expression analysis in 12 primary tumors showed that RAD51C and S6K were consistently expressed in all cases in which they were amplified and also in some tumors without amplification. These data suggest that 17q23 amplification results in simultaneous up-regulation of several genes, whose increased biological activity may jointly contribute to the more aggressive clinical course observed in patients with 17q23-amplified tumors.

Gains and losses of DNA sequences in osteosarcomas by comparative genomic hybridization.

Our aim was to identify chromosomal regions that are likely to harbor previously unknown genes with an important role in the genesis of osteosarcoma. Comparative genomic hybridization was used to screen for losses and gains of DNA sequences along all chromosome arms in 11 tumors. Extensive genetic aberrations, with an average of 11 changes/tumor (range, 1-20), were found in 10 of the 11 specimens. High level amplifications of small chromosomal regions were detected in eight tumors. These involved the 12q12-q13 region (known to contain the SAS-MDM2 locus) and several previously unreported amplification sites such as 17p11-p12, 3q26, and Xq12. When all DNA sequence gains were evaluated, the gains at 8q and Xp were most common (45%). The most common losses of DNA sequences were seen at 2q, 6q, 8p, and 10p (36%). In conclusion, despite the very complex pattern of genetic changes in osteosarcomas, certain chromosomal regions appear to be affected more often than others. Most of these regions have not previously been reported to be implicated in osteosarcomas and may thus highlight locations of novel genes with an important role in the development and progression of these tumors.

Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes.

Studies by comparative genomic hybridization have indicated that a major new locus for DNA amplification in breast cancer is 20q13 and suggested that this genetic event is associated with aggressive clinical behavior. We used interphase fluorescence in situ hybridization with anonymous cosmid probes and gene-specific P1 clones to determine the minimal common region of increased copy number and to study involvement of known genes at 20q13. Based on high-level copy number increases (3 to 10-fold) found with one or more probes in 5 of 14 (35%) breast cancer cell lines and in 3 of 36 (8%) primary tumors, the critical region was narrowed to approximately 1.5 megabases at 20q13.2 defined by fractional length pter values 0.81-0.84. Previously known genes were excluded as candidates, implying that this chromosomal region harbors a novel oncogene that contributes to the malignant progression of breast cancer.

Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization.

Genetic changes leading to the development of prostate cancer and factors that underlie the clinical progression of the disease are poorly characterized. Here, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes along all chromosomes in 31 primary and 9 recurrent uncultured prostate carcinomas. The aim of the study was to identify those chromosome regions that contain genes important for the development of prostate cancer and to identify genetic markers of tumor progression. CGH analysis indicated that 74% of primary prostate carcinoma showed DNA sequence copy number changes. Losses were 5 times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). Allelic loss studies with 5 polymorphic microsatellite markers for 4 different chromosomes were done from 13 samples and showed a 76% concordance with CGH results. In local recurrences that developed during endocrine therapy, there were significantly more gains (P < 0.001) and losses (P < 0.05) of DNA sequences than in primary tumors, with gains of 8q (found in 89% of recurrences versus 6% of primary tumors), X (56% versus 0%), and 7 (56% versus 10%), as well as loss of 8p (78% versus 32%), being particularly often involved. In conclusion, our CGH results indicate that losses of several chromosomal regions are common genetic changes in primary tumors, suggesting that deletional inactivation of putative tumor suppressor genes in these chromosomal sites is likely to underlie development of prostate cancer. Furthermore, the pattern of genetic changes seen in recurrent tumors with the frequent gains of 7, 8q, and X suggests that the progression of prostate cancer and development of hormone-independent growth may have a distinct genetic basis. These chromosome aberrations may have diagnostic utility as markers of prostate cancer progression.

Deletion of chromosome 17p loci in breast cancer cells detected by fluorescence in situ hybridization.

Allelic loss of tumor suppressor genes on chromosome 17p has been implicated in the progression of breast cancer. This is in principle detectable by fluorescence in situ hybridization if the loss occurs by deletion. In order to determine if detectable deletions occur in primary breast cancer, we used dual-color hybridization with chromosome 17 pericentromeric and region-specific DNA probes to study 19 primary breast cancers. The copy numbers of 17 centromere and 17p13.1 sequences were compared with the loss of heterozygosity (LOH) for probe YNZ22 at 17p13.3 detected by restriction fragment length polymorphism. Nine of 11 cases showing LOH also showed the major population of nuclei with a deletion. The remaining two tumors with LOH were trisomic for both the centromere and 17p13.1 cosmid. In contrast, seven of eight tumors without LOH had no deletions by fluorescence in situ hybridization. These data suggest that the dominant mechanism of allelic loss at 17p in breast cancer is a physical deletion and that analysis of deletions by fluorescence in situ hybridization is a rapid and sensitive approach to studying chromosomal aberrations.

Genetic heterogeneity and clonal evolution underlying development of asynchronous metastasis in human breast cancer.

To understand the genetic basis and clonal evolution underlying metastatic progression of human breast cancer in vivo, we analyzed the genetic composition of 29 primary breast carcinomas and their paired asynchronous metastases by comparative genomic hybridization and fluorescence in situ hybridization. The mean number of genetic changes by comparative genomic hybridization was 8.7 +/- 5.3 in primary tumors and 9.0 +/- 5.7 in their metastases. Although most of the genetic changes occurred equally often in the two groups, gains of the Xq12-q22 region were enriched in the metastases. According to a statistical analysis of shared genetic changes and breakpoints in paired specimens, 20 of the metastases (69%) showed a high degree of clonal relationship with the corresponding primary tumor, whereas the genetic composition of 9 metastases (31%) differed almost completely from that of the paired primary tumors. In both groups, however, chromosome X inactivation patterns suggested that the metastatic lesions originated from the same clone as the primary tumor. Fluorescence in situ hybridization analysis with probes specific to metastatic clones usually failed to find such cells in the primary tumor sample. In conclusion, detailed characterization of the in vivo progression pathways of metastatic breast cancer indicates that a linear progression model is unlikely to account for the progression of primary tumors to metastases. An early stem line clone apparently evolves independently in the primary tumor and its metastasis, eventually leading to multiple, genetically almost completely different, clones in the various tumor locations in a given patient. The resulting heterogeneity of metastatic breast cancer may underlie its poor responsiveness to therapy and explain why biomarkers of prognosis or therapy responsiveness measured exclusively from primary tumors give a restricted view of the biological properties of metastatic breast cancer.

Comparative genomic hybridization analysis of 38 breast cancer cell lines: a basis for interpreting complementary DNA microarray data.

Breast cancer cell lines provide a useful starting point for the discovery and functional analysis of genes involved in breast cancer. Here, we studied 38 established breast cancer cell lines by comparative genomic hybridization (CGH) to determine recurrent genetic alterations and the extent to which these cell lines resemble uncultured tumors. The following chromosomal gains were observed: 8q (75%), 1q (61%), 20q (55%), 7p (44%), 3q (39%), 5p (39%), 7q (39%), 17q (33%), 1p (30%), and 20p (30%), and the most common losses were: 8p (58%), 18q (58%), 1p (42%), Xp (42%), Xq (42%), 4p (36%), 11q (36%), 18p (33%), 10q (30%), and 19p (28%). Furthermore, 35 recurrent high-level amplification sites were identified, most often involving 8q23 (37%), 20q13 (29%), 3q25-q26 (24%), 17q22-q23 (16%), 17q23-q24 (16%), 1p13 (11%), 1q32 (11%), 5p13 (11%), 5p14 (11%), 11q13 (11%), 17q12-q21 (11%), and 7q21-q22 (11%). A comparison of DNA copy number changes found in the cell lines with those reported in 17 published studies (698 tumors) of uncultured tumors revealed a substantial degree of overlap. CGH copy number profiles may facilitate identification of important new genes located at the hotspots of such chromosomal alterations. This was illustrated by analyzing expression levels of 1236 genes using cDNA microarrays in four of the cell lines. Several highly overexpressed genes (such as RCH1 at 17q23, TOPO II at 17q21-q22, as well as CAS and MYBL2 at 20q13) were involved in these recurrent DNA amplifications. In conclusion, DNA copy number profiles were generated by CGH for most of the publicly available breast cancer cell lines and were made available on a web site (http://www.nhgri.nih.gov/DIR/CGB/++ +CR2000). This should facilitate the correlative analysis of gene expression and copy number as illustrated here by the finding by cDNA microarrays of several overexpressed genes that were amplified.

Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in human breast cancer.

DNA amplification at 20q13.2 is common in breast cancer, correlates with poor prognosis, and may reflect location of an important oncogene. Recently, other regions along 20q were also found to undergo amplification. Here, amplification levels and patterns of co-amplification were analyzed by interphase fluorescence in situ hybridization at 14 loci along 20q in 146 uncultured breast carcinomas and 14 cell lines. Three regions were independently amplified in uncultured tumors: RMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases), PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%). Co-amplifications involving two or three of these regions were seen in 11 of the 19 highly amplified tumors. The results suggest that three distinct nonsyntenic regions along 20q may be important and that complex chromosomal rearrangements underlie their frequent co-amplification in breast cancer.