Combined cDNA array comparative genomic hybridization and serial analysis of gene expression analysis of breast tumor progression.

To identify genetic changes involved in the progression of breast carcinoma, we did cDNA array comparative genomic hybridization (CGH) on a panel of breast tumors, including 10 ductal carcinoma in situ (DCIS), 18 invasive breast carcinomas, and two lymph node metastases. We identified 49 minimal commonly amplified regions (MCRs) that included known (1q, 8q24, 11q13, 17q21-q23, and 20q13) and several uncharacterized (12p13 and 16p13) regional copy number gains. With the exception of the 17q21 (ERBB2) amplicon, the overall frequency of copy number alterations was higher in invasive tumors than that in DCIS, with several of them present only in invasive cancer. Amplification of candidate loci was confirmed by quantitative PCR in breast carcinomas and cell lines. To identify putative targets of amplicons, we developed a method combining array CGH and serial analysis of gene expression (SAGE) data to correlate copy number and expression levels for each gene within MCRs. Using this approach, we were able to distinguish a few candidate targets from a set of coamplified genes. Analysis of the 12p13-p12 amplicon identified four putative targets: TEL/ETV6, H2AFJ, EPS8, and KRAS2. The amplification of all four candidates was confirmed by quantitative PCR and fluorescence in situ hybridization, but only H2AFJ and EPS8 were overexpressed in breast tumors with 12p13 amplification compared with a panel of normal mammary epithelial cells. These results show the power of combined array CGH and SAGE analysis for the identification of candidate amplicon targets and identify H2AFJ and EPS8 as novel putative oncogenes in breast cancer.

[1]  P. Sorensen,et al.  A fluorescence in situ hybridization study of ETV6‐NTRK3 fusion gene in secretory breast carcinoma , 2004, Genes, chromosomes & cancer.

[2]  Jun S. Liu,et al.  Clustering analysis of SAGE data using a Poisson approach , 2004, Genome Biology.

[3]  G. Sourvinos,et al.  Differential expression and mutation of the ras family genes in human breast cancer. , 1998, Biochemical and biophysical research communications.

[4]  R. Cardiff,et al.  c-MYC induces mammary tumorigenesis by means of a preferred pathway involving spontaneous Kras2 mutations , 2001, Nature Medicine.

[5]  Andrea Disanza,et al.  Eps8 controls actin-based motility by capping the barbed ends of actin filaments , 2004, Nature Cell Biology.

[6]  Hiroyuki Konishi,et al.  The PIK3CA gene is mutated with high frequency in human breast cancers , 2004, Cancer biology & therapy.

[7]  R. Zeillinger,et al.  Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups. , 1997, Cancer research.

[8]  S. Rodenhuis,et al.  Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung. , 1987, The New England journal of medicine.

[9]  M. Macleod,et al.  Effects of estrogen on global gene expression: identification of novel targets of estrogen action. , 2000, Cancer research.

[10]  N. Sorel,et al.  ETV6 (TEL) gene amplification in a myelodysplastic syndrome with excess of blasts , 2004, Leukemia.

[11]  Päivikki Kauraniemi,et al.  Amplification of a 280-kilobase core region at the ERBB2 locus leads to activation of two hypothetical proteins in breast cancer. , 2003, The American journal of pathology.

[12]  J. Rowley.  The role of chromosome translocations in leukemogenesis. , 1999, Seminars in hematology.

[13]  Wayne A. Phillips,et al.  Mutation of the PIK3CA Gene in Ovarian and Breast Cancer , 2004, Cancer Research.

[14]  W Godolphin,et al.  Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. , 1989, Science.

[15]  L. Chin,et al.  High-resolution characterization of the pancreatic adenocarcinoma genome , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Kylie L. Gorringe,et al.  Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers : a new approach for the molecular analysis of paraffin-embedded cancer tissue. , 2001, The American journal of pathology.

[17]  P. W. Janes,et al.  Structural Determinants of the Interaction between the erbB2 Receptor and the Src Homology 2 Domain of Grb7* , 1997, The Journal of Biological Chemistry.

[18]  Suk Woo Nam,et al.  PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas , 2005, Oncogene.

[19]  Kornelia Polyak,et al.  Molecular markers in ductal carcinoma in situ of the breast. , 2003, Molecular cancer research : MCR.

[20]  L. Minichiello,et al.  Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF‐dependent mitogenic signals. , 1993, The EMBO journal.

[21]  P. Hand,et al.  ras gene alterations and enhanced levels of ras p21 expression in a spectrum of benign and malignant human mammary tissues. , 1986, Laboratory investigation; a journal of technical methods and pathology.

[22]  M. Schwab,et al.  Amplification of oncogenes revisited: from expression profiling to clinical application. , 2001, Cancer letters.

[23]  Ash A. Alizadeh,et al.  Genome-wide analysis of DNA copy-number changes using cDNA microarrays , 1999, Nature Genetics.

[24]  D C Ward,et al.  Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[25]  C. Collins,et al.  High‐resolution array comparative genomic hybridization of chromosome arm 8q: Evaluation of genetic progression markers for prostate cancer , 2005, Genes, chromosomes & cancer.

[26]  Xin Huang,et al.  Somatic mutation and gain of copy number of PIK3CA in human breast cancer , 2005, Breast Cancer Research.

[27]  Daniel Pinkel,et al.  Genomic microarrays in human genetic disease and cancer. , 2003, Human molecular genetics.

[28]  P. Sorensen,et al.  A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma , 1998, Nature Genetics.

[29]  D. Ginzinger.  Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. , 2002, Experimental hematology.

[30]  M. Ringnér,et al.  Impact of DNA amplification on gene expression patterns in breast cancer. , 2002, Cancer research.

[31]  U. Weidle,et al.  Analysis of differential gene expression in human melanocytic tumour lesions by custom made oligonucleotide arrays , 2005, British Journal of Cancer.

[32]  D. Birnbaum,et al.  ETV6 gene rearrangements in invasive breast carcinoma , 2005, Genes, chromosomes & cancer.

[33]  P. Pelicci,et al.  Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation , 1995, Molecular and cellular biology.

[34]  B. Dutrillaux,et al.  The insulin‐like growth factor I receptor gene is the target for the 15q26 amplicon in breast cancer , 1994, Genes, chromosomes & cancer.

[35]  Rameen Beroukhim,et al.  Molecular characterization of the tumor microenvironment in breast cancer. , 2004, Cancer cell.

[36]  J. Baselga,et al.  Molecular approach to breast cancer treatment. , 2004, Seminars in oncology.

[37]  Patrick G Buckley,et al.  Genomic microarrays in the spotlight. , 2004, Drug discovery today.

[38]  R. Carbone,et al.  Regulation of the tyrosine kinase substrate Eps8 expression by growth factors, v-Src and terminal differentiation , 1997, Oncogene.

[39]  Christian A. Rees,et al.  Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Ptak,et al.  High Frequency of Mutations of the PIK3CA Gene in Human Cancers , 2004, Science.

[41]  Luke Hughes-Davies,et al.  Amplification of the BRCA2 Pathway Gene EMSY in Sporadic Breast Cancer Is Related to Negative Outcome , 2004, Clinical Cancer Research.

[42]  K. Chin,et al.  In situ analyses of genome instability in breast cancer , 2004, Nature Genetics.

[43]  M. Maa,et al.  Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation , 2001, Oncogene.

[44]  T. Tammela,et al.  RAD21 and KIAA0196 at 8q24 are amplified and overexpressed in prostate cancer , 2004, Genes, chromosomes & cancer.

[45]  S. Luoh,et al.  Overexpression of the amplified Pip4k2β gene from 17q11–12 in breast cancer cells confers proliferation advantage , 2004, Oncogene.

[46]  M. Wigler,et al.  Genomic amplification and oncogenic properties of the KCNK9 potassium channel gene. , 2003, Cancer cell.

[47]  Luke Hughes-Davies,et al.  EMSY Links the BRCA2 Pathway to Sporadic Breast and Ovarian Cancer , 2003, Cell.

[48]  P. Sorensen,et al.  Expression of the ETV6-NTRK3 gene fusion as a primary event in human secretory breast carcinoma. , 2002, Cancer cell.

[49]  M. Wigler,et al.  Circular binary segmentation for the analysis of array-based DNA copy number data. , 2004, Biostatistics.

[50]  P. Marynen,et al.  ETV6 gene rearrangements in hematopoietic malignant disorders. , 1996, Leukemia & lymphoma.

[51]  W. Kuo,et al.  High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays , 1998, Nature Genetics.

[52]  M. Rocchi,et al.  Identification of a commonly amplified 4.3 Mb region with overexpression of C8FW, but not MYC in MYC-containing double minutes in myeloid malignancies. , 2004, Human molecular genetics.

[53]  O. Monni,et al.  New amplified and highly expressed genes discovered in the ERBB2 amplicon in breast cancer by cDNA microarrays. , 2001, Cancer research.

[54]  G. Scita,et al.  The eps8 family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in the Ras/Rac pathway. , 2003, Molecular biology of the cell.

[55]  S. Orkin,et al.  Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner , 1993, Molecular and cellular biology.