A method for finding putative causes of gene expression variation

The majority of microarray studies evaluate gene ex- pression differences between various specimens or con- ditions. However, the causes of this variability often re- main unknown. Our aim is to identify underlying causes of these patterns, a process that would eventually enable a mechanistic understanding of the deregulation of gene expression in cancer. The procedure consists of three phases: pre-processing, data integration and statistical analysis. We have applied the strategy to identify genes that are overexpressed due to amplification in breast cancer. The data were obtained from 14 breast cancer cell lines, which were subjected to cDNA microarray based copy number and expression experiments. The re- sult of the analysis was a list that consisted of 92 genes. This set includes several genes that are known to be both overexpressed and amplified in breast cancer. The com- plete study was published in Journal of the Franklin In- stitute 2004, and in this paper we focus on the main issues of the study

Basal-like phenotype is not associated with patient survival in estrogen-receptor-negative breast cancers

INTRODUCTION: Basal-phenotype or basal-like breast cancers are characterized by basal epithelium cytokeratin (CK5/14/17) expression, negative estrogen receptor (ER) status and distinct gene expression signature. We studied the clinical and biological features of the basal-phenotype tumors determined by immunohistochemistry (IHC) and cDNA microarrays especially within the ER-negative subgroup. METHODS: IHC was used to evaluate the CK5/14 status of 445 stage II breast cancers. The gene expression signature of the CK5/14 immunopositive tumors was investigated within a subset (100) of the breast tumors (including 50 ER-negative tumors) with a cDNA microarray. Survival for basal-phenotype tumors as determined by CK5/14 IHC and gene expression signature was assessed. RESULTS: From the 375 analyzable tumor specimens, 48 (13%) were immunohistochemically positive for CK5/14. We found adverse distant disease-free survival for the CK5/14-positive tumors during the first years (3 years hazard ratio (HR) 2.23, 95% confidence interval (CI) 1.17 to 4.24, p = 0.01; 5 years HR 1.80, 95% CI 1.02 to 3.15, p = 0.04) but the significance was lost at the end of the follow-up period (10 years HR 1.43, 95% CI 0.84 to 2.43, p = 0.19). Gene expression profiles of immunohistochemically determined CK5/14-positive tumors within the ER-negative tumor group implicated 1,713 differently expressed genes (p < 0.05). Hierarchical clustering analysis with the top 500 of these genes formed one basal-like and a non-basal-like cluster also within the ER-negative tumor entity. A highly concordant classification could be constructed with a published gene set (Sorlie's intrinsic gene set, concordance 90%). Both gene sets identified a basal-like cluster that included most of the CK5/14-positive tumors, but also immunohistochemically CK5/14-negative tumors. Within the ER-negative tumor entity there was no survival difference between the non-basal and basal-like tumors as identified by immunohistochemical or gene-expression-based classification. CONCLUSION: Basal cytokeratin-positive tumors have a biologically distinct gene expression signature from other ER-negative tumors. Even if basal cytokeratin expression predicts early relapse among non-selected tumors, the clinical outcome of basal tumors is similar to non-basal ER-negative tumors. Immunohistochemically basal cytokeratin-positive tumors almost always belong to the basal-like gene expression profile, but this cluster also includes few basal cytokeratin-negative tumors

Molecular Consequences of Gene Amplification in Breast Cancer

Rintasyöpä on teollisuusmaiden naisten yleisin syöpämuoto ja koskettaa n. joka kahdeksatta naista heidän elinaikanaan. Rintasyöpä saa alkunsa solun perimäaineksen, DNA:n, muutoksista. Näiden DNA vaurioiden seurauksena solun kasvua edistävien syöpägeenien toiminta solussa voimistuu eli aktivoituu, kun taas solujen kasvua hillitsevien ns. kasvurajoitegeenien toiminta heikkenee. Muuttunut solu saavuttaa kasvuedun ja alkaa jakautua normaalisoluja nopeammin. Rintasyövässä syöpägeenien yleisin aktivoitumismekanismi on geenin monistuminen. Tähän mennessä tunnetuista rintasyövän geenimonistumista kliinisesti merkittävin on ERBB2-geenin monistuma, jota esiintyy n. 10-30 % rintasyövistä. Väitöskirjan ensimmäisessä ja toisessa osatyössä keskityttiin selvittämään ERBB2 geenimonistuma-alueen rakennetta ja tunnistamaan tältä alueelta uusia monistumalla aktivoituneita syöpägeenejä. Osoitimme, että monistuman ydinalue rajautuu 280 kb alueelle, jolta voidaan tunnistaa kymmenen geeniä niiden koodittamien lähetti-RNA-tuotteiden perusteella. Näistä geeneistä kuudella, ERBB2 mukaan luettuna, ilmentymistaso oli tilastollisesti merkittävästi kohonnut rintasyövässä vaikuttaen siten mahdollisesti ko. syövän kulkuun. ERBB2-geenimonistuman on aiemmin osoitettu olevan kliinisesti merkittävä, sillä sen esiintyminen on yhteydessä potilaiden huonoon ennusteeseen ja taudin uusiutumiseen. Hiljattain ERBB2 geenin koodaaman proteiinin toiminnan estämiseen kehitettiin spesifinen lääkeaine, Herceptin, jota käytetään ERBB2 geenin monistuman omaavilla potilailla. Tällä hetkellä Herceptin hoidon ja erityisesti hoitovasteissa havaittujen yksilöllisten erojen tuntemus on vielä puutteellista. Osatyössä III tarkastelimme Herceptin-hoidon vaikutuksia geenien ilmentymiseen koko genominlaajuista cDNA mikrosiruanalyysiä käyttäen. Tunnistimme joukon geenejä (439 geeniä), joiden ilmentymistaso oli sidoksissa ERBB2-monistuman olemassaoloon tai vastaavasti sen puuttumiseen. Toiminnaltaan nämä geenit edustivat useita keskeisiä biologisia prosesseja, kuten RNA:n muokkaus, DNA:n virheiden korjaus, solujen kiinnittyminen ja solujen kasvun säätely. Tulokset osoittivat myös, että rintasyövässä ERBB2 geenin ilmentymisasteella saattaa olla tärkeä merkitys Herceptin-hoidon vastetta ennustettaessa. ERBB2-geenimonistuma-alueen ohella rintasyövässä on tunnistettu useita muita monistuma-alueita kromosomaalista vertailevaa genomista hybridisaatiota (CGH) käyttämällä. Kuitenkin näiden monistumien geenitason vaikutuksista on vielä niukasti tietoa. Osatyössä IV käytimme aiempaa herkempää cDNA-mikrosiruun pohjautuvaa CGH-menetelmää, joka mahdollisti monistumien tarkemman kartoituksen sekä monistuma-alueilla sijaitsevien geenien ilmentymisen samanaikaisen määrityksen. Tämän menetelmän avulla tunnistimme 24 erillistä rintasyövän monistuma-aluetta eri puolelta genomia. Näiden joukossa oli mm. kaksi ennen kuuvaamatonta monistuma-aluetta kromosomeissa 9p13 ja 17q21.3. Samanaikaisen geenien ilmentymistasojen määrityksen avulla pystyimme tunnistamaan monistuma-alueilta yhteensä 270 geeniä, joiden ilmentyminen oli johdonmukaisesti kasvanut geeni-monistuman seurauksena. Yhteenvetona tutkimuksessa tunnistettiin useita monistumalla aktivoituneita geenejä, jotka ovat mahdollisesti osallisena rintasyövän synnyssä ja etenemisessä. Jatkotutkimuksissa keskitymme selvittämään näiden geenien toimintaa rintasyövässä.cDNA microarray technology was applied for a systematic survey of gene copy numbers and gene expression levels at the ERBB2 amplicon at 17q12. For this purpose a chromosome 17-specific cDNA microarray containing 217 clones from this region was constructed and used for parallel analysis of gene copy numbers and gene expression levels in seven breast cancer cell lines. Direct comparison of data from these two array-based analyses allowed identification of a small set of transcripts showing a consistent pattern of increased copy number and overexpression in the 17q12 amplified cell lines. Detailed characterization of the structure and extent of the 17q12 amplicon was performed using large insert size BAC clones and interphase fluorescence in situ hybridization (FISH). The amplicon mapping revealed a minimal common region of amplification that was restricted to a single BAC clone both in breast cancer cell lines and in primary breast tumors. This 280 kb minimal region contains ten transcripts, eight representing known genes and two encoding uncharacterised hypothetical proteins. The molecular consequences of amplification in primary breast tumors were determined using real time RT-PCR. Expression analysis identified six genes (ERBB2, GRB7, PNMT, MLN64, MGC9753, MGC14832) showing statistically highly significant correlation between gene copy number and expression levels. Global effects of Herceptin treatment on gene expression levels in ERBB2 amplified and non-amplified cell lines were explored using cDNA microarrays. In growth inhibition assays, Herceptin treatment induced a dose-dependent growth reduction in ERBB2 amplified breast cancer cell lines whereas non-amplified cell lines showed only a minimal effect. Using a step-wise gene selection algorithm a set of 439 genes that best separated the amplified and non-amplified cell lines from each other was identified. The discriminatory power of these genes was validated by both hierarchical clustering and self-organizing map analyses. In the ERBB2 amplified cell lines, Herceptin treatment induced the expression of several genes involved in RNA processing and DNA repair, while expression of cell adhesion mediators and known oncogenes such as c-FOS and c-KIT was downregulated. To explore the molecular consequences of gene amplification on a global level, a genome-wide gene copy number and expression analysis was performed in 14 breast cancer cell lines using a cDNA microarray containing 13,824 clones. High-resolution CGH microarray analysis identified 24 independent amplicons ranging in size from 0.2 to 12 Mb. Both low- and high-level copy number increases had a considerable effect on gene expression levels throughout the genome. Of the most highly amplified genes, 44 % showed overexpression whereas 10.5 % of the highly overexpressed genes were amplified. Statistical analysis with random permutation tests revealed a set of 270 genes whose expression levels were systematically attributable to gene amplification status. These included many previously described amplification target genes as well as several genes not previously shown to have a role in breast cancer

High-Resolution Genomic and Expression Profiling Reveals 105 Putative Amplification Target Genes . . .

Comparative genomic hybridization (CGH) studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4), which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer