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Supplementary Data from Tiling Path Genomic Profiling of Grade 3 Invasive Ductal Breast Cancers

Supplementary Table S1.

Purpose: To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer.Experimental Design: Twelve ovarian clear cell carcinoma cell lines were subjected to tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis. Regions of high-level amplification were defined and genes whose expression levels were determined by copy number and correlated with gene amplification were identified. The effects of inhibition of PPM1D were assessed using short hairpin RNA constructs and a small-molecule inhibitor (CCT007093). The prevalence of PPM1D amplification and mRNA expression was determined using chromogenic in situ hybridization and quantitative real-time reverse transcription-PCR in a cohort of pure ovarian clear cell carcinomas and on an independent series of unselected epithelial ovarian cancers.Results: Array-based comparative genomic hybridization analysis revealed regions of high-level amplification on 1q32, 1q42, 2q11, 3q24-q26, 5p15, 7p21-p22, 11q13.2-q13.4, 11q22, 17q21-q22, 17q23.2, 19q12-q13, and 20q13.2. Thirty-four genes mapping to these regions displayed expression levels that correlated with copy number gains/amplification. PPM1D had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines harboring gains/amplifications of 17q23.2. PPM1D inhibition revealed that PPM1D expression and phosphatase activity are selectively required for the survival of ovarian clear cell carcinoma cell lines with 17q23.2 amplification. PPM1D amplification was significantly associated with ovarian clear cell carcinoma histology (P = 0.0003) and found in 10% of primary ovarian clear cell carcinomas. PPM1D expression levels were significantly correlated with PPM1D gene amplification in primary ovarian clear cell carcinomas.Conclusion: Our data provide strong circumstantial evidence that PPM1D is a potential therapeutic target for a subgroup of ovarian clear cell carcinomas.

Supplementary Table S3.

Supplementary Data from PPM1D Is a Potential Therapeutic Target in Ovarian Clear Cell Carcinomas

Purpose: To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes.Experimental Design: Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs.Results: We show that basal-like and HER-2 tumors are characterized by “sawtooth” and “firestorm” genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification.Conclusions: Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

Purpose: As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors.Experimental Design: We carried out copy number profiling by array comparative genomic hybridization using a 32K bacterial artificial chromosome platform on 63 formalin-fixed paraffin-embedded cases of high-grade glioma arising in children and young people (Results: The genomic profiles of these tumors could be subclassified into four categories: those with stable genomes, which were associated with a better prognosis; those with aneuploid and those with highly rearranged genomes; and those with an amplifier genotype, which had a significantly worse clinical outcome. Independent of this was a clear segregation of cases with 1q gain (more common in children) from those with concurrent 7 gain/10q loss (a defining feature of adults). Detailed mapping of all the amplification and deletion events revealed numerous low-frequency amplifications, including IGF1R, PDGFRB, PIK3CA, CDK6, CCND1, and CCNE1, and novel homozygous deletions encompassing unknown genes, including those at 5q35, 10q25, and 22q13. Despite this, aberrations targeting the “core signaling pathways” in adult glioblastomas are significantly underrepresented in the pediatric setting.Conclusions: These data highlight that although there are overlaps in the genomic events driving gliomagenesis of all ages, the pediatric disease harbors a distinct spectrum of copy number aberrations compared with adults. Clin Cancer Res; 16(13); 3368–77. ©2010 AACR.

Expression, mutation and copy number analysis of platelet-derived growth factor receptor A (PDGFRA) and its ligand PDGFA in gliomas, BACKGROUND: Malignant gliomas are the most prevalent type of primary brain tumours but the therapeutic armamentarium for these tumours is limited. Platelet-derived growth factor (PDGF) signalling has been shown to be a key regulator of glioma development. Clinical trials evaluating the efficacy of anti-PDGFRA therapies on gliomas are ongoing. In this study, we intended to analyse the expression of PDGFA and its receptor PDGFRA, as well as the underlying genetic (mutations and amplification) mechanisms driving their expression in a large series of human gliomas. METHODS: PDGFA and PDGFRA expression was evaluated by immunohistochemistry in a series of 160 gliomas of distinct World Health Organization (WHO) malignancy grade. PDGFRA-activating gene mutations (exons 12, 18 and 23) were assessed in a subset of 86 cases by PCR-single-strand conformational polymorphism (PCR-SSCP), followed by direct sequencing. PDGFRA gene amplification analysis was performed in 57 cases by quantitative real-time PCR (QPCR) and further validated in a subset of cases by chromogenic in situ hybridisation (CISH) and microarray-based comparative genomic hybridisation (aCGH). RESULTS: PDGFA and PDGFRA expression was found in 81.2% (130 out of 160) and 29.6% (48 out of 160) of gliomas, respectively. Its expression was significantly correlated with histological type of the tumours; however, no significant association between the expression of the ligand and its receptor was observed. The absence of PDGFA expression was significantly associated with the age of patients and with poor prognosis. Although PDGFRA gene-activating mutations were not found, PDGFRA gene amplification was observed in 21.1% (12 out of 57) of gliomas. No association was found between the presence of PDGFRA gene amplification and expression, excepting for grade II diffuse astrocytomas. CONCLUSION: The concurrent expression of PDGFA and PDGFRA in different subtypes of gliomas, reinforce the recognised significance of this signalling pathway in gliomas. PDGFRA gene amplification rather than gene mutation may be the underlying genetic mechanism driving PDGFRA overexpression in a portion of gliomas. Taken together, our results could provide in the future a molecular basis for PDGFRA-targeted therapies in gliomas., OM is a recipient of a PhD fellowship (SFRH/BD/36463/2007) from Fundação para a Ciência e a Tecnologia (FCT), Portugal. JSRF,MBKL, AM, NT, KF and AA are funded by Breakthrough BreastCancer. This study was supported in part by Pfizer/Sociedade de Ciências dicas de Lisboa with the prize: Research in oncology diseases, Professor Francisco Gentil’ and Breakthrough Breast Cancer., info:eu-repo/semantics/publishedVersion

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2-amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual-colour FISH, were studied by microarray-based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling-path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray-based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted.

Purpose: To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer. Experimental Design: Twelve ovarian clear cell carcinoma cell lines were subjected to tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis. Regions of high-level amplification were defined and genes whose expression levels were determined by copy number and correlated with gene amplification were identified. The effects of inhibition of PPM1D were assessed using short hairpin RNA constructs and a small-molecule inhibitor (CCT007093). The prevalence of PPM1D amplification and mRNA expression was determined using chromogenic in situ hybridization and quantitative real-time reverse transcription-PCR in a cohort of pure ovarian clear cell carcinomas and on an independent series of unselected epithelial ovarian cancers. Results: Array-based comparative genomic hybridization analysis revealed regions of high-level amplification on 1q32, 1q42, 2q11, 3q24-q26, 5p15, 7p21-p22, 11q13.2-q13.4, 11q22, 17q21-q22, 17q23.2, 19q12-q13, and 20q13.2. Thirty-four genes mapping to these regions displayed expression levels that correlated with copy number gains/amplification. PPM1D had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines harboring gains/amplifications of 17q23.2. PPM1D inhibition revealed that PPM1D expression and phosphatase activity are selectively required for the survival of ovarian clear cell carcinoma cell lines with 17q23.2 amplification. PPM1D amplification was significantly associated with ovarian clear cell carcinoma histology (P = 0.0003) and found in 10% of primary ovarian clear cell carcinomas. PPM1D expression levels were significantly correlated with PPM1D gene amplification in primary ovarian clear cell carcinomas. Conclusion: Our data provide strong circumstantial evidence that PPM1D is a potential therapeutic target for a subgroup of ovarian clear cell carcinomas.

HER2 and TOP2A are targets for the therapeutic agents trastuzumab and anthracyclines and are frequently amplified in breast cancers. The aims of this study were to provide a detailed molecular genetic analysis of the 17q12-q21 amplicon in breast cancers harbouring HER2/TOP2A co-amplification and to investigate additional recurrent co-amplifications in HER2/TOP2A-co-amplified cancers. In total, 15 breast cancers with HER2 amplification, 10 of which also harboured TOP2A amplification, as defined by chromogenic in situ hybridisation, and 6 breast cancer cell lines known to be amplified for HER2 were subjected to high-resolution microarray-based comparative genomic hybridisation analysis. This revealed that the genomes of 12 cases were characterised by at least one localised region of clustered, relatively narrow peaks of amplification, with each cluster confined to a single chromosome arm (ie 'firestorm' pattern) and 3 cases displayed many narrow segments of duplication and deletion affecting the vast majority of chromosomes (ie 'sawtooth' pattern). The smallest region of amplification (SRA) on 17q12 in the whole series extended from 34.73 to 35.48 Mb, and encompassed HER2 but not TOP2A. In HER2/TOP2A-co-amplified samples, the SRA extended from 34.73 to 36.54 Mb, spanning a region of approximately 1.8 Mb. Apart from HER2 and TOP2A, this region encompassed four additional genes whose expression levels as defined by quantitative real-time PCR are significantly higher in HER2/TOP2A-co-amplified vs HER2-amplified breast cancers: CASC3, CDC6, RARA and SMARCE1. Of the cell lines studied, SKBR3 and UACC812 showed HER2/TOP2A co-amplification. In conclusion, this is the first detailed genome-wide characterisation of HER2/TOP2A-amplified breast cancers; cell lines were identified that can be used to model these cancers in vitro. The 17q12 amplicon is complex and harbours multiple genes that may be associated with breast cancer development and progression, and potentially exploitable as therapeutic targets.

To establish a panel of highly radiation responsive genes suitable for biological dosimetry and to explore inter-individual variation in response to ionising radiation exposure.Analysis of gene expression in response to radiation was carried out using three independent techniques (Microarray, Multiplex Quantitative Real-Time Polymerase Chain Reaction (MQRT- PCR) and nCounter® Analysis System) in human dividing lymphocytes in culture and peripheral blood leukocytes exposed ex vivo from the same donors.Variations in transcriptional response to exposure to ionising radiation analysed by microarray allowed the identification of genes which can be measured accurately using MQRT PCR and another technique allowing direct count of mRNA copies. We have identified genes which are consistently up-regulated following exposure to 2 or 4 Gy of X-rays at different time points, for all individuals in blood and cultured lymphocytes. Down-regulated genes including cyclins, centromeric and mitotic checkpoint genes, particularly those associated with chromosome instability and cancer could be detected in dividing lymphocytes only.The data provide evidence that there are a number of genes which seem suitable for biological dosimetry using peripheral blood, including sestrin 1 (SESN1), growth arrest and DNA damage inducible 45 alpha (GADD45A), cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin G1 (CCNG1), ferredoxin reductase (FDXR), p53 up-regulated mediator of apoptosis (BBC3) and Mdm2 p53 binding protein homolog (MDM2). These biomarkers could potentially be used for triage after large-scale radiological incidents and for monitoring radiation exposure during radiotherapy.

As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors. Purpose: As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors. Experimental Design: We carried out copy number profiling by array comparative genomic hybridization using a 32K bacterial artificial chromosome platform on 63 formalin-fixed paraffin-embedded cases of high-grade glioma arising in children and young people (, National Health Service funding to the NIHR Biomedical Research Centre. This work was supported by The Royal Marsden Children's Department Fund, Fundação para a Ciência e Tecnologia, Portugal, and Breakthrough Breast Cancer

Aims: Microglandular adenosis (MGA) is a rare breast lesion, which has long been considered to be hyperplastic. However, atypical forms of MGA (AMGA) and invasive carcinomas arising in the background of MGA are recorded. Recent studies have suggested that MGA may be a non-obligate precursor of invasive carcinomas that are negative for hormone receptors and lack HER-2 overexpression (triple-negative phenotype). The aim of this study was to determine whether MGA is clonal and whether it harbours chromosomal aberrations similar to those found in matched invasive ductal carcinoma of no special type (IDC-NST). Methods and results: We report on a case comprising MGA, AMGA and a high-grade IDC-NST. The three components were separately microdissected and subjected to genetic analysis with high-resolution microarray comparative genomic hybridisation. Identical genetic changes were detected in all components with subsequent acquisition of additional genetic aberrations in the invasive component, suggesting that MGA was the substrate for the development of the invasive carcinoma. Immunohistochemistry revealed concordant profiles across all components, characterized by triple-negative phenotype and variable positivity for basal markers. Conclusions: Similar to adenomas, MGA is, at least in some cases, a clonal lesion and may be a non-obligate precursor of a subgroup of high-grade triple-negative and basal-like breast carcinomas.

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP173.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2-amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual-colour FISH, were studied by microarray-based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling-path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray-based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted.

Background: Secretory breast cancer (SBC) is a rare entity characterised by indolent clinical behaviour, distinctive histological features and the presence of a recurrent chromosomal translocation t(12;15)(p13;q25), leading to the formation of the ETV6–NTRK3 fusion gene. Aim: To describe the molecular genetic features of a case of SBC which harbours a duplication of the t(12;15) translocation. Methods: Tiling path array comparative genomic hybridisation (aCGH) analysis and fluorescence in situ hybridisation (FISH) using in-house-generated probes for ETV6 , NTRK3 and the fusion genes, centromeric probes for chromosomes 12 and 15, and a commercially available split-apart ETV6/NTRK3 probe. Results: FISH revealed the presence of a duplication of the translocation t(12;15), which resulted from the gain of one copy of the derivative chromosome der(15)t(12;15), retention of one normal copy of both ETV6 and NTRK3 genes and deletion of the derivative chromosome der(12)t(12;15). Consistent with FISH findings, aCGH revealed copy number gains of ETV6 and NTRK3 and deletions encompassing the regions centromeric to ETV6 and telomeric to NTRK3 . Additional regions of copy number changes included gains of 10q21, 10q26.3, 12p13.3–p13.31 15q11–q25.3 and 16pq and losses of 6q24.1–q27, 12p13.2–q12 and 15q25.3–q26.3. Conclusions: To the best of our knowledge, this is the first time a carcinoma has been shown to harbour a duplication of the ETV6–NTRK3 translocation. The presence of an additional copy of the derivative chromosome der(15)t(12;15) coupled with deletion of the other derivative der(12)t(12;15) in the modal population of cancer cells suggests that this was either an early phenomenon or conferred additional growth advantage on neoplastic cells.

The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer.

Purpose: To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes. Experimental Design: Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs. Results: We show that basal-like and HER-2 tumors are characterized by “sawtooth” and “firestorm” genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification. Conclusions: Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

Micropapillary carcinomas (MPCs) can present as a rare histological special type of breast cancer; however, this histological type is more frequently found admixed with invasive ductal carcinomas of no special type (IDC-NSTs). We have previously demonstrated that pure MPCs constitute a distinct entity at the morphological and genetic levels. Here, we sought to determine whether mixed MPCs have genomic aberrations similar to those found in pure MPCs, and to investigate whether the distinct morphological components of MPCs harbour different genetic aberrations. Using high-resolution microarray comparative genomic hybridization (aCGH), we profiled a series of 10 MPCs of mixed histology and 20 IDC-NSTs matched for grade and oestrogen receptor (ER) status. In addition, we generated tissue microarrays containing a series of 24 pure and 40 mixed MPCs and performed immunohistochemical analysis with ER, progesterone receptor (PR), Ki-67, HER2, cytokeratin (CK) 5/6, CK14, CK17, EGFR, topoisomerase-IIalpha, cyclin D1, caveolin-1 and E-cadherin antibodies. In situ hybridization was employed to evaluate the prevalence of HER2, TOP2A, EGFR, CCND1, MYC and FGFR1 gene amplification. Our results demonstrate that mixed MPCs harbour similar patterns of genomic aberrations and phenotype (82.5% luminal and 17.5% HER2) compared to pure MPCs. A comparison between the distinct morphological components of mixed MPCs in a pairwise fashion revealed that both components harbour strikingly similar genomic profiles. When compared to grade- and ER-matched IDC-NSTs, mixed MPCs significantly more frequently harboured amplification of multiple regions on 8q (adjusted Fisher's p value < 0.05). Furthermore, mixed MPCs displayed higher proliferative rates than grade- and ER-matched IDC-NSTs. Our results suggest that micropapillary differentiation in breast cancer may identify a subgroup of more aggressive ER-positive breast carcinomas, even in those featuring a mixed histology, and that mixed MPCs are more closely related to pure MPCs than to IDC-NSTs.

Tumour cell lines derived from breast cancer patients constitute one of the cornerstones of breast cancer research. To characterise breast cancer cell lines at the genetic level, we have developed a full tiling path bacterial artificial chromosome (BAC) array collection for comparative genomic hybridisation (aCGH). This aCGH BAC collection covers 98% of the entire human genome at a resolution of 40-60 kbp. We have used this platform alongside an in-house produced 17 K cDNA microarray set to characterise the genetic and transcriptomic profiles of 24 breast cancer cell lines, as well as cell types derived from non-diseased breast. We demonstrate that breast cancer cell lines have genomic and transcriptomic features that recapitulate those of primary breast cancers and can be reliably subclassified into basal-like and luminal subgroups. By overlaying aCGH and transcriptomic data, we have identified 753 genes whose expression correlate with copy number; this list comprised numerous oncogenes recurrently amplified and overexpressed in breast cancer (e.g., HER2, MYC, CCND1 and AURKA). Finally, we demonstrate that although breast cancer cell lines have genomic features usually found in grade III breast cancers (i.e., gains of 1q, 8q and 20q), basal-like and luminal cell lines are characterised by distinct genomic aberrations.

Expression profiling studies have suggested that HER2-amplified breast cancers constitute a heterogeneous group that may be subdivided according to their ER status: HER2-amplified ER-positive breast carcinomas that fall into the luminal B cluster; and HER2-amplified ER-negative cancers which form a distinct molecular subgroup, known as the erbB2 or HER2 subgroup. ER-negative breast cancer differs significantly from ER-positive disease in the pattern, type, and complexity of genetic aberrations. Here we have compared the genomic profiles of ER-positive and ER-negative HER2-amplified cancers using tiling path microarray-based comparative genomic hybridization (aCGH). Validation of the differentially amplified regions was performed in an independent series of 70 HER2-amplified breast cancers. Although HER2-amplified cancers had remarkably complex patterns of molecular genetic aberrations, ER-positive and ER-negative HER2-amplified breast carcinomas shared most molecular genetic features as defined by aCGH. Genome-wide Fisher's exact test analysis revealed that less than 1.5% of the genome was significantly differentially gained or lost in ER-positive versus ER-negative HER2-amplified cancers. However, two regions of amplification were significantly associated with ER-positive carcinomas, one of which mapped to 17q21.2 and encompassed GJC1, IGFBP4, TNS4, and TOP2A. Chromogenic in situ hybridization analysis of an independent validation series confirmed the association between ER status and TOP2A amplification. In conclusion, although hormone receptor status does not determine the overall genetic profile of HER2-amplified breast cancers, specific genetic aberrations may be characteristic of subgroups of HER2 breast cancers.

Pure invasive micropapillary carcinoma (MPC) is a special histological type that accounts for 0.7-3% of all breast cancers. MPC has a distinctive growth pattern and a more aggressive clinical behaviour than invasive ductal carcinomas of no special type (IDC-NSTs). To define the molecular characteristics of MPCs, we profiled a series of 12 MPCs and 24 grade and oestrogen receptor (ER)-matched IDC-NSTs using high-resolution microarray comparative genomic hybridization (aCGH). In addition, we generated a tissue microarray containing a series of 24 MPCs and performed immunohistochemical analysis with ER, PR, Ki-67, HER2, CK5/6, CK14, CK17, EGFR, topoisomerase-IIalpha, cyclin D1, caveolin-1, E-cadherin, and beta-catenin antibodies. In situ hybridization probes were employed to evaluate the prevalence of amplification of HER2, TOP2A, EGFR, CCND1, MYC, ESR1, and FGFR1 genes. aCGH analysis demonstrated that MPCs significantly differed from IDC-NSTs at the genomic level. Gains of 1q, 2q, 4p, 6p, 6q23.2-q27, 7p, 7q, 8p, 8q, 9p, 10p, 11q, 12p, 12q, 16p, 17p, 17q, 19p, 20p, 20q, and 21q, and losses of 1p, 2p, 6q11.1-q16.3, 6q21-q22.1, 9p, 11p, 15q, and 19q were more prevalent in MPCs. High-level gains/amplifications of 8p12-p11, 8q12, 8q13, 8q21, 8q23, 8q24, 17q21, 17q23, and 20q13 were significantly associated with MPCs. A comparison between 24 MPCs and a series of 48 grade and ER-matched IDC-NSTs revealed that high cyclin D1 expression, high proliferation rates, and MYC (8q24) amplification were significantly associated with MPCs. Our results demonstrate that MPCs have distinct histological features and molecular genetic profiles supporting the contention that they constitute a distinct pathological entity.

For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. Following microdissection, most samples fail to produce sufficient quantities of DNA for genome-wide experiments. Various PCR-based amplification methods have been used, but these usually produce nonuniform representations of the genome. Bacteriophage Phi29 DNA polymerase random-primed DNA amplification is based on isothermal multiple displacement amplification. We sought to define the genome representation of this method in a bacterial artificial chromosome microarray comparative genomic hybridisation (aCGH) platform. Test genomic female DNA was amplified using Phi29 amplification at four different starting concentrations (0.5, 5, 10 and 50 ng). These products were combined with unamplified and amplified genomic female DNA as reference. In addition, 50 ng of DNA from five microdissected breast cancer frozen samples, were amplified using the same method. Three combinations were performed: unamplified test with unamplified reference, amplified test with unamplified reference and both amplified tumour and reference DNA. aCGH was performed with an in-house 16 K BAC platform (a resolution of approximately 100 Kb). Pearson's correlation tests and hierarchical clustering were performed to compare the profiles obtained. aCGH profiles obtained with amplified test and unamplified reference female genomic DNA showed copy number biases throughout the genome. These biases were more conspicuous with smaller amounts of starting material and mapped to regions of known copy number polymorphisms. When similar concentrations of test and reference DNA were amplified, the biases were significantly reduced, rendering accurate profiles. For the tumours, representative profiles were obtained when both test and reference DNA were amplified. Phi29 amplification induces copy number biases and unamplified material remains the gold standard for copy number analysis. For accurate results using Phi29 amplification, samples subjected to aCGH analysis should be combined with reference DNA amplified with the same method, using similar amounts of starting template.