Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors.

Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme­the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors­most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases.

Development of a robust RNA-based classifier to accurately determine ER, PR, and HER2 status in breast cancer clinical samples.

Breast cancers are categorized into three subtypes based on protein expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/ERBB2). Patients enroll onto experimental clinical trials based on ER, PR, and HER2 status and, as receptor status is prognostic and defines treatment regimens, central receptor confirmation is critical for interpreting results from these trials. Patients enrolling onto experimental clinical trials in the metastatic setting often have limited available archival tissue that might better be used for comprehensive molecular profiling rather than slide-intensive reconfirmation of receptor status. We developed a Random Forests-based algorithm using a training set of 158 samples with centrally confirmed IHC status, and subsequently validated this algorithm on multiple test sets with known, locally determined IHC status. We observed a strong correlation between target mRNA expression and IHC assays for HER2 and ER, achieving an overall accuracy of 97 and 96%, respectively. For determining PR status, which had the highest discordance between central and local IHC, incorporation of expression of co-regulated genes in a multivariate approach added predictive value, outperforming the single, target gene approach by a 10% margin in overall accuracy. Our results suggest that multiplexed qRT-PCR profiling of ESR1, PGR, and ERBB2 mRNA, along with several other subtype associated genes, can effectively confirm breast cancer subtype, thereby conserving tumor sections and enabling additional biomarker data to be obtained from patients enrolled onto experimental clinical trials.

A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes.

Recent studies suggest that thousands of genes may contribute to breast cancer pathophysiologies when deregulated by genomic or epigenomic events. Here, we describe a model "system" to appraise the functional contributions of these genes to breast cancer subsets. In general, the recurrent genomic and transcriptional characteristics of 51 breast cancer cell lines mirror those of 145 primary breast tumors, although some significant differences are documented. The cell lines that comprise the system also exhibit the substantial genomic, transcriptional, and biological heterogeneity found in primary tumors. We show, using Trastuzumab (Herceptin) monotherapy as an example, that the system can be used to identify molecular features that predict or indicate response to targeted therapies or other physiological perturbations.

Genomic and transcriptional aberrations linked to breast cancer pathophysiologies.

This study explores the roles of genome copy number abnormalities (CNAs) in breast cancer pathophysiology by identifying associations between recurrent CNAs, gene expression, and clinical outcome in a set of aggressively treated early-stage breast tumors. It shows that the recurrent CNAs differ between tumor subtypes defined by expression pattern and that stratification of patients according to outcome can be improved by measuring both expression and copy number, especially high-level amplification. Sixty-six genes deregulated by the high-level amplifications are potential therapeutic targets. Nine of these (FGFR1, IKBKB, ERBB2, PROCC, ADAM9, FNTA, ACACA, PNMT, and NR1D1) are considered druggable. Low-level CNAs appear to contribute to cancer progression by altering RNA and cellular metabolism.

Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3.

CpG islands are present in one-half of all human and mouse genes and typically overlap with promoters or exons. We developed a method for high-resolution analysis of the methylation status of CpG islands genome-wide, using arrays of BAC clones and the methylation-sensitive restriction enzyme NotI. Here we demonstrate the accuracy and specificity of the method. By computationally mapping all NotI sites, methylation events can be defined with single-nucleotide precision throughout the genome. We also demonstrate the unique expandability of the array method using a different methylation-sensitive restriction enzyme, BssHII. We identified and validated new CpG island loci that are methylated in a tissue-specific manner in normal human tissues. The methylation status of the CpG islands is associated with gene expression for several genes, including SHANK3, which encodes a structural protein in neuronal postsynaptic densities. Defects in SHANK3 seem to underlie human 22q13 deletion syndrome. Furthermore, these patterns for SHANK3 are conserved in mice and rats.

Therapy-induced malignant neoplasms in Nf1 mutant mice.

Therapy-induced cancers are a severe complication of genotoxic therapies. We used heterozygous Nf1 mutant mice as a sensitized genetic background to investigate tumor induction by radiation (RAD) and cyclophosphamide (CY). Mutagen-exposed Nf1(+/-) mice developed secondary cancers that are common in humans, including myeloid malignancies, sarcomas, and breast cancers. RAD cooperated strongly with heterozygous Nf1 inactivation in tumorigenesis. Most of the solid tumors showed loss of the wild-type Nf1 allele but retained two Trp53 alleles. Comparative genomic hybridization demonstrated distinct patterns of copy number aberrations in sarcomas and breast cancers from Nf1 mutant mice, and tumor cell lines showed deregulated Ras signaling. Nf1(+/-) mice provide a tractable model for investigating the pathogenesis of common mutagen-induced cancers and for testing preventive strategies.

Statistical techniques to construct assays for identifying likely responders to a treatment under evaluation from cell line genomic data.

BACKGROUND: Developing the right drugs for the right patients has become a mantra of drug development. In practice, it is very difficult to identify subsets of patients who will respond to a drug under evaluation. Most of the time, no single diagnostic will be available, and more complex decision rules will be required to define a sensitive population, using, for instance, mRNA expression, protein expression or DNA copy number. Moreover, diagnostic development will often begin with in-vitro cell-line data and a high-dimensional exploratory platform, only later to be transferred to a diagnostic assay for use with patient samples. In this manuscript, we present a novel approach to developing robust genomic predictors that are not only capable of generalizing from in-vitro to patient, but are also amenable to clinically validated assays such as qRT-PCR. METHODS: Using our approach, we constructed a predictor of sensitivity to dacetuzumab, an investigational drug for CD40-expressing malignancies such as lymphoma using genomic measurements of cell lines treated with dacetuzumab. Additionally, we evaluated several state-of-the-art prediction methods by independently pairing the feature selection and classification components of the predictor. In this way, we constructed several predictors that we validated on an independent DLBCL patient dataset. Similar analyses were performed on genomic measurements of breast cancer cell lines and patients to construct a predictor of estrogen receptor (ER) status. RESULTS: The best dacetuzumab sensitivity predictors involved ten or fewer genes and accurately classified lymphoma patients by their survival and known prognostic subtypes. The best ER status classifiers involved one or two genes and led to accurate ER status predictions more than 85% of the time. The novel method we proposed performed as well or better than other methods evaluated. CONCLUSIONS: We demonstrated the feasibility of combining feature selection techniques with classification methods to develop assays using cell line genomic measurements that performed well in patient data. In both case studies, we constructed parsimonious models that generalized well from cell lines to patients.

Integration of genomic analysis and in vivo transfection to identify sprouty 2 as a candidate tumor suppressor in liver cancer.

UNLABELLED: Hepatocellular carcinoma (HCC) is 1 of the leading causes of cancer-related deaths worldwide, yet the molecular genetics underlying this malignancy are still poorly understood. In our study, we applied statistical methods to correlate human HCC gene expression data obtained from complementary DNA (cDNA) microarrays and corresponding DNA copy number variation data obtained from array-based comparative genomic hybridization. We have thus identified 76 genes that are up-regulated and show frequent DNA copy number gain, and 37 genes that are down-regulated and show frequent DNA copy loss in human HCC samples. Among these down-regulated genes is Sprouty2 (Spry2), a known inhibitor of receptor tyrosine kinases. We investigated the potential role of Spry2 in HCC by expressing dominant negative Spry2 (Spry2Y55F) and activated beta-catenin (DeltaN90-beta-catenin) in the mouse liver through hydrodynamic injection and sleeping beauty-mediated somatic integration. When stably expressed in mouse hepatocytes, Spry2Y55F cooperates with DeltaN90-beta-catenin to confer a neoplastic phenotype in mice. Tumor cells show high levels of expression of phospho-extracellular signal-regulated kinase (ERK), as well as deregulation of genes involved in cell proliferation, apoptosis, and angiogenesis. CONCLUSION: We identified a set of candidate oncogenes and tumor suppressor genes for human HCC. Our study provides evidence that inhibition of Spry activity cooperates with other oncogenes to promote liver cancer in mouse models, and Spry2 may function as a candidate tumor suppressor for HCC development in vivo. In addition, we demonstrate that the integration of genomic analysis and in vivo transfection is a powerful tool to identify genes that are important during hepatic carcinogenesis.

High-resolution genomic and expression analyses of copy number alterations in breast tumors.

Analysis of recurrent DNA amplification can lead to the identification of cancer driver genes, but this process is often hampered by the low resolution of existing copy number analysis platforms. Fifty-one breast tumors were profiled for copy number alterations (CNAs) with the high-resolution Affymetrix 500K SNP array. These tumors were also expression-profiled and surveyed for mutations in selected genes commonly mutated in breast cancer (TP53, CDKN2A, ERBB2, KRAS, PIK3CA, PTEN). Combined analysis of common CNAs and mutations revealed putative associations between features. Analysis of both the prevalence and amplitude of CNAs defined regions of recurrent alteration. Compared with previous array comparative genomic hybridization studies, our analysis provided boundaries for frequently altered regions that were approximately one-fourth the size, greatly reducing the number of potential alteration-driving genes. Expression data from matched tumor samples were used to further interrogate the functional relevance of genes located in recurrent amplicons. Although our data support the importance of some known driver genes such as ERBB2, refined amplicon boundaries at other locations, such as 8p11-12 and 11q13.5-q14.2, greatly reduce the number of potential driver genes and indicate alternatives to commonly suggested driver genes in some cases. For example, the previously reported recurrent amplification at 17q23.2 is reduced to a 249 kb minimal region containing the putative driver RPS6KB1 as well as the putative oncogenic microRNA mir-21. High-resolution copy number analysis provides refined insight into many breast cancer amplicons and their relationships to gene expression, point mutations and breast cancer subtype classifications. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Deletion of chromosome 11q predicts response to anthracycline-based chemotherapy in early breast cancer.

Despite the recent consensus on the eligibility of adjuvant systemic therapy in patients with lymph node-negative breast cancer (NNBC) based on clinicopathologic criteria, specific biological markers are needed to predict sensitivity to the different available therapeutic options. We examined the feasibility of developing a genomic predictor of chemotherapy response and recurrence risk in 185 patients with NNBC using assembled arrays containing 2,460 bacterial artificial chromosome clones for scanning the genome for DNA copy number changes. After surgery, 90 patients received anthracycline-based chemotherapy, whereas 95 did not. Tamoxifen was administered to patients with hormone receptor-positive tumors. The association of genomic and clinicopathologic data and outcome was computed using Cox proportional hazard models and multiple testing adjustment procedures. Analysis of NNBC genomes revealed a common genomic signature. Specific DNA copy number aberrations were associated with hormonal receptor status, but not with other clinicopathologic variables. In patients treated with chemotherapy, none of the genomic changes were significantly correlated with recurrence. In patients not receiving chemotherapy, deletion of eight bacterial artificial chromosome clones clustered to chromosome 11q was independently associated with relapse (disease-free survival at 10 years+/-SE, 40%+/-14% versus 86%+/-6%; P<0.0001). The 54 patients with deletion of 11q (29%) did not present more aggressive clinicopathologic features than those without 11q loss. The adverse influence of 11q deletion on clinical outcome was confirmed in an independent validation series of 88 patients with NNBC. Our data suggests that patients with NNBC with the 11q deletion might benefit from anthracycline-based chemotherapy despite other clinical, pathologic, or genetic features. However, these initial findings should be evaluated in randomized clinical trials.

Improving melanoma classification by integrating genetic and morphologic features.

BACKGROUND: In melanoma, morphology-based classification systems have not been able to provide relevant information for selecting treatments for patients whose tumors have metastasized. The recent identification of causative genetic alterations has revealed mutations in signaling pathways that offer targets for therapy. Identifying morphologic surrogates that can identify patients whose tumors express such alterations (or functionally equivalent alterations) would be clinically useful for therapy stratification and for retrospective analysis of clinical trial data. METHODOLOGY/PRINCIPAL FINDINGS: We defined and assessed a panel of histomorphologic measures and correlated them with the mutation status of the oncogenes BRAF and NRAS in a cohort of 302 archival tissues of primary cutaneous melanomas from an academic comprehensive cancer center. Melanomas with BRAF mutations showed distinct morphological features such as increased upward migration and nest formation of intraepidermal melanocytes, thickening of the involved epidermis, and sharper demarcation to the surrounding skin; and they had larger, rounder, and more pigmented tumor cells (all p-values below 0.0001). By contrast, melanomas with NRAS mutations could not be distinguished based on these morphological features. Using simple combinations of features, BRAF mutation status could be predicted with up to 90.8% accuracy in the entire cohort as well as within the categories of the current World Health Organization (WHO) classification. Among the variables routinely recorded in cancer registries, we identified age < 55 y as the single most predictive factor of BRAF mutation in our cohort. Using age < 55 y as a surrogate for BRAF mutation in an independent cohort of 4,785 patients of the Southern German Tumor Registry, we found a significant survival benefit (p < 0.0001) for patients who, based on their age, were predicted to have BRAF mutant melanomas in 69% of the cases. This group also showed a different pattern of metastasis, more frequently involving regional lymph nodes, compared to the patients predicted to have no BRAF mutation and who more frequently displayed satellite, in-transit metastasis, and visceral metastasis (p < 0.0001). CONCLUSIONS: Refined morphological classification of primary melanomas can be used to improve existing melanoma classifications by forming subgroups that are genetically more homogeneous and likely to differ in important clinical variables such as outcome and pattern of metastasis. We expect this information to improve classification and facilitate stratification for therapy as well as retrospective analysis of existing trial data.

Distinct sets of genetic alterations in melanoma.

BACKGROUND: Exposure to ultraviolet light is a major causative factor in melanoma, although the relationship between risk and exposure is complex. We hypothesized that the clinical heterogeneity is explained by genetically distinct types of melanoma with different susceptibility to ultraviolet light. METHODS: We compared genome-wide alterations in the number of copies of DNA and mutational status of BRAF and N-RAS in 126 melanomas from four groups in which the degree of exposure to ultraviolet light differs: 30 melanomas from skin with chronic sun-induced damage and 40 melanomas from skin without such damage; 36 melanomas from palms, soles, and subungual (acral) sites; and 20 mucosal melanomas. RESULTS: We found significant differences in the frequencies of regional changes in the number of copies of DNA and mutation frequencies in BRAF among the four groups of melanomas. Samples could be correctly classified into the four groups with 70 percent accuracy on the basis of the changes in the number of copies of genomic DNA. In two-way comparisons, melanomas arising on skin with signs of chronic sun-induced damage and skin without such signs could be correctly classified with 84 percent accuracy. Acral melanoma could be distinguished from mucosal melanoma with 89 percent accuracy. Eighty-one percent of melanomas on skin without chronic sun-induced damage had mutations in BRAF or N-RAS; the majority of melanomas in the other groups had mutations in neither gene. Melanomas with wild-type BRAF or N-RAS frequently had increases in the number of copies of the genes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1), downstream components of the RAS-BRAF pathway. CONCLUSIONS: The genetic alterations identified in melanomas at different sites and with different levels of sun exposure indicate that there are distinct genetic pathways in the development of melanoma and implicate CDK4 and CCND1 as independent oncogenes in melanomas without mutations in BRAF or N-RAS.

Amplification of PVT1 contributes to the pathophysiology of ovarian and breast cancer.

PURPOSE: This study was designed to elucidate the role of amplification at 8q24 in the pathophysiology of ovarian and breast cancer because increased copy number at this locus is one of the most frequent genomic abnormalities in these cancers. EXPERIMENTAL DESIGN: To accomplish this, we assessed the association of amplification at 8q24 with outcome in ovarian cancers using fluorescence in situ hybridization to tissue microarrays and measured responses of ovarian and breast cancer cell lines to specific small interfering RNAs against the oncogene MYC and a putative noncoding RNA, PVT1, both of which map to 8q24. RESULTS: Amplification of 8q24 was associated with significantly reduced survival duration. In addition, small interfering RNA-mediated reduction in either PVT1 or MYC expression inhibited proliferation in breast and ovarian cancer cell lines in which they were both amplified and overexpressed but not in lines in which they were not amplified/overexpressed. Inhibition of PVT1 expression also induced a strong apoptotic response in cell lines in which it was overexpressed but not in lines in which it was not amplified/overexpressed. Inhibition of MYC, on the other hand, did not induce an apoptotic response in cell lines in which MYC was amplified and overexpressed. CONCLUSIONS: These results suggest that MYC and PVT1 contribute independently to ovarian and breast pathogenesis when overexpressed because of genomic abnormalities. They also suggest that PVT1-mediated inhibition of apoptosis may explain why amplification of 8q24 is associated with reduced survival duration in patients treated with agents that act through apoptotic mechanisms.

Aging impacts transcriptomes but not genomes of hormone-dependent breast cancers.

INTRODUCTION: Age is one of the most important risk factors for human malignancies, including breast cancer; in addition, age at diagnosis has been shown to be an independent indicator of breast cancer prognosis. Except for inherited forms of breast cancer, however, there is little genetic or epigenetic understanding of the biological basis linking aging with sporadic breast cancer incidence and its clinical behavior. METHODS: DNA and RNA samples from matched estrogen receptor (ER)-positive sporadic breast cancers diagnosed in either younger (age or= 70 years) Caucasian women were analyzed by array comparative genomic hybridization and by expression microarrays. Array comparative genomic hybridization data were analyzed using hierarchical clustering and supervised age cohort comparisons. Expression microarray data were analyzed using hierarchical clustering and gene set enrichment analysis; differential gene expression was also determined by conditional permutation, and an age signature was derived using prediction analysis of microarrays. RESULTS: Hierarchical clustering of genome-wide copy-number changes in 71 ER-positive DNA samples (27 younger women, 44 older women) demonstrated two age-independent genotypes; one with few genomic changes other than 1q gain/16q loss, and another with amplifications and low-level gains/losses. Age cohort comparisons showed no significant differences in total or site-specific genomic breaks and amplicon frequencies. Hierarchical clustering of 5.1 K genes variably expressed in 101 ER-positive RNA samples (53 younger women, 48 older women) identified six transcriptome subtypes with an apparent age bias (P < 0.05). Samples with higher expression of a poor outcome-associated proliferation signature were predominantly (65%) younger cases. Supervised analysis identified cancer-associated genes differentially expressed between the cohorts; with younger cases expressing more cell cycle genes and more than threefold higher levels of the growth factor amphiregulin (AREG), and with older cases expressing higher levels of four different homeobox (HOX) genes in addition to ER (ESR1). An age signature validated against two other independent breast cancer datasets proved to have >80% accuracy in discerning younger from older ER-positive breast cancer cases with characteristic differences in AREG and ESR1 expression. CONCLUSION: These findings suggest that epigenetic transcriptome changes, more than genotypic variation, account for age-associated differences in sporadic breast cancer incidence and prognosis.

Evaluation of whole genome amplification protocols for array and oligonucleotide CGH.

Genome-based technologies such as genomic arrays and next generation sequencing are poised to make significant contributions to clinical oncology. However, translation of these technologies to the clinic will require that they produce high-quality reproducible data from small archived tumor specimens and biopsies. Herein, we report on a systematic and comprehensive microarray analysis comparing multiple whole genome amplification methods using a variety of difficult clinical specimens, including formalin-fixed and paraffin-embedded tissues. Quantitative analysis and clustering suggest that Sigma's whole genome amplification protocol performed best on all specimens and, moreover, worked well with a formalin-fixed, paraffin-embedded biopsy.

Chromosomal instability in microsatellite-unstable and stable colon cancer.

PURPOSE: The genomic instability in colon cancer can be divided into at least two major types, microsatellite instability (MSI) or chromosomal instability (CIN). Although initially felt to be mutually exclusive, recent evidence suggests that there may be overlap between the two. The aim of this study was to identify chromosomal alterations at high resolution in sporadic colon cancers with high-level microsatellite instability (MSI-H) and to compare them to those present in a set of matched microsatellite stable (MSS) tumors. EXPERIMENTAL DESIGN: Array-based comparative genomic hybridization was used to analyze a set of 23 sporadic MSI-H and 23 MSS colon cancers matched for location, gender, stage, and age. The arrays consisted of 2,464 bacterial artificial chromosome clones. RESULTS: MSI and MSS colon cancers differed significantly with respect to frequency and type of chromosomal alterations. The median fraction of genome altered was lower among MSI-H tumors than MSS tumors (2.8% versus 30.7%, P=0.00006). However, the MSI-H tumors displayed a range of genomic alterations, from the absence of detectable alterations to extensive alterations. Frequent alterations in MSI-H tumors included gains of chromosomes 8, 12, and 13, and loss of 15q14. In contrast, the most frequent alterations in MSS tumors were gains of 7, 13, 8q, and 20, and losses of 8p, 17p, and 18. A small, previously uncharacterized, genomic deletion on 16p13.2, found in 35% of MSI-H and 21% of MSS tumors, was confirmed by fluorescence in situ hybridization. CONCLUSION: MSI and CIN are not mutually exclusive forms of genomic instability in sporadic colon cancer, with MSI tumors also showing varying degrees of CIN.

Integrating data on DNA copy number with gene expression levels and drug sensitivities in the NCI-60 cell line panel.

Chromosome rearrangement, a hallmark of cancer, has profound effects on carcinogenesis and tumor phenotype. We used a panel of 60 human cancer cell lines (the NCI-60) as a model system to identify relationships among DNA copy number, mRNA expression level, and drug sensitivity. For each of 64 cancer-relevant genes, we calculated all 4,096 possible Pearson's correlation coefficients relating DNA copy number (assessed by comparative genomic hybridization using bacterial artificial chromosome microarrays) and mRNA expression level (determined using both cDNA and Affymetrix oligonucleotide microarrays). The analysis identified an association of ERBB2 overexpression with 3p copy number, a finding supported by data from human tumors and a mouse model of ERBB2-induced carcinogenesis. When we examined the correlation between DNA copy number for all 353 unique loci on the bacterial artificial chromosome microarray and drug sensitivity for 118 drugs with putatively known mechanisms of action, we found a striking negative correlation (-0.983; 95% bootstrap confidence interval, -0.999 to -0.899) between activity of the enzyme drug L-asparaginase and DNA copy number of genes near asparagine synthetase in the ovarian cancer cells. Previous analysis of drug sensitivity and mRNA expression had suggested an inverse relationship between mRNA levels of asparagine synthetase and L-asparaginase sensitivity in the NCI-60. The concordance of pharmacogenomic findings at the DNA and mRNA levels strongly suggests further study of L-asparaginase for possible treatment of a low-synthetase subset of clinical ovarian cancers. The DNA copy number database presented here will enable other investigators to explore DNA transcript-drug relationships in their own domains of research focus.

Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma.

Genomes of solid tumors are characterized by gains and losses of regions, which may contribute to tumorigenesis by altering gene expression. Often the aberrations are extensive, encompassing whole chromosome arms, which makes identification of candidate genes in these regions difficult. Here, we focused on narrow regions of gene amplification to facilitate identification of genetic pathways important in oral squamous cell carcinoma (SCC) development. We used array comparative genomic hybridization (array CGH) to define minimum common amplified regions and then used expression analysis to identify candidate driver genes in amplicons that spanned <3 Mb. We found genes involved in integrin signaling (TLN1), survival (YAP1, BIRC2), and adhesion and migration (TLN1, LAMA3, MMP7), as well as members of the hedgehog (GLI2) and notch (JAG1, RBPSUH, FJX1) pathways to be amplified and overexpressed. Deregulation of these and other members of the hedgehog and notch pathways (HHIP, SMO, DLL1, NOTCH4) implicates deregulation of developmental and differentiation pathways, cell fate misspecification, in oral SCC development.

Breast tumor copy number aberration phenotypes and genomic instability.

BACKGROUND: Genomic DNA copy number aberrations are frequent in solid tumors, although the underlying causes of chromosomal instability in tumors remain obscure. Genes likely to have genomic instability phenotypes when mutated (e.g. those involved in mitosis, replication, repair, and telomeres) are rarely mutated in chromosomally unstable sporadic tumors, even though such mutations are associated with some heritable cancer prone syndromes. METHODS: We applied array comparative genomic hybridization (CGH) to the analysis of breast tumors. The variation in the levels of genomic instability amongst tumors prompted us to investigate whether alterations in processes/genes involved in maintenance and/or manipulation of the genome were associated with particular types of genomic instability. RESULTS: We discriminated three breast tumor subtypes based on genomic DNA copy number alterations. The subtypes varied with respect to level of genomic instability. We find that shorter telomeres and altered telomere related gene expression are associated with amplification, implicating telomere attrition as a promoter of this type of aberration in breast cancer. On the other hand, the numbers of chromosomal alterations, particularly low level changes, are associated with altered expression of genes in other functional classes (mitosis, cell cycle, DNA replication and repair). Further, although loss of function instability phenotypes have been demonstrated for many of the genes in model systems, we observed enhanced expression of most genes in tumors, indicating that over expression, rather than deficiency underlies instability. CONCLUSION: Many of the genes associated with higher frequency of copy number aberrations are direct targets of E2F, supporting the hypothesis that deregulation of the Rb pathway is a major contributor to chromosomal instability in breast tumors. These observations are consistent with failure to find mutations in sporadic tumors in genes that have roles in maintenance or manipulation of the genome.

Bladder cancer stage and outcome by array-based comparative genomic hybridization.

PURPOSE: Bladder carcinogenesis is believed to follow alternative pathways of disease progression driven by an accumulation of genetic alterations. The purpose of this study was to evaluate associations between measures of genomic instability and bladder cancer clinical phenotype. EXPERIMENTAL DESIGN: Genome-wide copy number profiles were obtained for 98 bladder tumors of diverse stages (29 pT(a), 14 pT1, 55 pT(2-4)) and grades (21 low-grade and 8 high-grade superficial tumors) by array-based comparative genomic hybridization (CGH). Each array contained 2,464 bacterial artificial chromosome and P1 clones, providing an average resolution of 1.5 Mb across the genome. A total of 54 muscle-invasive cases had follow-up information available. Overall outcome analysis was done for patients with muscle-invasive tumors having "good" (alive >2 years) versus "bad" (dead in <2 years) prognosis. RESULTS: Array CGH analysis showed significant increases in copy number alterations and genomic instability with increasing stage and with outcome. The fraction of genome altered (FGA) was significantly different between tumors of different stages (pT(a) versus pT1, P = 0.0003; pT(a) versus pT(2-4), P = 0.02; and pT1 versus pT(2-4), P = 0.03). Individual clones that differed significantly between different tumor stages were identified after adjustment for multiple comparisons (false discovery rate < 0.05). For muscle-invasive tumors, the FGA was associated with patient outcome (bad versus good prognosis patients, P = 0.002) and was identified as the only independent predictor of overall outcome based on a multivariate Cox proportional hazards method. Unsupervised hierarchical clustering separated "good" and "bad" prognosis muscle-invasive tumors into clusters that showed significant association with FGA and survival (Kaplan-Meier, P = 0.019). Supervised tumor classification (prediction analysis for microarrays) had a 71% classification success rate based on 102 unique clones. CONCLUSIONS: Array-based CGH identified quantitative and qualitative differences in DNA copy number alterations at high resolution according to tumor stage and grade. Fraction genome altered was associated with worse outcome in muscle-invasive tumors, independent of other clinicopathologic parameters. Measures of genomic instability add independent power to outcome prediction of bladder tumors.