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Acquired genomic aberrations associated with methotrexate resistance vary with background genomic instability

Snijders, Antoine M; Hermsen, Mario A; Baughman, Joshua; Buffart, Tineke E; Huey, Bing; Gajduskova, Pavla; Roydasgupta, Ritu; Tokuyasu, Taku; Meijer, Gerrit A; Fridlyand, Jane; Albertson, Donna G
Tumors vary widely in chromosomal level genome instability. To gain a better understanding of the underlying defects which foster specific types of aberrations, we investigated the response of cells of related genetic backgrounds to challenge with methotrexate. We studied mismatch repair deficient HCT116 cells, two derivatives also deficient in XRCC5 (HCT116 Ku86+/-) or BLM (HCT116 BLM-/-), and mismatch repair competent HCT116+chr3 cells. We show that colony formation occurred at a significantly higher frequency in HCT116 cells and HCT116 Ku86+/- cells compared to HCT116 BLM-/- and HCT116+chr3 cells. Visible colonies arose most rapidly in HCT116 Ku86+/- cells, whereas they formed most slowly in HCT116+chr3 cells. Copy number changes acquired by the methotrexate resistant HCT116 and HCT116 BLM-/- cells most often included whole chromosome gains or losses or no acquired copy number changes, whereas resistance in HCT116+chr3 and HCT116 Ku86+/- cells was associated with amplification of DHFR and copy number transitions leading to increased copy number of DHFR, respectively. The additional copies of DHFR were present on unstable chromosomes and organized as inverted repeats in HCT116+chr3 cells, while they were most often present as direct repeats in HCT116 Ku86+/- cells. These observations suggest that different mutational mechanisms promote drug resistance in these genetic backgrounds; mismatch repair deficiency in HCT116, high rates of chromosomal instability in HCT116 Ku86+/-, and low rates of chromosomal instability in HCT116+chr3. On the other hand, it appears that loss of BLM function suppresses the mismatch repair mutator mechanism in mismatch repair and BLM deficient HCT116 BLM-/- cells.
PMID: 17943968
ISSN: 1045-2257
CID: 372592

Genomic approaches to breast cancer subset identification and treatment [Meeting Abstract]

Albertson, D; Chin, K; Devries, S; Feiler, H; Pinkel, D; Spellman, P; Waldman, F; Wang, N; Hennessy, B; Mills, G; Barcellos, HMH; Bissell, M; Guan, Y; Hu, Z; Kuo, WL; McCormick, F; Neve, R; Stampfer, M; Wooster, R; Yaswen, P; Das, D; Fridlyand, J; Correll, E; Jin, J; Nordmeyer, B; Sudar, D; Chew, K; Dairkee, S; Ljung, BM; Hwang, S; Esserman, L; Arbushites, M; Benz, C; Koehler, M; Marks, JD; Zhou, Y; Park, J; Weber, B; Gray, J
ISI:000251398500026
ISSN: 0167-6806
CID: 104667

DNA profiling of primary serous ovarian and fallopian tube carcinomas with array comparative genomic hybridization and multiplex ligation-dependent probe amplification

Nowee, M E; Snijders, A M; Rockx, D A P; de Wit, R M; Kosma, V M; Hamalainen, K; Schouten, J P; Verheijen, R H M; van Diest, P J; Albertson, D G; Dorsman, J C
Primary serous ovarian carcinoma (OVCA) and serous Fallopian tube carcinoma (FTC), both belonging to the BRCA-linked tumour spectrum, share many properties and are treated similarly. However, a detailed molecular comparison has been lacking. We hypothesized that comparative genomic studies of serous OVCAs and FTCs should point to gene regions critically involved in their tumorigenesis. Array comparative genomic hybridization (array CGH) analysis indicated that serous OVCAs and serous FTCs displayed common but also more distinctive patterns of recurrent changes. Targeted gene identification using a dedicated multiplex ligation-dependent probe amplification (MLPA) probe set directly identified EIF2C2 on 8q as a potentially important driver gene. Other previously unappreciated gained/amplified genes included PSMB4 on 1q, MTSS1 on 8q, TEAD4 and TSPAN9 on 12p, and BCAS4 on 20q. SPINT2 and ACTN4 on 19q were predominantly found in FTCs. Gains/amplifications of CCNE1 and MYC, often in conjunction with changes in genes of the AKT pathway, EVI1 and PTK2, seemed to be involved at earlier stages, whereas changes of ERBB2 were associated with advanced stages. The only BRCA1-mutated FTC shared common denominators with the sporadic tumours. In conclusion, the data suggest that serous OVCAs and FTCs, although related, exhibit differences in genomic profiles. In addition to known pathways, new genes/pathways are likely to be involved, with changes in an miRNA-associated gene, EIF2C2, as one important new feature. Dedicated MLPA sets constitute potentially important tools for differential diagnosis and may provide footholds for tailored therapy.
PMID: 17668415
ISSN: 0022-3417
CID: 880752

Detection of single clone deletions using array CGH: identification of submicroscopic deletions in the 22q11.2 deletion syndrome as a model system

Tokuyasu, Taku A; Cotter, Philip D; Segraves, Richard; Harris, Jeffrey; Elder, Melissa E; Gonzales, Marcos; Pinkel, Daniel; Albertson, Donna G; Rauen, Katherine A
Constitutional submicroscopic DNA copy number alterations have been shown to cause numerous medical genetic syndromes, and are suspected to occur in a portion of cases for which the causal events remain undiscovered. Array comparative genomic hybridization (array CGH) allows high-throughput, high-resolution genome scanning for DNA dosage aberrations and thus offers an attractive approach for both clinical diagnosis and discovery efforts. Here we assess this capability by applying array CGH to the analysis of copy number alterations in 44 patients with a phenotype of the 22q11.2 deletion syndrome. Twenty-five patients had the deletion on chromosome 22 characteristic of this syndrome as determined by fluorescence in situ hybridization (FISH). The array measurements were in complete concordance with the FISH analysis, supporting their diagnostic utility. These data show that a genome-scanning microarray has the level of sensitivity and specificity required to prospectively interrogate and identify single copy number aberrations in a clinical setting. We demonstrate that such technology is ideally suited for microdeletion syndromes such as 22q11.2.
PMID: 17394204
ISSN: 1552-4825
CID: 372632

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

Climent, Joan; Dimitrow, Peter; Fridlyand, Jane; Palacios, Jose; Siebert, Reiner; Albertson, Donna G; Gray, Joe W; Pinkel, Daniel; Lluch, Ana; Martinez-Climent, Jose A
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.
PMID: 17234794
ISSN: 0008-5472
CID: 372642

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

Yau, Christina; Fedele, Vita; Roydasgupta, Ritu; Fridlyand, Jane; Hubbard, Alan; Gray, Joe W; Chew, Karen; Dairkee, Shanaz H; Moore, Dan H; Schittulli, Francesco; Tommasi, Stefania; Paradiso, Angelo; Albertson, Donna G; Benz, Christopher C
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.
PMCID:2216076
PMID: 17850661
ISSN: 1465-5411
CID: 372602

Genome position and gene amplification

Gajduskova, Pavla; Snijders, Antoine M; Kwek, Serena; Roydasgupta, Ritu; Fridlyand, Jane; Tokuyasu, Taku; Pinkel, Daniel; Albertson, Donna G
BACKGROUND: Amplifications, regions of focal high-level copy number change, lead to overexpression of oncogenes or drug resistance genes in tumors. Their presence is often associated with poor prognosis; however, the use of amplification as a mechanism for overexpression of a particular gene in tumors varies. To investigate the influence of genome position on propensity to amplify, we integrated a mutant form of the gene encoding dihydrofolate reductase into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells. RESULTS: We observed site-specific differences in methotrexate sensitivity, amplicon organization and amplification frequency. One site was uniquely associated with a significantly enhanced propensity to amplify and recurrent amplicon boundaries, possibly implicating a rare folate-sensitive fragile site in initiating amplification. Hierarchical clustering of gene expression patterns and subsequent gene enrichment analysis revealed two clusters differing significantly in expression of MYC target genes independent of integration site. CONCLUSION: These studies suggest that genome context together with the particular challenges to genome stability experienced during the progression to cancer contribute to the propensity to amplify a specific oncogene or drug resistance gene, whereas the overall functional response to drug (or other) challenge may be independent of the genomic location of an oncogene.
PMCID:2394771
PMID: 17584934
ISSN: 1474-7596
CID: 372622

A critical role for FBXW8 and MAPK in cyclin D1 degradation and cancer cell proliferation

Okabe, Hiroshi; Lee, Sang-Hyun; Phuchareon, Janyaporn; Albertson, Donna G; McCormick, Frank; Tetsu, Osamu
Cyclin D1 regulates G1 progression. Its transcriptional regulation is well understood. However, the mechanism underlying cyclin D1 ubiquitination and its subsequent degradation is not yet clear. We report that cyclin D1 undergoes increased degradation in the cytoplasm during S phase in a variety of cancer cells. This is mediated by phosphorylation at Thr286 through the activity of the Ras/Raf/MEK/ERK cascade and the F-box protein FBXW8, which is an E3 ligase. The majority of FBXW8 is expressed in the cytoplasm during G1 and S phase. In contrast, cyclin D1 accumulates in the nucleus during G1 phase and exits into the cytoplasm in S phase. Increased cyclin D1 degradation is linked to association with FBXW8 in the cytoplasm, and enhanced phosphorylation of cyclin D1 through sustained ERK1/2 signaling. Depletion of FBXW8 caused a significant accumulation of cyclin D1, as well as sequestration of CDK1 in the cytoplasm. This resulted in a severe reduction of cell proliferation. These effects could be rescued by constitutive nuclear expression of cyclin D1-T286A. Thus, FBXW8 plays an essential role in cancer cell proliferation through proteolysis of cyclin D1. It may present new opportunities to develop therapies targeting destruction of cyclin D1 or its regulator E3 ligase selectively.
PMCID:1762433
PMID: 17205132
ISSN: 1932-6203
CID: 372652

Genomic DNA microarray for comparative genomic hybridization

Chapter by: Snijders, Antoine M.; Segraves, Richard L.; Blackwood, Stephanie; Pinkel, Daniel; Albertson, Donna G.
in: Cell Biology, Four-Volume Set by
[S.l. : s.n.], 2006
pp. 445-454
ISBN: 9780121647308
CID: 2785522

Genomic and transcriptional aberrations linked to breast cancer pathophysiologies

Chin, Koei; DeVries, Sandy; Fridlyand, Jane; Spellman, Paul T; Roydasgupta, Ritu; Kuo, Wen-Lin; Lapuk, Anna; Neve, Richard M; Qian, Zuwei; Ryder, Tom; Chen, Fanqing; Feiler, Heidi; Tokuyasu, Taku; Kingsley, Chris; Dairkee, Shanaz; Meng, Zhenhang; Chew, Karen; Pinkel, Daniel; Jain, Ajay; Ljung, Britt Marie; Esserman, Laura; Albertson, Donna G; Waldman, Frederic M; Gray, Joe W
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.
PMID: 17157792
ISSN: 1535-6108
CID: 372662