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

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.

FBXW7 and DNA copy number instability.

SKP1-cullin-F-box protein (SCF) type ubiquitin ligases degrade proteins controlling the G1/S transition. Deficiency for FBXW7 (also known as hCDC4), which encodes the F-box protein of the SCF type ubiquitin ligase is associated with genomic instability. Here, we investigated the association of FBXW7 deficiency with chromosomal instability in breast cancer. We screened 49 tumors previously profiled by array CGH for mutations in conserved regions of FBXW7, but found no mutations. Copy number loss of FBXW7, however was associated with enhanced genomic instability in the Complex breast tumor subtype, but instability may not be due to FBXW7 haploinsufficiency, since transcript levels were not reduced in tumors with loss of the locus, whereas reduced expression was observed for other neighboring genes involved in maintenance of genome stability. We also investigated whether cells deficient for FBXW7 showed enhanced instability by challenging cells with methotrexate and assessing numbers of genomic alterations arising in resistant cells. Although methotrexate resistant colonies formed at high frequencies in HCT116 FBXW7+/- and HCT116 FBXW7-/- cells compared to parental HCT116, few copy number alterations were detected in the resistant cells. Taken together these studies suggest that FBXW7 deficiency is unlikely to contribute to the extensive copy number aberrations associated with breast and possibly other tumor types.

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.

Genome position and gene amplification.

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.

Chromosomal aberrations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma unspecified: A matrix-based CGH approach.

Angioimmunoblastic T-cell lymphoma (AILT) is a histopathologically well-defined entity. However, despite a number of cytogenetic studies, the genetic basis of this lymphoma entity is not clear. Moreover, there is an overlap to some cases of peripheral T-cell lymphoma unspecified (PTCL-u) in respect to morphological and genetic features. We used array-based comparative genomic hybridization (CGH) to study genetic imbalances in 39 AILT and 20 PTCL-u. Array-based CGH revealed complex genetic imbalances in both AILT and PTCL-u. Chromosomal imbalances were more frequent in PTCL-u than in AILT and gains exceeded the losses. The most recurrent changes in AILT were gains of 22q, 19, and 11p11-q14 (11q13) and losses of 13q. The most frequent changes in PTCL-u were gains of 17 (17q11-q25), 8 (involving the MYC locus at 8q24), and 22q and losses of 13q and 9 (9p21-q33). Interestingly, gains of 4q (4q28-q31 and 4q34-qtel), 8q24, and 17 were significantly more frequent in PTCL-u than in AILT. The regions 6q (6q16-q22) and 11p11 were predominantly lost in PTCL-u. Moreover, we could identify a recurrent gain of 11q13 in both AILT and PTCL-u, which has previously not been described in AILT. Trisomies 3 and 5, which have been described as typical aberrations in AILT, were identified only in a small number of cases. In conclusion, CGH revealed common genetic events in peripheral T-cell lymphomas as well as peculiar differences between AILT and PTCL-u.

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.

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.

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.