Faculty Bibliography

Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb(f), collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb(f) loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb(f) function; Rb(f) inactivation by T(121), a fragment of SV40 T antigen that binds to and inactivates pRb(f) proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.

Gene amplification is an important mechanism of oncogene activation in breast and other cancers. Characterization of amplified regions of the genome in breast cancer has led to the identification of important oncogenes including erbB-2/HER-2, C-MYC, and fibroblast growth factor receptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10-15% of human breast cancers. The putative oncogene FGFR1 localizes to this region; however, we show evidence that FGFR inhibition fails to slow growth of three breast cancer cell lines with 8p11-p12 amplification. We present a detailed analysis of this amplicon in three human breast cancer cell lines using comparative genomic hybridization, traditional Southern and Northern analysis, and chromosome 8 cDNA microarray expression profiling. This study has identified new candidate oncogenes within the 8p11-p12 region, supporting the hypothesis that genes other than FGFR1 may contribute to oncogenesis in breast cancers with proximal 8p amplification.

The development of solid tumors is associated with acquisition of complex genetic alterations, indicating that failures in the mechanisms that maintain the integrity of the genome contribute to tumor evolution. Thus, one expects that the particular types of genomic alterations seen in tumors reflect underlying failures in maintenance of genetic stability, as well as selection for changes that provide growth advantage. In order to investigate genomic alterations we are using microarray-based comparative genomic hybridization (array CGH). The computational task is to map and characterize the number and types of copy number alterations present in the tumors, and so define copy number phenotypes and associate them with known biological markers. To utilize the spatial coherence between nearby clones, we use an unsupervised hidden Markov models approach. The clones are partitioned into the states which represent the underlying copy number of the group of clones. The method is demonstrated on the two cell line datasets, one with known copy number alterations. The biological conclusions drawn from the analyses are discussed. © 2004 Elsevier Inc. All rights reserved.

BACKGROUND & AIMS: Pathogenesis of gastric cancer is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. In order to investigate the patterns of chromosomal aberrations in gastric carcinomas, we performed genome-wide microarray based comparative genomic hybridisation (microarray CGH). With this recently developed technique chromosomal aberrations can be studied with high resolution and sensitivity. METHODS: Array CGH was applied to a series of 35 gastric adenocarcinomas using a genome-wide scanning array with 2275 BAC and P1 clones spotted in triplicate. Each clone contains at least one STS for linkage to the sequence of the human genome. These arrays provide an average resolution of 1.4 Mb across the genome. DNA copy number changes were correlated with clinicopathological tumour characteristics as well as survival. RESULTS: All thirty-five cancers showed chromosomal aberrations and 16 of the 35 tumours showed one or more amplifications. The most frequent aberrations are gains of 8q24.2, 8q24.1, 20q13.12, 20q13.2, 7p11.2, 1q32.3, 8p23.1-p23.3, losses of 5q14.1, 18q22.1, 19p13.12-p13.3, 9p21.3-p24.3, 17p13.1-p13.3, 13q31.1, 16q22.1, 21q21.3, and amplifications of 7q21-q22, and 12q14.1-q21.1. These aberrations were correlated to clinicopathological characteristics and survival. Gain of 1q32.3 was significantly correlated with lymph node status (p=0.007). Tumours with loss of 18q22.1, as well as tumours with amplifications were associated with poor survival (p=0.02, both). CONCLUSIONS: Microarray CGH has revealed several chromosomal regions that have not been described before in gastric cancer at this frequency and resolution, such as amplification of at 7q21-q22 and 12q14.1-q21.1, as well gains at 1q32.3, 7p11.2, and losses at 13q13.1. Interestingly, gain of 1q32.3 and loss of 18q22.1 are associated with a bad prognosis indicating that these regions could harbour gene(s) that may determine aggressive tumour behaviour and poor clinical outcome.

INTRODUCTION: Cyclin E, a G1 cyclin essential for G1-S phase transition, is known to have a profound effect on tumorigenesis. Elevated levels of cyclin E have been associated with breast cancer, and chromosomal instability observed in breast cancer is suggested to be associated with constitutive expression of cyclin E. It was previously demonstrated that SUM149PT human breast cancer cells show very high levels of cyclin E expression by western analysis and that they express a nonfunctional cyclin E ubiquitin ligase due to a mutation in the F-box protein hCdc4. METHODS: We examined cyclin E expression in both MCF10A and SUM149PT cells using western blot analysis and flow cytometry. Immunofluorescence was utilized for the localization of cyclin E in both normal and breast cancer cells. In addition, array comparative genomic hybridization analysis was performed to compare chromosome copy number alterations with levels of cyclin E expression among a panel of breast cancer cell lines. RESULTS: SUM149PT cells overexpress cyclin E on a cell per cell basis for the duration of the cell cycle. High cyclin E levels are maintained throughout the S phase, and SUM149PT cells exhibit an S phase delay or arrest probably due to cyclin E overexpression. In addition, comparative genomic hybridization indicated that SUM149PT cells exhibit many chromosome copy number alterations, which may reflect prior or ongoing genomic instability. However, no direct correlation was observed between cyclin E levels and genomic copy number alteration in a panel of human breast cancer cell lines. CONCLUSIONS: Cyclin E is overexpressed at high levels throughout the cell cycle in SUM149PT cells, which is in stark contrast to cyclin E degradation observed in the mid to late S phase of normal cells. SUM149PT cells are unable to regulate cyclin E and also exhibit many copy number alterations. However, there was a lack of direct correlation between cyclin E overexpression and chromosomal instability across a panel of other breast cancer cell lines examined.

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32 855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human fingerprint map, 99% of the current assembled sequence and has an effective resolving power of 79 kb. We have made the clone set publicly available, anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease.

The RAS/mitogen-activated protein kinase pathway sends external growth-promoting signals to the nucleus. BRAF, a critical serine/threonine kinase in this pathway, is frequently activated by somatic mutation in melanoma. Using a cohort of 115 patients with primary invasive melanomas, we show that BRAF mutations are statistically significantly more common in melanomas occurring on skin subject to intermittent sun exposure than elsewhere (23 of 43 patients; P<.001, two-sided Fisher's exact test). By contrast, BRAF mutations in melanomas on chronically sun-damaged skin (1 of 12 patients) and melanomas on skin relatively or completely unexposed to sun, such as palms, soles, subungual sites (6 of 39 patients), and mucosal membranes (2 of 21 patients) are rare. We found no association of mutation status with clinical outcome or with the presence of an associated melanocytic nevus. The mutated BRAF allele was frequently found at an elevated copy number, implicating BRAF as one of the factors driving selection for the frequent copy number increases of chromosome 7q in melanoma. In summary, the uneven distribution of BRAF mutations strongly suggests distinct genetic pathways leading to melanoma. The high mutation frequency in melanomas arising on intermittently sun-exposed skin suggests a complex causative role of such exposure that mandates further evaluation.

Alterations in the genome that lead to changes in DNA sequence copy number are a characteristic of solid tumors and are found in association with developmental abnormalities and/or mental retardation. Comparative genomic hybridization (CGH) can be used to detect and map these changes. Recent improvements in the resolution and sensitivity of CGH have been possible through implementation of microarray-based CGH (array CGH). Here we discuss the performance characteristics of different array platforms and review some of the recent applications of array CGH in cancer and medical genetics.

BACKGROUND: Gastric cancer is one of the most frequent malignancies in the world, ranking fifth in the Netherlands as a cause of cancer death. Surgery is the only curative treatment for advanced cases, but results of gastrectomy largely depend on the stage of the disease. A better understanding of the mechanisms of progression from a preneoplastic condition through intraepithelial neoplasia to invasive cancer may provide information relevant to designing focused prevention strategies. METHODS: Because the pattern of chromosomal aberrations in precursors of gastric cancer is unclear, 11 gastric polyps with intraepithelial neoplasia (three hyperplastic polyps and eight adenomas) were analysed by microarray comparative genomic hybridisation to study chromosomal instability in precursors of gastric cancer. RESULTS: Chromosomal aberrations were detected in all specimens. Adenomas showed no more chromosomal aberrations than did the hyperplastic polyps. The most frequent aberrations were gain of 7q36 and 20q12, and loss of 5q14-q21 in the adenomas, and loss of 15q11-14, 1p21-31, and 21q11-21.2 in the hyperplastic polyps. The most frequent chromosomal aberration in common to both types was loss of 9p21.3. CONCLUSION: Hyperplastic polyps showed many chromosomal aberrations, confirming that neoplastic transformation can occur in these lesions. These observations are consistent with the existence of two morphologically and genetically distinct pathways to gastric cancer-the hyperplastic polyp pathway and the (intestinal type) adenoma pathway. The relative contribution of each to gastric carcinogenesis in general, and how they compare to patterns of chromosomal aberrations in the more prevalent flat foci of intraepithelial neoplasia remain to be determined.