Faculty Bibliography

Fallopian tube carcinoma (FTC) is a rare, poorly studied and aggressive cancer, associated with poor survival. Since tumorigenesis is related to the acquisition of genetic changes, we used genome-wide array comparative genomic hybridization to analyse copy number aberrations occurring in FTC in order to obtain a better understanding of FTC carcinogenesis and to identify prognostic events and targets for therapy. We used arrays of 2464 genomic clones, providing approximately 1.4 Mb resolution across the genome to map genomic DNA copy number aberrations quantitatively from 14 FTC onto the human genome sequence. All tumors showed a high frequency of copy number aberrations with recurrent gains on 3q, 6p, 7q, 8q, 12p, 17q, 19 and 20q, and losses involving chromosomes 4, 5q, 8p, 16q, 17p, 18q and X. Recurrent regions of amplification included 1p34, 8p11-q11, 8q24, 12p, 17p13, 17q12-q21, 19p13, 19q12-q13 and 19q13. Candidate, known oncogenes mapping to these amplicons included CMYC (8q24), CCNE1 (19q12-q21) and AKT2 (19q13), whereas PIK3CA and KRAS, previously suggested to be candidate driver genes for amplification, mapped outside copy number maxima on 3q and 12p, respectively. The FTC were remarkably homogeneous, with some recurrent aberrations occurring in more than 70% of samples, which suggests a stereotyped pattern of tumor evolution.

Genome-wide copy number profiles were characterized in 41 primary bladder tumors using array-based comparative genomic hybridization (array CGH). In addition to previously identified alterations in large chromosomal regions, alterations were identified in many small genomic regions, some with high-level amplifications or homozygous deletions. High-level amplifications were detected for 192 genomic clones, most frequently at 6p22.3 (E2F3), 8p12 (FGFR1), 8q22.2 (CMYC), 11q13 (CCND1, EMS1, INT2), and 19q13.1 (CCNE). Homozygous deletions were detected in 51 genomic clones, with four showing deletions in more than one case: two clones mapping to 9p21.3 (CDKN2A/p16, in nine cases), one at 8p23.1 (three cases), and one at 11p13 (two cases). Significant correlations were observed between copy number gain of clones containing CCNE1 and gain of ERBB2, and between gain of CCND1 and deletion of TP53. In addition, there was a significant complementary association between gain of CCND1 and gain of E2F3. Although there was no significant relationship between copy number changes and tumor stage or grade, the linked behavior among genomic loci suggests that array CGH will be increasingly important in understanding pathways critical to bladder tumor biology.

Breast tumors display a wide variety of genomic alterations. This review focuses on DNA copy number variations in these tumors as measured by the recently developed microarray-based form of comparative genomic hybridization. The capabilities of this new technology are reviewed. Initial applications of array CGH to the analysis of breast cancer, and the mechanisms by which the particular types of copy number changes might arise are discussed.

Membrane type serine protease 1 (MT-SP1) is a representative member of a large family of related enzymes known as type II transmembrane serine proteases or membrane type serine proteases. MT-SP1 has been implicated in the selective proteolysis of key extracellular substrates but its physiological role is still not fully understood. MT-SP1 expression at the protein and RNA level has been previously examined by nonquantitative methods such as in situ hybridization, Northern blotting and immunohistochemistry. To establish an introductory understanding of the quantitative mRNA expression of MT-SP1 and to correlate these levels with urokinase-type plasminogen activator receptor (uPAR), a key component of extracellular proteolysis, quantitative RT-PCR was carried out. RNA expression was analyzed in 34 human cancer cell lines, 26 human tissues and 18 primary human breast cancer tissue samples. MT-SP1 mRNA is highly expressed in many breast, ovarian, prostate and colon cancer cell lines and normal human tissues of endodermal origin. At the transcript level, MT-SP1 shows a highly statistically significant correlation (Pearson's product moment correlation r = 0.784, p < 0.001) with uPAR in human breast cancer tissue. The exact role of MT-SP1 in concert with proteins such as uPAR and other members of the plasminogen activator cascade has yet to be ascertained. However, the significant correlation between MT-SP1 and uPAR transcript levels in this initial study suggests further work to establish the role of MT-SP1 as a possible prognostic, diagnostic or therapeutic target for breast cancer.

Gastric carcinogenesis is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. We analysed the patterns of chromosomal instability in 35 gastric carcinomas and their clinical correlations. With microarray competitive genomic hybridization, genomewide chromosomal copy number changes can be studied with high resolution and sensitivity. A genomewide scanning array with 2275 BAC and P1 clones spotted in triplicate was used. This array provided an average resolution of 1.4 Mb across the genome. Patterns of chromosomal aberrations were analysed by hierarchical cluster analysis of the normalized log(2) tumour to normal fluorescence ratios of all clones, and cluster membership was correlated to clinicopathological data including survival. Hierarchical cluster analysis revealed three groups with different genomic profiles that correlated significantly with lymph node status (P=0.02). Moreover, gastric cancer cases from cluster 3 showed a significantly better prognosis than those from clusters 1 and 2 (P=0.02). Genomic profiling of gastric adenocarcinomas based on microarray analysis of chromosomal copy number changes predicted lymph node status and survival. The possibility to discriminate between patients with a high risk of lymph node metastasis could clinically be helpful for selecting patients for extended lymph node resection.

We have used prostate cancer, the most commonly diagnosed noncutaneous neoplasm among men, to investigate the feasibility of performing genomic array analyses of archival tissue. Prostate-specific antigen and a biopsy Gleason grade have not proven to be accurate in predicting clinical outcome, yet they remain the only accepted biomarkers for prostate cancer. It is likely that distinct spectra of genomic alterations underlie these phenotypic differences, and that once identified, may be used to differentiate between indolent and aggressive tumors. Array comparative genomic hybridization allows quantitative detection and mapping of copy number aberrations in tumors and subsequent associations to be made with clinical outcome. Archived tissues are needed to have patients with sufficient clinical follow-up. In this report, 20 formalin-fixed and paraffin-embedded prostate cancer samples originating from 1986 to 1996 were studied. We present a straightforward protocol and demonstrate the utility of archived tissue for array comparative genomic hybridization with a 2400 element BAC array that provides high-resolution detection of both deletions and amplifications.

Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array-CGH (Comparative Genomic Hybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array-CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.

The MALT1 gene was identified through its involvement in t(11;18)(q21;q21), seen in 30% of cases of mucosa-associated lymphoid tissue (MALT) lymphoma. Here, we show that deregulated MALT1 expression may occur in B-cell non-Hodgkin lymphoma (B-NHL) of various histologic subtypes either through translocation to the immunoglobulin heavy chain (IGH) locus or by genomic amplification. First, 2 cases, one case of MALT lymphoma and another of aggressive marginal zone lymphoma (MZL) with t(14;18)(q32;q21), cytogenetically identical to the translocation involving BCL2, were shown by fluorescence in situ hybridization (FISH) to involve MALT1, which lies about 5 Mb centromeric of BCL2. Molecular cloning of both by long-distance inverse polymerase chain reaction showed breakpoints lying 1 to 2 kilobase (kb) centromeric of the first 5' MALT1 exon; both cases showed MALT1 overexpression at either RNA or protein levels. Second, we examined the structure and gene expression profile of genomic amplifications involving 18q21 in a panel of 40 B-NHL cell lines using comparative genomic hybridization to microarrays (array CGH) and gene expression profiling techniques. Using array CGH, 2 peaks of genomic amplification were observed, one centered around BCL2 and the other around MALT1. Ofthe 3 cell lines with MALT1 amplification, 2 showed MALT1 overexpression as assessed by gene profiling, quantitative reverse transcription-polymerase chain reaction (QRT-PCR), and Western blotting. To determine if comparable events occurred in primary MALT and splenic MZL tumors, 40 cases were analyzed by FISH or QRT-PCR; genomic amplification and MALT1 overexpression were seen in 2 cases. Together, these data implicate MALT1 as a dominant oncogene that may play a role in the pathogenesis of B-NHL.

Metastasis is responsible for most deaths from cancer. Currently, little is known about the early genetic events in the metastatic evolution. Here we describe the application of a newly developed strategy for an in-depth characterization of genomic changes in micrometastatic cells. Unique tumor cell lines were established from bone marrow of patients with cancer of the prostate and analyzed by multiplex-FISH (M-FISH) and array CGH. M-FISH revealed that the occult disseminated cells were characterized by very complex numerical and structural aberrations. Many of these aberrations resulted in chromosomal gains and losses, such as losses of 8p, 13q, and 18q and gains of 8q, 9q, 20, and the X chromosome, which are typically observed in prostate cancer. Array CGH allowed an unprecedented high-resolution assessment of copy number changes, pinpointing commonly gained or lost regions, which should narrow down the identification of regions critically involved in metastasis. Thus, occult micrometastatic cells are now amenable to detailed analyses of their genome. Markers for prognosis and treatment decisions can now be established.