Tissue microarrays for high-throughput molecular profiling of tumor specimens

The development and progression of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. Microarrays of cDNA provide a powerful tool for studying these complex phenomena. The tumorigenic properties of a human melanoma cell line, UACC-903, can be suppressed by introduction of a normal human chromosome 6, resulting in a reduction of growth rate, restoration of contact inhibition, and suppression of both soft agar clonogenicity and tumorigenicity in nude mice. We used a high density microarray of 1,161 DNA elements to search for differences in gene expression associated with tumour suppression in this system. Fluorescent probes for hybridization were derived from two sources of cellular mRNA [UACC-903 and UACC-903(+6)] which were labelled with different fluors to provide a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene. The fluorescence signals representing hybridization to each arrayed gene were analysed to determine the relative abundance in the two samples of mRNAs corresponding to each gene. Previously unrecognized alterations in the expression of specific genes provide leads for further investigation of the genetic basis of the tumorigenic phenotype of these cells.

We illustrate the use of fluorescence in situ hybridization (FISH) for analysis of ERBB2 oncogene copy number, the level of amplification (here defined as the ratio of ERBB2 copy number to copy number of chromosome 17 centromeres), and the distribution of amplified genes in breast cancer cell lines and uncultured primary breast carcinomas. The relative ERBB2 copy number determined by FISH in 10 breast cancer cell lines correlated strongly with Southern blot results (r = 0.98) when probes for an identical reference locus were used in the two methods. Metaphase analysis of cell lines showed that amplified ERBB2 copies always occurred in intrachromosomal clusters but that the number and chromosomal location of these clusters varied among the cell lines. In interphase nuclei of primary tumors showing ERBB2 amplification (10/44), ERBB2 copies were seen as one to four clusters, also suggesting intrachromosomal localization. Regardless of the average level of amplification, all these tumors contained highly amplified cell subpopulations with at least 25, and sometimes more than 100, ERBB2 copies per cell. Tumors that did not show amplification by FISH (34/44) had an average of one to five ERBB2 copies scattered randomly in the nuclei and completely lacked cells with high copy levels. FISH results on primary tumors were concordant with slot blot results on amplification and with immunohistochemical detection of overexpression. Quantitative analysis of ERBB2 amplification by FISH may improve prognostic assessments based on the pattern of amplification and detection of heavily amplified tumor cell subpopulations.

A modified comparative genomic hybridization (mCGH) technique was applied to a series of 17 primary breast carcinomas in which cytogenetic study (CG) demonstrated the presence of homogeneously staining region(s), suggesting the occurrence of DNA amplification. mCGH demonstrated recurrent amplifications of the whole chromosome arms 8q (9 times) and 1q (7 times) and of DNA loci in the following bands: 11q13 (6 times), 9p13 and 17q21.1 (4 times), 1q21.1 and 16p11.2 (3 times), and 8q22, 8q24.1, 10q22, 15q26, 17q23, and 20q13.3 (twice). Amplification of whole chromosome arms is likely to have resulted from unbalanced translocations or isochromosomes, whereas amplifications of smaller chromosomal segments probably arose through real DNA amplification processes. In all tumors but one, more than one amplified locus was detected. The fact that many chromosomal sites were involved suggests that the process of amplification is complex and that many genes are potential targets.