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Genetic instability results, in a large majority of solid tumors, in deep chromosomal rearrangements. However, because chromosomal instability produces highly complex caryotypes, rarely showing stereotypic aberrations, it has not been possible to characterize solid cancers according to specific patterns of chromosomal rearrangements. This contrasts with the situation in hematological malignancies, where cytogenetics has allowed to lay out the basis of a renewed classification. New insights have been brought by the development of comparative genomic hybridization (CGH). This molecular cytogenetics approach was originally devised to detect regions in the genome of tumor cells undergoing quantitative changes, i.e. gains or losses of copy numbers. The large body of studies based on CGH show that solid tumors undergo frequent gains and losses and that every chromosomes show at least one region of anomaly. Furthermore, different tumor types present distinct CGH patterns of gains and losses. These observations favor the idea that it may be possible to type human solid cancers according to their patterns of genomic aberrations. However, despite the fact that a number of CGH based studies present data suggesting that different tumor types or cancers at different stages of evolution show distinct patterns of gains and losses, it has proven difficult to be conclusive. This can be mainly attributed to the lack of spatial resolution of CGH. Indeed, CGH uses metaphase chromosomes as hybridization targets and therefore its resolution is at the level of chromosomal banding. The recent adaptation of DNA array technology to CGH will allow to pass this limitation. In DNA array based CGH (array-CGH) metaphase chromosomes have been replaced by spots of cloned DNA. These DNA clones may either be genomic (BACs, YACs or cosmids) or coding (cDNAs). The resolution of array-CGH is therefore determined by the size of the cloned DNA insert (100 Kb for BACs, 1-2 kb for cDNAs). Data corresponding to each of these clones is or will be in a near future linked to DNA sequence data. Hence, in a near future, array-CGH will allow to increase the resolution from a cytogenetic level to a molecular level. Finally, because array technology is highly adaptable to automation, going from classical CGH to array-CGH will produce a quantum leap in throughput.

Chromosome 17q is frequently rearranged in breast cancer. Allelotyping studies have proposed the existence of at least four regions of allelic imbalance (AI). Here we present a study combining allelotyping using 19 CA repeat markers mapping in the 17q21-25 region and molecular cytogenetics (CGH and FISH). Allelotyping was undertaken on 178 pairs of cognate tumor and normal DNA in order to determine the number of regions of AI and define the shortest overlaps. AI ranged from 34-54% of the informative cases according to the marker and, overall, 66% of the tumors presented AI at one of the markers tested. Analysis of the patterns of imbalances revealed at least five common regions of imbalance respectively defined by markers: D17S855, which is intragenic of BRCA1 (SRO 1), D17S1607 (SRO 2), D17S1855 (SRO 3), between D17S789 and D17S785 (SRO 4) and D17S784 (SRO 5). In order to characterize the nature of the genetic events revealed by allelotyping we performed CGH analysis on a subset of 43 tumors presenting variable patterns of imbalance. CGH showed that AI at 17q could represent four different types of genetic events: loss of chromosome 17, gain of 17q, gain of 17q22-q24, loss of 17q11-q21 and/or 17q25-qter. Some of these anomalies could occur concomitantly within the same tumor. Since 35% of the tumors analysed by CGH presented gains, these data indicated that AI at 17q were not solely indicative of losses of genetic material and could also represent DNA amplification. Gains were most commonly observed in the 17q23-q24 regions. This suggested that AI in SRO 2 and SRO 3 corresponded to DNA amplification. To assess this, we isolated BAC clones by PCR screening for markers D17S1607 and D17S1855 and used these in FISH experiments on six breast tumor cell lines and 14 breast cancer specimens. FISH results showed that both D17S1607 and D17S1855 were frequently involved in DNA amplification (8-30 copies). Altogether, our data show that allelotyping can be efficiently used in amplicon mapping. Clinico-pathological correlations indicated that imbalance at 17q preferentially occurred in high grade, PR- and ERBB2 amplified tumors.

Both detection and chromosomal analysis of fetal cells present in the maternal circulation can be performed using Histopaque double density gradient centrifugation followed by primed in situ (PRINS) labeling technique. This approach has been tested on blood samples from 15 pregnant women and 6 control donors with primers specific for chromosomes 9, X and Y. The cell separation technique allows recovery of both mononuclear cells and polynuclear cells with a 97% efficiency. PRINS labeling was successful in 100% cells from control blood samples. Among patient samples, 2 "false-negative" results were observed. These preliminary results suggest that the present protocol might be efficient for non-invasive prenatal chromosome analysis.

Prenatal diagnosis is presently performed following invasive procedures with variable risks of fetal loss; non-invasive procedures using fetal cells in maternal blood would be welcome for the early detection of fetal sex or aneuploidy. We describe a simple and rapid protocol to detect fetal cells and thus to assess fetal sex. In a first step, nucleated blood cells were separated into mononuclear and polynuclear cells using a double density gradient centrifugation. In a second step primed in situ (PRINS) labelling technique was performed to label Y-chromosomes. 15 samples were studied and correct gender assignment was made in 13/15. The number of labelled nuclei was higher in polynuclear cell phases than in mononuclear cell phases. Moreover, the polylobular aspect of labelled nuclei from polynuclear cell phases strongly suggested that they could belong to fetal polynuclear cells. The PRINS technique combines some advantages of FISH, such as visual assessment of in situ chromosome labelling and the powerful specificity and sensitivity of PCR. In association with a simple enrichment procedure it constitutes a rapid protocol for fetal cell detection, non-invasive early prenatal sex assessment, and could further be applied to detect the main viable aneuploidies.