Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth.

Representational Oligonucleotide Microarray Analysis (ROMA) detects genomic amplifications and deletions with boundaries defined at a resolution of ∼50 kb. We have used this technique to examine 243 breast tumors from two separate studies for which detailed clinical data were available. The very high resolution of this technology has enabled us to identify three characteristic patterns of genomic copy number variation in diploid tumors and to measure correlations with patient survival. One of these patterns is characterized by multiple closely spaced amplicons, or “firestorms,” limited to single chromosome arms. These multiple amplifications are highly correlated with aggressive disease and poor survival even when the rest of the genome is relatively quiet. Analysis of a selected subset of clinical material suggests that a simple genomic calculation, based on the number and proximity of genomic alterations, correlates with life-table estimates of the probability of overall survival in patients with primary breast cancer. Based on this sample, we generate the working hypothesis that copy number profiling might provide information useful in making clinical decisions, especially regarding the use or not of systemic therapies (hormonal therapy, chemotherapy), in the management of operable primary breast cancer with ostensibly good prognosis, for example, small, node-negative, hormone-receptor-positive diploid cases.

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We have isolated cDNAs for four human genes (DPDE1 through DPDE4) closely related to the dnc learning and memory locus ofDrosophila melanogaster. The deduced amino acid sequences of the Drosophila and human proteins have considerable homology, extending beyond the putative catalytic region to include two novel, highly conserved, upstream conserved regions (UCR1 and UCR2). The upstream conserved regions are located in the amino-terminal regions of the proteins and appear to be unique to these genes. Polymerase chain reaction analysis suggested that these genes encoded the only homologs of dnc in the human genome. Three of the four genes were expressed in Saccharomyces cerevisiae and shown to encode cyclic AMP-specific phosphodiesterases. The products of the expressed genes displayed the pattern of sensitivity to inhibitors expected for members of the type IV, cyclic AMP-specific class of phosphodiesterases. Each of the four genes demonstrated a distinctive pattern of expression in RNA from human cell lines.Regulation of cyclic AMP (cAMP) is important in central nervous system (CNS) function in both invertebrates and mammals. Studies of two different invertebrates in particular have demonstrated a role for cAMP in learning and memory. InAplysia snails, long-term facilitation of neurons in the gill retraction reflex occurs with learning. These neuronal changes are associated with alterations in activity of several components of the cAMP signalling pathway, particularly adenylyl cyclase and a cAMP-dependent protein kinase (32). In Drosophila melanogaster, numerous mutations in genes affecting learning have been isolated, including dunce (dnc), which encodes a cAMP-specific phosphodiesterase (PDE), and rutabaga (rut), which encodes a calcium-calmodulinstimulated adenylyl cyclase (10,16,28). Electrophysiologic studies of neurons from dnc-and rut-flies have shown alterations in synaptic plasticity, suggesting that the functional changes occurring in learning in wild-type flies are similar to those seen in Aplysia snails (40). These observations suggest that modulation of the cAMP pathway can influence learning and memory in organisms widely separated in evolution.Evidence for a functional role of dunce-like PDEs in the mammalian CNS initially came from the development of specific inhibitors. Inhibitors of cAMP-specific PDEs, including the drug rolipram, have clinical activity as antidepressants (11). The initial biochemical analysis of mammalian CNS PDEs, and of these inhibitors, was limited to testing in partially purified preparations, which can contain multiple PDE isoforms (1). More recently, cDNAs for homologs of dnc have been cloned from rats (5,7,9,(36)(37)(38) and shown to be rolipram sensitive (14,37,38

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