Cytogenetic and flow cytometric studies in a variety of human solid tumors have suggested that gross aneuploidy may arise by a process of abrupt chromosome complement doubling followed by gradual chromosome loss. However, this sequence has not been demonstrated directly in serial studies in individual patients in vivo. The purpose of this study was to search for evidence of chromosome complement doubling and subsequent chromosome loss in flow cytometric ploidy patterns in serial bladder washings and/or biopsies from individual patients with early bladder cancer. Fifty-two patients with noninvasive bladder cancer were followed with serial flow cytometric DNA studies for periods ranging from 5.1 to 42.7 months (median 15.1 months). Serial changes in DNA ploidy and S phase fractions were recorded and correlated with histologic and/or cytologic findings, response to treatment and clinical outcome. The data suggest a series of genetic evolutionary changes in early bladder cancer that include the initial development of peridiploid aneuploidy and repeated rounds of DNA content doubling with chromosome loss in patients with progressive disease. It is likely that gross DNA aneuploidy, and more specifically, DNA multiploidy and DNA hypertetraploidy, all arise by this mechanism. The sequence of DNA diploidy, peridiploid aneuploidy, neartetraploidy, hypotetraploidy and hypertetraploidy is associated with a progressive increase in S phase fraction, and with increasing tumor grade; late steps in this ploidy sequence were often present in tumors that were refractory to local therapeutic measures and tumors that developed deep tumor invasion and/or distant metastases. We conclude that DNA multiploidy and hypertetraploidy are markers of advanced stages of genetic evolution in human bladder cancer. o 1995 Wiley-Liss, Inc.Key terms: Aneuploidy, bladder cancer, prognosis, flow cytometry, tetraploidization Unlike the leukemias, human solid tumors frequently exhibit gross aneuploidy, with chromosome counts often in the range of 60-90 per cell (or a DNA index of 1.3 or greater by flow cytometric DNA analysis). While gross aneuploidy has been implicated in the development and progression of many human solid tumors, its value as a clinical prognostic factor has been controversial. In a recent consensus conference sponsored by the National Cancer Institute, a panel of experts concluded that aneuploidy is a useful prognostic factor in such tumors as ical methods might be helpful in uncovering statistical interactions among various clinical prognostic factors. However, a more fundamental understanding of the biology of the various genetic processes that drive tumor evolution and the mechanisms that underlie the interactions among them may ultimately provide more useful clinical prognostic information.Received for publication June 21, 1994; accepted May 1, 1995. This work was supported by Allegheny-Singer Research Institute grant superficial bladder cancer (34) and in prostate cancer (26), but is not an independent prognostic factor in br...

Human epidermal growth factor receptor-2 (HER2) and epidermal growth factor receptor (EGFR) heterodimerize to activate mitogenic signaling pathways. We have shown previously, using MCF7 subcloned cell lines with graded levels of HER2 expression, that responsiveness to trastuzumab and AG1478 (an anti-EGFR agent), varied directly with levels of HER2 expression. HER2 and EGFR upregulate vascular endothelial growth factor (VEGF), a growth factor that promotes angiogenesis and participates in autocrine growth-stimulatory pathways that might be active in vitro. Here, we show that trastuzumab, erlotinib, and bevacizumab, individually and in combination, inhibit cell proliferation in a panel of unrelated human breast cancer cell lines, in proportion to their levels of HER2 expression. The combination of all three drugs provided a greater suppression of growth than any single drug or twodrug combination in the high HER2 -expressing cell lines (P < 0.001). Combination index analysis suggested that the effects of these drugs in combination were additive. The pretreatment net level of VEGF production in each cell line was correlated with the level of HER2 expression (r = 0.883, P = 0.016). Trastuzumab and erlotinib each reduced total net VEGF production in all cell lines. Multiparameter flow cytometry studies indicated that erlotinib alone and the triple drug combination produced a prolonged but reversible blockade of cells in G 1 , but did not increase apoptosis substantially. These studies suggest that the effects of two and three-drug combinations of trastuzumab, erlotinib, and bevacizumab might offer potential therapeutic advantages in HER2-overexpressing breast cancers, although these effects are of low magnitude, and are likely to be transient. [Mol Cancer Ther 2007;6(10):2664 -74]