Thomas M. Baer

Gene expression profiles of human breast cancer progression

Although distinct pathological stages of breast cancer have been described, the molecular differences among these stages are largely unknown. Here, through the combined use of laser capture microdissection and DNA microarrays, we have generated in situ gene expression profiles of the premalignant, preinvasive, and invasive stages of human breast cancer. Our data reveal extensive similarities at the transcriptome level among the distinct stages of progression and suggest that gene expression alterations conferring the potential for invasive growth are already present in the preinvasive stages. In contrast to tumor stage, different tumor grades are associated with distinct gene expression signatures. Furthermore, a subset of genes associated with high tumor grade is quantitatively correlated with the transition from preinvasive to invasive growth.

Single-frequency operation in solid-state laser materials with short absorption depths

Single-frequency output power of 35 mW has been observed in 3.0% doped Nd:YVO/sub 4/ pumped with a 200-mW laser diode. The single-frequency operation is due to the spatial overlap of longitudinal modes at the surface of a mirror in the laser cavity where the material is pumped. The strong absorption coefficient at 808.7 nm and narrow bandwidth of the Nd:YVO/sub 4/ only allow a single laser mode to operate. The effect is modeled theoretically and compared with the experiment. >

Synchronously pumped dye laser using ultrashort pump pulses

The output of a continuous wave (cw) mode-locked Nd:YAG laser has been compressed and then frequency doubled to provide a new pumping source for a synchronously pumped dye laser. Using Rhodamine 6G, tunable subpicosecond pulses have been generated in both synchronously pumped and synchronously pumped cavity dumped configurations.

Performance of diode-pumped Nd:YAG and Nd:YLF lasers in a tightly folded resonator configuration

The performance of diode bar pumped Nd:YAG and Nd:YLF lasers in a novel tightly folded resonator configuration is described. For Nd:YLF at a pump level of 9.2 W a TEM/sub 00/ output power of 3.1 W with a slope efficiency of 47% was observed. In Q-switched operation a pulse energy of 0.7 mJ and a pulsewidth of 5 ns were obtained. The same resonator design configured to operate as an optical amplifier provides small-signal gain of 28 dB and average output powers of greater than 1 W. >

Peak power fluctuations in optical pulse compression

The effects of fluctuations in input intensity and pulsewidth on the output pulse of an optical pulse compressor are investigated. The performance of the fiber grating pulse compressor is modeled in the absence of group velocity dispersion. This model is verified by comparison to experimental results obtained with a compressed, continuous-wave, mode-locked Nd:YAG laser. An optimized servosystem is designed for the pulse-compressed laser system which results in as much as 50-dB noise reduction at low frequencies. >

High-speed laser microsurgery of alert fruit flies for fluorescence imaging of neural activity.

Significance Microscopy and neurophysiology experiments in live animals commonly involve complex surgical preparations, which are often time-consuming, demand considerable manual dexterity, and can sharply limit experimental throughput. Here we present a spatially precise laser microsurgical technique using a pulsed UV laser. Our approach reduces surgical time by up to two orders of magnitude while substantially improving reproducibility. Using the fruit fly as a model, we show that laser microsurgery leaves complex behaviors intact and allows us to visualize brain activity in live flies for up to 18 h, more than four times longer than reported previously using hand dissection. We also demonstrate laser microsurgery on nematodes, ants, and the mouse cranium, illustrating broad potential utility for both optical and electrophysiological studies. Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-µm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12- to 350-µm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca2+ signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5–20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation.

Discovery of new gene expression predictors for adjuvant tamoxifen outcome for breast cancer patients

9503 Background: A better understanding of tamoxifen resistance and accurately targeting tamoxifen therapy to responsive patients remain critical challenges in breast cancer treatment. Tamoxifen acts primarily by competing with estrogens for binding to the estrogen receptor (ER). Consequently, the presence of ER and PR (progesterone receptor) is currently the best predictor for tamoxifen response. However, 25%, 66% and 55% of ER+/PR+, ER+/PR- and ER-/PR+ tumors, respectively, fail to respond or develop resistance to tamoxifen through mechanisms that remain largely unclear. Therefore, it is currently an unmet medical need to better predict response to tamoxifen. METHODS Using high-density oligonucleotide microarrays, we analyzed gene expression profiles of hormone receptor-positive breast cancers from 60 patients with similar tumor size, nodal status, and tumor grade. These 60 patients were uniformly treated with systemic adjuvant tamoxifen only. In addition, we validated gene expression differences in an independent cohort of 20 hormone-receptor positive, patients treated only with tamoxifen. RESULTS We identified 19 genes (gene names will be disclosed at the presentation) whose expression levels were correlated with treatment outcome. Expression ratios of pairs of these genes were simple and powerful predictors of treatment outcome both in the 60 patients used for discovery and in the independent cohort of 20 patients. Expression of one of the genes in the mammary epithelial cell line MCF10A resulted in increased cell motility and invasion in a manner that is synergistic with EGF, suggesting functional interaction with the EGFR signaling pathway. CONCLUSIONS These results demonstrate the potential for a gene expression-based test for selecting patients for tamoxifen treatment and provide new insight into the mechanisms of tamoxifen resistance. No significant financial relationships to disclose.