High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P ≤ 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 × 108 transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.

Detection and characterization of carcinoma cells in the blood

A highly sensitive assay combining immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis has been developed to detect, enumerate, and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood. Peripheral blood (10–20 ml) from 30 patients with carcinoma of the breast, from 3 patients with prostate cancer, and from 13 controls was examined by flow cytometry for the presence of circulating epithelial cells defined as nucleic acid+, CD45−, and cytokeratin+. Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with cancer including 17 with organ-confined disease. To determine whether the circulating epithelial cells in the cancer patients were neoplastic cells, cytospin preparations were made after immunomagnetic enrichment and were analyzed. Epithelial cells from patients with breast cancer generally stained with mAbs against cytokeratin and 3 of 5 for mucin-1. In contrast, no cells that stained for these antigens were observed in the blood from normal controls. The morphology of the stained cells was consistent with that of neoplastic cells. Of 8 patients with breast cancer followed for 1–10 months, there was a good correlation between changes in the level of tumor cells in the blood with both treatment with chemotherapy and clinical status. The present assay may be helpful in early detection, in monitoring disease, and in prognostication.

Biochemical changes in the frontal lobe of HIV-infected individuals detected by magnetic resonance spectroscopy

We have developed a proton magnetic resonance spectroscopy method that selectively can sample cortical gray matter and adjacent white matter in the frontal lobe. We have used this approach to study a group of patients (n = 7) infected with HIV and clinical manifestations of the AIDS dementia complex (ADC), a group of patients (n = 8) infected with HIV without any indications of ADC, and seven controls. The patients without ADC had a statistically significant increase in the ratio of myo-inositol to creatine in white matter compared with normal controls. In contrast, the group of patients with ADC had almost normal levels of myo-inositol to creatine in both gray matter and white matter and showed a statistically significant decrease in the N-acetylaspartate to creatine ratio in gray matter compared with either the normal controls or the patients without ADC. Patterns of spectral abnormalities correlated with neuropsychological measures of frontal lobe dysfunction, suggesting that the evaluation of frontal lobe metabolism by magnetic resonance spectroscopy can play a role in the early detection of ADC, in determining its progression, and in assessing responses to therapeutic interventions.

In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D–Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D–Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Neuronal coincidence detection by voltage-sensitive electrical synapses

Coincidence detection is important for functions as diverse as Hebbian learning, binaural localization, and visual attention. We show here that extremely precise coincidence detection is a natural consequence of the normal function of rectifying electrical synapses. Such synapses open to bidirectional current flow when presynaptic cells depolarize relative to their postsynaptic targets and remain open until well after completion of presynaptic spikes. When multiple input neurons fire simultaneously, the synaptic currents sum effectively and produce a large excitatory postsynaptic potential. However, when some inputs are delayed relative to the rest, their contributions are reduced because the early excitatory postsynaptic potential retards the opening of additional voltage-sensitive synapses, and the late synaptic currents are shunted by already opened junctions. These mechanisms account for the ability of the lateral giant neurons of crayfish to sum synchronous inputs, but not inputs separated by only 100 μsec. This coincidence detection enables crayfish to produce reflex escape responses only to very abrupt mechanical stimuli. In light of recent evidence that electrical synapses are common in the mammalian central nervous system, the mechanisms of coincidence detection described here may be widely used in many systems.

Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.

Fluorescence tomographic imaging in turbid media using early-arriving photons and Laplace transforms

We present a multichannel tomographic technique to detect fluorescent objects embedded in thick (6.4 cm) tissue-like turbid media using early-arriving photons. The experiments use picosecond laser pulses and a streak camera with single photon counting capability to provide short time resolution and high signal-to-noise ratio. The tomographic algorithm is based on the Laplace transform of an analytical diffusion approximation of the photon migration process and provides excellent agreement between the actual positions of the fluorescent objects and the experimental estimates. Submillimeter localization accuracy and 4- to 5-mm resolution are demonstrated. Moreover, objects can be accurately localized when fluorescence background is present. The results show the feasibility of using early-arriving photons to image fluorescent objects embedded in a turbid medium and its potential in clinical applications such as breast tumor detection.

Detection of targeted GFP-Hox gene fusions during mouse embryogenesis

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13GFPneo embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13lacZneo embryos. Similarly, Hoxa1GFPneo expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1GFPneo embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

Detection of neuritic plaques in Alzheimer’s disease by magnetic resonance microscopy

Magnetic resonance microscopy (MRM) theoretically provides the spatial resolution and signal-to-noise ratio needed to resolve neuritic plaques, the neuropathological hallmark of Alzheimer’s disease (AD). Two previously unexplored MR contrast parameters, T2* and diffusion, are tested for plaque-specific contrast to noise. Autopsy specimens from nondemented controls (n = 3) and patients with AD (n = 5) were used. Three-dimensional T2* and diffusion MR images with voxel sizes ranging from 3 × 10−3 mm3 to 5.9 × 10−5 mm3 were acquired. After imaging, specimens were cut and stained with a microwave king silver stain to demonstrate neuritic plaques. From controls, the alveus, fimbria, pyramidal cell layer, hippocampal sulcus, and granule cell layer were detected by either T2* or diffusion contrast. These structures were used as landmarks when correlating MRMs with histological sections. At a voxel resolution of 5.9 × 10−5 mm3, neuritic plaques could be detected by T2*. The neuritic plaques emerged as black, spherical elements on T2* MRMs and could be distinguished from vessels only in cross-section when presented in three dimension. Here we provide MR images of neuritic plaques in vitro. The MRM results reported provide a new direction for applying this technology in vivo. Clearly, the ability to detect and follow the early progression of amyloid-positive brain lesions will greatly aid and simplify the many possibilities to intervene pharmacologically in AD.

Early physiological abnormalities after simian immunodeficiency virus infection

Central nervous system (CNS) damage and dysfunction are devastating consequences of HIV infection. Although the CNS is one of the initial targets for HIV infection, little is known about early viral-induced abnormalities that can affect CNS function. Here we report the detection of early physiological abnormalities in simian immunodeficiency virus-infected monkeys. The acute infection caused a disruption of the circadian rhythm manifested by rises in body temperature, observed in all five individuals between 1 and 2 weeks postinoculation (p.i.), accompanied by a reduction in daily motor activity to 50% of control levels. Animals remained hyperthermic at 1 and 2 months p.i. and returned to preinoculation temperatures at 3 months after viral inoculation. Although motor activity recovered to baseline values at 1 month p.i., activity levels then decreased to approximately 50% of preinoculation values over the next 2 months. Analysis of sensory-evoked responses 1 month p.i. revealed distinct infection-induced changes in auditory-evoked potential peak latencies that persisted at 3 months after viral inoculation. These early physiological abnormalities may precede the development of observable cognitive or motor deficiencies and can provide an assay to evaluate agents to prevent or alleviate neuronal dysfunction.

Parallel detection of violations of color constancy

The perceived colors of reflecting surfaces generally remain stable despite changes in the spectrum of the illuminating light. This color constancy can be measured operationally by asking observers to distinguish illuminant changes on a scene from changes in the reflecting properties of the surfaces comprising it. It is shown here that during fast illuminant changes, simultaneous changes in spectral reflectance of one or more surfaces in an array of other surfaces can be readily detected almost independent of the numbers of surfaces, suggesting a preattentive, spatially parallel process. This process, which is perfect over a spatial window delimited by the anatomical fovea, may form an early input to a multistage analysis of surface color, providing the visual system with information about a rapidly changing world in advance of the generation of a more elaborate and stable perceptual representation.

Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.