A History of Caricature and Grotesque in Literature and Art by Thomas Wright
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Accordingly, the United States Government may have baldus correction bit for horses rights in the invention. The present invention generally relates to a baldus correction bit for horses of biomarkers for predicting survival. More specifically, the present invention relates to methods of predicting survival of a subject with cancer. Lung cancer is the leading cause of cancer death in men and women in the United States.
This high mortality is mainly due to the absence of an effective screening strategy to identify lung cancer at an early curable stage. There are no reliable clinical or molecular predictors for identifying those at high risk for developing recurrent therapy. There is, therefore, a critical need in the art to identify a reliable molecular signature in the tumor that could identify those who are likely to develop recurrent disease.
If such biomarkers are identified, adjuvant therapy could be selectively administered to those at high risk for relapse. Conversely, the low risk group can be baldus correction bit for horses the side effects of adjuvant therapy. Among the several aspects of the invention is provided a method for predicting survival of a subject with lung cancer.
The method comprises measuring the expression of a plurality of biomarkers in a sample of cells from the subject. The expression value of each biomarker is determined, and the expression values are analyzed to generate a risk score of survival.
A risk score with a positive value is indicative of a short survival time and a risk score with a negative value is indicative of a long survival time. An additional aspect is a plurality of biomarkers for predicting survival of a subject with a lung cancer.
In yet another aspect, the invention provides a kit for predicting survival of a subject with a lung cancer. The kit comprises a plurality of agents for measuring the expression of a plurality of biomarkers, means for converting the expression of each biomarker into an expression value, and means for analyzing the expression values to generate a risk score that predicts survival time. In still another aspect, the invention provides an array comprising nucleic acids capable of hybridizing to the mRNAs of the following biomarkers: Positive fold change represents up regulation and negative fold change represent down regulation in short-term survival patients.
Positive CRAPB1 immunoreactivity was observed in the cytoplasm of tumor baldus correction bit for horses from a short-term survivor Bbut not in tumor cells from baldus correction bit for horses long-term survivor Baldus correction bit for horses. The intensity of staining was scored as 1 weak2 intermediateor 3 strong.
The protein score was calculated by multiplying the positive cell score by baldus correction bit for horses intensity score, with 12 being the maximum score.
Panel B presents Kaplan-Meier survival curves for stage IA and IB patients classified as having positive high risk or negative low risk scores based upon the gene expression signature. Panel C presents the area under the curve AUC for survival models based on stage information solid line or expression data dashed line.
Panel A presents Kaplan-Meier survival curves for dataset 6 Gerald et al. The present invention provides methods for predicting survival of a subject with cancer. The prediction method is based upon the differential expression of a plurality of biomarkers in cancer cells. It was discovered that some biomarkers tend to be over-expressed in short-term cancer survivors, whereas other biomarkers tend to be over-expressed in long-term cancer survivors. The unique pattern of expression of these biomarkers in a sample of cells from a subject with cancer may be used to predict relative survival time, and ultimately the prognosis, for that subject.
One aspect of the invention provides a method for predicting cancer survival. The method comprises determining the differential expression of a plurality of biomarkers in a sample of cells from a subject with cancer. The biomarker expression signature of the cancer may be used to derive a risk score that is predictive of survival from that cancer. The score may indicate low risk, such that the subject may survive a long time i. A meta-analysis of data sets from five different microarray studies on lung cancer revealed 64 biomarkers whose expression is highly related to cancer survival see Table A.
Some of the biomarkers are over-expressed in long-term survivors Table Band some of the biomarkers are over-expressed in short-term survivors Table C. A biomarker may play a role in cancer metastasis by affecting cell adhesion e.
A biomarker may also be involved in apoptosis e. A biomarker may play a role in transport mechanisms e.
A biomarker may also be associated with survival in other types of cancer including breast cancer, brain cancer, and gastric cancer e. The method entails measuring the differential expression of a plurality of survival-related biomarkers in a sample of cells from a subject with cancer. The differential pattern of expression in each cancer—or gene expression baldus correction bit for horses then be used to generate a risk score that is predictive of cancer survival.
The level of expression of a biomarker may be increased or decreased in a subject relative to other subjects with cancer. The expression of a biomarker may be higher in long-term survivors than in short-term survivors. Alternatively, the expression of a biomarker may be higher in short-term survivors than in long-term survivors. The differential expression of a plurality of biomarkers may be measured by a variety of techniques that are well known in the art.
Quantifying the levels of the messenger RNA mRNA of a biomarker may be used to measure the expression of the biomarker. Alternatively, quantifying the levels of the protein product of a biomarker may be to measure the expression of the biomarker. Additional information regarding the methods discussed below may be found in Ausubel et al.
One skilled in the art will know which parameters may be manipulated to optimize detection of the mRNA or protein of interest. A nucleic acid microarray may be used to quantify the differential expression of a plurality of biomarkers. Typically, single-stranded nucleic acids e. The arrayed sequences are then hybridized with specific nucleic acid probes from the cells of interest. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescently labeled deoxynucleotides by reverse transcription of RNA extracted from the cells of interest.
Alternatively, the RNA may be baldus correction bit for horses by in vitro transcription and labeled with a marker, such as biotin. The labeled probes are then hybridized to the immobilized nucleic acids on the microchip under highly stringent conditions.
After stringent washing to remove the non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. The raw fluorescence intensity data in the hybridization files are generally preprocessed with the robust multichip average RMA algorithm to generate expression values.
The amount of PCR product is followed cycle-by-cycle in real time, which allows for determination of the initial concentrations of mRNA. The reaction may also be performed with a fluorescent reporter probe that is specific for the DNA being amplified.
The fluorescent reporter probe fluoresces when the quencher is removed during the PCR extension cycle. Muliplex QRT-PCR may be performed by using multiple gene-specific reporter probes, each of which contains a different fluorophore.
Fluorescence values are recorded during each cycle and represent the amount of product amplified to that point in the amplification reaction. To minimize errors and reduce any sample-to-sample variation, QRT-PCR is typically performed using a baldus correction bit for horses standard.
The ideal reference standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. Suitable reference standards include, but are not limited to, mRNAs for the housekeeping genes glyceraldehydephosphate-dehydrogenase GAPDH and beta-actin.
The level of mRNA in the original sample or the fold change baldus correction bit for horses expression of each biomarker may be determined using calculations well known in the art. Immunohistochemical staining may also be used to measure the differential expression of a plurality of biomarkers. This method enables the localization of a protein in the cells of a tissue section by interaction of the protein with a specific antibody. For this, the tissue may be fixed in formaldehyde or another suitable fixative, embedded in wax or plastic, and cut into thin sections from about 0.
Alternatively, the tissue may be frozen and cut into thin sections using a cryostat. The sections of tissue may be arrayed onto and affixed to a solid surface i. The sections of tissue are incubated with a primary antibody against the antigen of interest, followed by washes to remove the unbound antibodies. The primary antibody may be coupled to a detection system, or the primary antibody may be detected with a secondary antibody that is coupled to a detection system.
The detection system may be a fluorophore or it may be an enzyme, such as horseradish peroxidase or alkaline phosphatase, which can convert a substrate into a colorimetric, fluorescent, or chemiluminescent product. The stained tissue sections are baldus correction bit for horses scanned under a microscope.
Because a sample of tissue from a subject with cancer may be heterogeneous, i. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for the biomarker. An enzyme-linked immunosorbent assay, or ELISA, may be used to measure the differential expression of a plurality of biomarkers. All are baldus correction bit for horses on the immobilization of an antigen or antibody on a solid surface, generally a microtiter plate.
The original ELISA method comprises preparing a sample containing the biomarker proteins of interest, coating the wells of a microtiter plate with the sample, incubating each well with a primary antibody that recognizes a specific antigen, washing away the unbound antibody, and then detecting the antibody-antigen complexes.
The antibody-antibody complexes may be detected directly. For this, the primary antibodies are conjugated to a detection system, such as an enzyme that produces a detectable product. The antibody-antibody complexes may be baldus correction bit for horses indirectly. For this, the primary antibody is detected by a secondary antibody that is conjugated to a detection system, as described above.
The microtiter plate is then scanned and the raw intensity data may be converted into expression values using means known in the art.
An antibody microarry may also be used to measure the differential expression of a plurality of biomarkers. For this, a plurality of antibodies is arrayed and covalently attached to the surface of the microarray or biochip. A protein extract containing the biomarker proteins of interest is generally labeled with a fluorescent dye. The labeled biomarker proteins are baldus correction bit for horses with the antibody microarry.
After washes to remove the unbound proteins, the microarray is scanned. The raw fluorescent intensity data maybe converted into expression baldus correction bit for horses using means known in the art.
Luminex multiplexing microspheres may also be used to measure the differential expression of a plurality of biomarkers. Baldus correction bit for horses microscopic polystyrene beads are internally color-coded with fluorescent baldus correction bit for horses, such that each bead has a unique spectral signature of which there are up to Beads with the same signature are tagged with a specific oligonucleotide or specific antibody that will bind the target of interest i.
The target, in turn, is also tagged with a fluorescent reporter. Hence, there are two sources of color, one from the bead and the other from the reporter molecule on the target. The beads are then incubated with the sample containing the targets, of which up may baldus correction bit for horses detected in one well. The captured targets are detected by high-tech fluidics based upon flow cytometry in which lasers excite the internal dyes that identify each bead and also any reporter dye captured during baldus correction bit for horses assay.
The data from the acquisition files may baldus correction bit for horses converted into expression values using means known in the art. In situ hybridization may also be used to measure the differential expression baldus correction bit for horses a plurality of biomarkers.
